Analysis of the Tribolium homeotic complex: insights into mechanisms constraining insect Hox clusters

The remarkable conservation of Hox clusters is an accepted but little understood principle of biology. Some organizational constraints have been identified for vertebrate Hox clusters, but most of these are thought to be recent innovations that may not apply to other organisms. Ironically, many model organisms have disrupted Hox clusters and may not be well-suited for studies of structural constraints. In contrast, the red flour beetle, Tribolium castaneum, which has a long history in Hox gene research, is thought to have a more ancestral-type Hox cluster organization. Here, we demonstrate that the Tribolium homeotic complex (HOMC) is indeed intact, with the individual Hox genes in the expected colinear arrangement and transcribed from the same strand. There is no evidence that the cluster has been invaded by non-Hox protein-coding genes, although expressed sequence tag and genome tiling data suggest that noncoding transcripts are prevalent. Finally, our analysis of several mutations affecting the Tribolium HOMC suggests that intermingling of enhancer elements with neighboring transcription units may constrain the structure of at least one region of the Tribolium cluster. This work lays a foundation for future studies of the Tribolium HOMC that may provide insights into the reasons for Hox cluster conservation.
Introduction
Hox clusters arose near the origins of the animal kingdom (Larroux et al. 2007; Ryan et al. 2007). The last common ancestor of the protostomes and deuterostomes is thought to have had a cluster of at least seven genes characterized by a common transcriptional orientation and by colinearity in the order of the genes and their expression domains along the anterior–posterior axis (reviewed in Garcia-Fernandez 2005).
In various metazoan lineages, Hox clusters have gained or lost genes by duplication and deletion but often have maintained their chromosomal order, transcriptional orientation, and both spatial and temporal colinearity of expression patterns (reviewed in Ferrier and Minguillon 2003), suggesting that Hox cluster organization has been subject to strong constraints during evolution. Classical model systems, such as Drosophila and Caenorhabditis elegans, have provided many important insights into the developmental functions of Hox genes but do not provide particularly good examples of Hox cluster conservation. The Hox cluster of Drosophila melanogaster is split into two parts (the Antennapedia (ANTC) and bithorax (BXC) complexes), shows changes in transcriptional orientation of some genes, and includes interspersed genes of independent origin as well as Hox-derived genes that have evolved novel developmental roles (reviewed in Ferrier and Minguillon 2003). These alterations suggest that the constraints keeping the Hox cluster intact may have been lost in the lineage leading to Drosophila. Additional Hox cluster rearrangements (breaks, microinversions, and gene transpositions) have been found in other Drosophila species (Negre et al. 2003; Negre and Ruiz 2007; Von Allmen et al. 1996) as well as in the silk moth Bombyx mori (Yasukochi et al. 2004). The Hox genes of C. elegans (reviewed in Aboobaker and Blaxter 2003) and the tunicate Oikopleura dioica (Seo et al. 2004) have undergone even more extreme loss and rearrangement such that none of their remaining Hox genes are clustered. In most cases, the Hox genes of these organisms still show spatial but not temporal colinearity. Rapid development seems to be the common denominator among most of these organisms, perhaps making temporal colinearity of Hox genes unnecessary, or even undesirable (Ferrier and Holland 2002; Ferrier and Minguillon 2003; Negre et al. 2005).
While studies of disrupted Hox clusters have provided some insights into Hox cluster maintenance, a more complete understanding will require analysis of organisms where they are still intact. Studies of vertebrate Hox clusters have uncovered several potential mechanisms that may promote temporal colinearity and therefore constrain the organization of these clusters (reviewed in Kmita and Duboule 2003). These include progressive changes in chromatin state along the length of the cluster, varying affinity of regulatory elements to a gradient of signal, and the presence of global enhancer elements outside the cluster that regulate multiple genes within the cluster. However, it is not clear whether these mechanisms apply to other organisms.
Duboule (2007) has suggested that the modern vertebrate Hox clusters are actually more organized than the ancestral cluster. Some of the mechanisms constraining the organization of vertebrate Hox clusters likely evolved concomitant with the co-option of Hox genes for functions such as limb development (Duboule 2007; Kmita and Duboule 2003) and, therefore, may not be applicable to other lineages. Based on this model, we might expect to gain a better understanding of the ancestral constraints on Hox clusters by studying a less organized but still intact cluster. Such clusters have been described in organisms as diverse as the cephalochordate amphioxus (Garcia-Fernandez and Holland 1994; Minguillon et al. 2005), sea urchins (Cameron et al. 2006), and the insects Apis (Honey Bee Genome Sequencing Consortium 2006; Dearden et al. 2006) and Anopheles (Holt et al. 2002; Negre and Ruiz 2007). Evidence also suggests that the red flour beetle, Tribolium castaneum, has an intact Hox cluster. Conventional cloning and sequencing of the portion of the cluster corresponding to the Drosophila Antennapedia complex has shown that this region of the homeotic complex (HOMC) is intact in Tribolium (Brown et al. 2002). Genetic mapping also suggests that the integrity of the Tribolium Hox cluster has been maintained (Beeman 1987). Moreover, the genetic methodologies possible with Tribolium, as well as the application of RNAi, have provided a comprehensive description of the full repertoire of Hox genes and their functions (e.g., Beeman et al. 1993; Beeman et al. 1989; Brown et al. 2000; Shippy et al. 2000; Shippy et al. 2006; Stuart et al. 1991; Stuart et al. 1993; Tomoyasu et al. 2005). The Tribolium genome has recently been sequenced, giving us the opportunity to explore the structure of its Hox cluster in detail. Here, we present an analysis of several Hox mutations along with the transcriptional profile of the cluster during embryonic development. We discuss these results with respect to potential mechanisms of Hox cluster organization and constraint.
Materials and methods
Sequence and transposable element analysis
Sequence analysis was performed using Vector NTI Advance 10 (Invitrogen). Basic Local Alignment Search Tools (BLASTs) against Tribolium genome sequence (Tcas_2.0) were performed at http://www.hgsc.bcm.tmc.edu/blast/blast.cgi?organism= Tcastaneum or http://www.ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi?taxid= 7070, and subsequent analysis was performed using Genboree (http://www.genboree.org/java-bin/login.jsp) or NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). The entire HOMC sequence was used as a BLASTn query against a collection of expressed sequence tags (ESTs) provided by Dr. Yoonseong Park (Department of Entomology, Kansas State University, Manhattan, KS, USA). Transposable elements were identified and classified using Censor to search the arthropod subset of Repbase (Kohany et al. 2006).
Array design and probe synthesis
Sequence for the Tribolium HOMC was taken from the Tcas_2.0 Baylor HSGC assembly. The tiled region consists of ∼810,000 bases from LG2 (2,290,000 to 301,000) stretching between the two non-Hox genes flanking the complex. NimbelGen designed ∼50 mer oligos covering this region at two densities: (1) one feature per 91 bp and (2) one feature per 70 bp. An additional region spanning 5 kb on either side of the putative homolog of dme-miR-iab-4 was tiled at a higher density of one feature every 10 bp. Visualization and scaling of tiling data was performed using Integrated Genome Browser (Affymetrix, http://www.affymetrix.com/support/developer/tools/download_igb.affx).
Tribolium 0–72-h-old embryos, grown at 30°C in standard media, were collected by sieving, dechorionated for 2 min in 100% bleach, and homogenized in 200 μl of Trizol using a teflon pestle. Total RNA was then extracted using the standard Trizol protocol (Invitrogen). dsDNA was prepared from ∼10 μg of total RNA with random hexamers according to Kapranov et al. (2002), with the following modifications. Primers were annealed using a 20-min ramp to 15°C, and the first strand reaction was not subdivided for second strand synthesis. The resulting cDNA was used as template by NimbelGen for labeling and hybridization (Squazzo et al. 2006).
Fluorescent in situ hybridization
Probe labeling, embryo fixation, and RNA FISH were performed according to Kosman et al. (2004) with the following modifications. Tribolium embryos were dechorionated in 100% bleach for 2 min and agitated for 45 min to 1 h. Embryos were devitellinized by alternating 1 min vortexing with 1 min shaking for 5 min after the addition of cold methanol, followed by passage through an 18-gauge syringe three to five times. Primers used for making the TcNC-1 and iab-4 probes are as follows: TcNC5′:AGATAAGATATAATGAGGTGTAGAGTTG, TcNC3′: TGATTAACATGGACGGCTTCATTAG, iab-45′: CATCCTATGCACATGCGTTC, iab-43′: CGTTTTAATGGGTGCATCGT. Dig-labeled RNA probes were detected using sheepαDIG (Roche) primary and donkeyαsheep Alexa Fluor 555 (Molecular Probes) secondary antibodies.
Genetics
Beetles were cultured at 30°C on whole wheat flour supplemented with 5% brewer’s yeast as described by Beeman et al. (1989). Strains used were: Ga-1 and Ga-2 (wild type); mxpDch-3/Ey; Cx6/AEs; ptlKT76/+; Cx61/AEs; ptlD60/Ey and Dfd1/AEs. Eyeless (Ey; Beeman et al. 1996) and AbdominalExtra sclerite (AEs; Beeman et al. 1989) are dominantly marked balancer chromosomes that suppress crossing over within the HOMC.
Cuticle preparations were performed as described by Shippy et al. (2000). For documentation, cuticles were placed in 9:1 lactic acid/ethanol on a depression slide and covered with a coverslip. Images were captured at several focal planes using a Nikon DXM1200F digital camera and combined into a single image using Auto-Montage software (Syncrosopy).
RNAi
Parental RNAi for ptl/Tc-Antp was performed by injection of dsRNA into the abdomens of female pupae. Eggs were collected from injected females at 3-day intervals, aged to hatching, and subjected to cuticle preparation.
Analysis of ptlD60 breakpoints
Eggs were collected after overnight incubation at 30°C and allowed to develop for 3 days. Genomic DNA was isolated from individual ptlD60 homozygous larvae as described by Gloor et al. (1993).
Polymerase chain reaction (PCR) surveys of the HOMC were used to identify likely breakpoint positions, and fragments spanning these putative breakpoints were amplified using universal PCR (Beeman and Stauth 1997; Sarkar et al. 1993). PCR products were cloned and sequenced at the Kansas State University DNA Sequencing Center. The resulting sequences were compared to the Tribolium genome sequence to characterize the breakpoints. GenBank accession numbers for these sequences are as follows: ptlD60 A (EF591668) and ptlD60 B (EF591669).
Analysis of mxpDch-3 breakpoints
Tribolium genomic DNA was isolated from mxpDch-3/Ey, AEs/Ey, and Ga-1 pupae as described by Brown et al. (1990), with the exception that DNA was not purified on a CsCl gradient. Digested DNAs were separated on a 0.7% agarose gel by field inversion gel electrophoresis and transferred to GeneScreen nylon membrane (NEN Life Sciences). To look for restriction fragment length polymorphisms associated with mxpDch-3, the blot was probed with pBmxp2.1, a 5.2 kb HindIII fragment containing the 5′ end of the mxp/Tc-pb coding region.
Inverse PCR (Ochman et al. 1988) was used to clone breakpoints associated with mxpDch-3. mxpDch-3/Ey genomic DNA was digested with a restriction enzyme (EcoRI, HindIII, and RsaI for breakpoint fragments A, B, and C, respectively). After circularization of the fragments, two rounds of PCR were performed with primers designed from known sequence. Resulting fragments were cloned using the TOPO-TA Cloning Kit (Invitrogen), sequenced, and submitted to GenBank under the following accession numbers: Dch3 A (EF591670), Dch3 B (EF591671), Dch3 C (EF591672). Sequences were compared to the Tribolium genome sequence to determine the location of breakpoints.
Analysis of ptlKT76 transposon insertion site
The ptlKT76 piggyBac-insertion site was amplified by vectorette PCR as described by Lorenzen et al. (2007). The resulting product was sequenced by Elim Biopharmaceuticals, Inc. (Hayward, CA, USA) and the sequence was submitted to GenBank as accession number EU056827.
Results
The Tribolium Hox cluster has retained an ancestral organization
Several bacterial artificial chromosome (BAC) clones encompassing the ANTC-like region of the Tribolium Hox cluster were previously sequenced and annotated (Brown et al. 2002). Using the newly assembled Tribolium genome sequence, we have performed a similar analysis of the BXC-like portion of the cluster and find that this region contains the Tribolium orthologs of Ultrabithorax (Ubx; Bennett et al. 1999), abdominal-A (abd-A; Shippy et al. 1998) and Abdominal-B (Abd-B). As in Drosophila, the transcription units in this part of the complex are larger than those of the ANTC-like portion due to the presence of longer introns.
As expected from previous molecular and genetic studies (Beeman 1987; Brown et al. 2002), all of the Tribolium Hox genes map to a single cluster on LG2. This cluster spans approximately 756 Kb within a single scaffold of the assembled genome sequence. A few small sequencing gaps are present in the assembly, but more than half can be filled by other available sequences (i.e., the three BAC clones previously sequenced for the ANTC-like portion of the cluster and four BACs from the BXC-like region sequenced for verification of the shotgun genome assembly; Tribolium Genome Consortium 2008). The total length of the filled gaps is approximately 2,635 bp (mismatches in the sequence flanking the gaps lead to some ambiguity), which is only slightly longer than the estimated total length of these gaps (1,938 bp). Thus, estimation of sequencing gaps in the HOMC region appears to be quite accurate.
Two of these gaps are immediately adjacent to transposable element insertion sites and may result from difficulties in assembling repetitive DNA. These two sites account for about 1,300 bp of the total gaps in the HOMC. In two other cases, gaps in the genome assembly are associated with tandem duplications that are not present in the BAC assemblies: an approximately 160-bp duplication between Tc-Deformed (Tc-Dfd) and Tc-zen1 and an approximately 8.5-kb duplication between prothoraxless/Tc-Antennapedia (ptl/Tc-Antp) and Tc-fushi tarazu (Tc-ftz). We designed primers to amplify across the regions in question using Ga-2 genomic DNA (the same inbred strain that was used for the Tribolium genome sequence). In both cases, the size of the resulting fragment is consistent with that predicted from the BAC sequence (data not shown), suggesting that these gaps and duplications are artifacts of the genome assembly process. It is important to note that these artifacts affect only a small fraction of the HOMC sequence, but they underscore the increased quality of finished versus draft sequences.
The single Tribolium Hox cluster contains orthologs of all eight Drosophila Hox genes, as well as orthologs of the Hox-derived genes, fushi tarazu and zerknüllt (zen; Tribolium Genome Consortium 2008 and Fig. 1). (In the case of zen, Tribolium has two paralogs apparently resulting from a recent duplication in the beetle lineage, independent of the zen duplication that occurred in the Drosophila lineage (Brown et al. 2002)). These genes are arranged in the same order on the chromosome as their counterparts in other insects. As in Apis (Dearden et al. 2006; Negre and Ruiz 2007), but in contrast to Drosophila and Anopheles (Negre and Ruiz 2007), the Hox and Hox-derived genes in the Tribolium cluster are all oriented in the same direction (Fig. 1). In addition, the two miRNA genes (miR-10 and miR-iab-4) that have been described in other insect Hox clusters are found at conserved positions in the Tribolium HOMC (Tanzer et al. 2005 and Fig. 1).
Fig. 1Embryonic transcription across the complete Tribolium Hox complex. The tiling array consists of ∼50,000 50 bp probes that estimate degree of transcription. Relative intensities for each probe are represented as peaks correlated with a consensus annotation of the Tribolium Hox complex (below). Peak height, shown as Percentile Probe Intensity (PPI), corresponds to the level of transcription for a particular probe. The nucleotide position for each segment is displayed in the upper left and right corners of the panel (numbers correspond with linkage group 2, release Tcas_2.0). New ESTs (cyan) are displayed in the annotation track along with transposable elements (gray). For annotated genes and ESTs, the arrow indicates the direction of transcription. Red arrows indicate the location of two RNA-FISH probes
The ANTC and BXC clusters of Drosophila melanogaster contain a number of non-Hox, protein-coding genes. In contrast, there is no evidence for non-Hox, protein-coding genes in the Tribolium HOMC (The Tribolium Genome Consortium 2008). Here, we corroborate those findings by using several methods to address whether unrelated genes might be interspersed among the Tribolium Hox genes. First, we searched the Tribolium genome for orthologs of genes that are located within the D. melanogaster clusters and determined that none of these genes are located within the Tribolium HOMC. Second, we analyzed predicted proteins within the region to determine whether any of them have recognizable orthologs in other species. Other than the Hox and Hox-derived genes, we found no evolutionarily conserved proteins among either the GLEAN predictions or the GNOMON ab initio predictions that map to the Hox cluster. Third, we searched a collection of Tribolium ESTs for expressed sequences within the HOMC. By this approach, we identified three non-Hox EST clusters that appear to represent noncoding transcripts as well as evidence for a mariner transposase gene (see below), but no other protein-coding genes were found. Finally, we analyzed the embryonically transcribed sequences identified by a tiling array to determine if any were likely to encode proteins. Again, we found no evidence of non-Hox-related protein-coding genes other than those within transposable elements. Although there are caveats to these analyses (e.g., gene prediction methods are imperfect, the tiling array represents only the embryonic transcriptome and EST coverage is incomplete), our results strongly suggest that the protein-coding genes in the Tribolium Hox complex (excluding genes within transposable elements) are all either Hox or Hox-derived genes.
Comparison of transposable element density in the Hox complexes of various animals has led to the suggestion that higher abundance of transposable elements in Hox clusters is correlated with loss of structural integrity. Mammalian Hox complexes have a reduced number of transposons compared to other regions of the genome (Ferrier and Minguillon 2003). Moreover, when transposons are present, they seem to be preferentially inserted into nontranscribed regions of the clusters (Mainguy et al. 2007). In contrast, transposons occur fairly frequently in the split Drosophila clusters (Fried et al. 2004). Though the prediction of three transposable elements in the Tribolium Hox complex (Fig. 1) may be an underestimate, the same method predicts fivefold more in the Drosophila Hox clusters. Additionally, only three transposable elements (all mariners) have been found in the larger but intact Apis Hox complex (Dearden et al. 2006). These numbers are consistent with the apparent inverse correlation between transposon number and the level of Hox cluster organization.
Taken together, these observations suggest that with respect to gene content, order, and orientation, the Tribolium Hox cluster closely resembles the putative ancestral Hox cluster (Garcia-Fernandez 2005). Thus, the constraints preserving the integrity of the Hox cluster may still be in force in Tribolium.
To determine whether these constraints extend outside the Tribolium Hox cluster, we examined synteny beyond the cluster itself. As previously described, Tc-chaoptic partially overlaps the 3′ UTR of Tc-labial (Tc-lab) on the opposite strand (Nie et al. 2001). Working outward, the first gene on the same strand as the Hox genes is Tc_00927, a dolichyl glycosyltransferase orthologous to D. melanogaster CG4542. Beyond the Tc-Abd-B locus is a cluster of putative serine carboxypeptidase genes (Tc_00887, Tc_00664, Tc_00665, and Tc_00666) and the ortholog of D. melanogaster CG3909 (Tc_00886). We identified the orthologs of these genes in D. melanogaster, Anopheles gambiae, and Apis mellifera and determined their map positions. None of these genes map near the Hox cluster in any of the other insects. Likewise, orthologs of the genes adjacent to lab and Abd-B in D. melanogaster do not map near the Hox clusters of the other three insects. These results suggest that the constraints preserving the Hox cluster act only on the Hox genes themselves and not the surrounding region.
The Tribolium Hox cluster produces numerous noncoding transcripts
We developed a tiling microarray covering the Tribolium Hox complex to identify the transcription units active during a broad window of Tribolium embryonic development. Tiling array signal intensity profiles were compared with previously described Hox cDNA structures. Though the tiling density is not fine-scaled enough to effectively resolve intron–exon boundaries, there is a near-perfect correlation between tiling array-predicted transcription and the position of exons in the well-characterized Hox genes. The only caveat is that the 5′ exons of maxillopedia/Tc-proboscipedia (mxp/Tc-pb) and Tc-Abd-B exhibit weaker signals than the other exons of these genes. The most likely explanation is that the 5′ exon is present only in a small subset of the transcripts derived from the gene (i.e., a minor spliceoform).
During the first 3 days of development, numerous regions of the Hox complex, including intergenic and intronic noncoding regions, are actively transcribed (Fig. 1). Interestingly, neither of the two most likely noncoding candidates, the previously described miRNAs, is robustly identified on the tiling array. Transcription at the tca-miR-10 locus is not detected, and transcription at the tca-miR-iab-4 locus is weak. It may be that tca-miR-10 is not expressed during the stages examined, whereas in situ hybridization assays show that tca-miR-iab-4 is strongly expressed during part of the developmental window examined (Fig. 2b).
Fig. 2Expression pattern of two HOMC noncoding transcripts in Tribolium embryos. Probe positions are shown in Fig. 1. a Expression pattern from a 1-kb probe located ∼86 kb 5′ of the start of ptl/Tc-Antp. b The expression pattern of the Tribolium homolog of the iab-4 miRNA (tca-miR-iab-4)
The most intense hybridization signals are detected in the central 250 kb of the Hox complex, encompassing the ptl/Tc-Antp and Ultrathorax/Tc-Ultrabithorax (Utx/Tc-Ubx) genes. Strikingly, transcription in this region is almost equally intense for coding and noncoding loci, and for both the introns and exons of the protein-coding genes. There are hundreds of discrete regions (500 bp or longer) where signal intensity is many times greater than for verified Hox gene exons. It is not possible to determine from the single time point we have analyzed if any of these discrete regions are part of larger transcripts. To verify that the observed signals in the tiling array represent authentic transcription, RNA fluorescent in situ hybridization (FISH) was performed with a representative 1-kb region between ptl/Tc-Antp and Utx/Tc-Ubx (TcNC-1 in Fig. 1). This region is expressed in a Hox-like pattern with distinct anterior and posterior borders in the posterior region of the elongating germ band (Fig. 2a). Interestingly, signal is detected primarily in two spots per nucleus, presumably at the sites of nascent transcription. This suggests either rapid degradation or processing of a primary transcript as would be seen for a pri-miRNA or an intron.
In our search for additional genes within the Tribolium Hox cluster, we identified three ESTs that appear to represent noncoding transcripts. One seems to be a chimeric artifact, arising from the fusion of a tca-miR-10 precursor and part of a 28s rRNA gene. The second, represented by two independent cDNAs, maps between ptl/Tc-Antp and Utx/Tc-Ubx while the third is located within the first intron of Utx/Tc-Ubx. The last two are transcribed from the strand opposite the Hox genes and are correlated with regions of strong signal in the tiling array analysis.
The mxpDch-3 mutation affects regulation of both mxp/Tc-pb and Cx/Tc-Scr
Because complex regulatory regions may act as a constraining force keeping Hox clusters intact, we analyzed the mxpDch-3 mutation, which was shown to have unusual effects on the expression of mxp/Tc-pb (Shippy et al. 2000); mxpDch-3 homozygotes lack most, if not all, normal mxp/Tc-pb expression, but both heterozygotes and homozygotes display strong ectopic mxp/Tc-pb expression in a pattern reminiscent of Cephalothorax/Tc-Sex combs reduced (Cx/Tc-Scr) expression (albeit with an apparent posterior shift of some domains). This ectopic expression is sufficient to rescue some aspects of mxp/Tc-pb function so mxpDch-3 is not an mxp/Tc-pb null (Shippy et al. 2000). Interestingly, we find that mxpDch-3 fails to complement a null allele of Cx/Tc-Scr (and, in fact, appears to be null for Cx/Tc-Scr) but complements null alleles of Tc-Dfd and ptl/Tc-Antp (data not shown).
To better understand this complex mutation, we characterized the breakpoints associated with mxpDch-3. mxpDch-3 is associated with a chromosomal rearrangement involving at least four breakpoints. Although we have not ruled out the presence of additional breakpoints, the simplest interpretation of our data is that a fragment of the HOMC (including Tc-zen1, Tc-zen2, Tc-Dfd, Cx/Tc-Scr, and Tc-ftz) has been removed from the HOMC and inserted between a fragment of LG9 and a non-HOMC fragment of LG2 (Fig. 3a). This scenario is consistent with previously reported pseudo-linkage of LG2 and LG9 associated with mxpDch-3 (Beeman et al. 1996) and provides an explanation for the mid-embryonic lethality of the mxpDch-3 homozygotes (Shippy et al. 2000). That is, non-HOMC breakpoints interrupt both the TFIIA-L ortholog, which is a component of the transcriptional machinery (Yokomori et al. 1993), and a homolog of groucho, which is an important transcriptional corepressor in Drosophila (Jimenez et al. 1997; Paroush et al. 1994). We conclude that the breakpoint between Tc-ftz and ptl/Tc-Antp is likely to account for the loss of Cx/Tc-Scr function associated with mxpDch-3, probably by separating the Cx/Tc-Scr transcription unit from some or all of its regulatory units. The rearrangement probably juxtaposes these regulatory elements with the mxp/Tc-pb transcription unit, providing a likely explanation for the Cx/Tc-Scr-like expression of mxp/Tc-pb in mxpDch-3 embryos.
Fig. 3Rearrangements of the HOMC. In these schematic diagrams, the positions of cloned breakpoint fragments are underlined. Wild-type chromosomal position (not to scale) on LG2 (purple) is indicated by a gradient of color to illustrate the effects of inversions. a In the mxpDch-3 rearrangement, an approximately 150-kb fragment of the HOMC has been transposed between fragments of LG9 and LG2. b The ptlD60 mutation is a large inversion that splits the Hox cluster into two parts. Small fragments at each end of the inversion appear to have been deleted, including part of the ptl/Tc-Antp locus
Cx/Tc-Scr regulatory elements map near or within ptl/Tc-Antp
Beeman et al. (1993) observed that mutations in Cx/Tc-Scr and ptl/Tc-Antp often partially fail to complement one another. Because this is precisely the type of genetic interaction that would be predicted if the separation of Hox genes has deleterious effects, we decided to analyze ptlD60, an allele of ptl/Tc-Antp that shows such effects. The ptlD60 mutation, which results in transformation of the larval legs toward antennae as well as reductions of some labial and thoracic tissue, has been proposed to be a null allele of ptl/Tc-Antp (Beeman et al. 1993). However, adults transheterozygous for ptlD60/ptlD2 have a very different phenotype from that produced by larval ptl/Tc-Antp RNAi (Tomoyasu et al. 2005) and, instead, resemble adults in which both ptl/Tc-Antp and Cx/Tc-Scr have been knocked down (Tomoyasu, personal communication). This result raises the possibility that the ptlD60 mutation affects the function of both genes. To address this issue, we performed parental RNAi with ptl/Tc-Antp and found that the resulting larvae (Fig. 4b) show a phenotype almost identical to that of ptlD60 homozygotes (Fig. 4c), suggesting that the ptlD60 mutation primarily affects the function of ptl/Tc-Antp during embryonic development. However, there is a subtle difference between the ptlD60 and ptl/Tc-Antp RNAi phenotypes in the positioning of the T1 appendages, which are located near the ventral midline in the RNAi larvae but more laterally in the mutants. This difference is likely attributable to partial loss of Cx/Tc-Scr function in ptlD60 because Cx/Scr is responsible for the midline position of the labial appendages in Tribolium (Shippy et al. 2006) and other insects (Hughes and Kaufman 2000; Pattatucci et al. 1991; Rogers et al. 1997). Thus, ptlD60 appears to be not only a null allele of ptl/Tc-Antp but also a hypomorphic allele of Cx/Tc-Scr.
Fig. 4Cuticle and enhancer trap phenotypes of ptl/Tc-Antp mutations. The antennae (ant) and the labial (lab) and thoracic (T1–T3) segments are denoted where relevant. a–e Cuticle preps displaying the phenotypes of wild-type (Ga-1; a), ptl/Tc-Antp RNAi (b), ptlD60/ptlD60 (c), ptlKT76/ptlKT76 (d), and ptlKT76/ptlD60 (e) first instar larvae. Enhancer trap-driven EGFP expression in a ptlKT76 embryo (f) appears in a very similar pattern to Cx/Tc-Scr expression (purple) in a wild-type embryo (g). A ptlKT76 larva (h) and pupa (i) also display EGFP enhancer trap expression in parts of the labial and first thoracic segments
To understand why the ptlD60 mutation affects both ptl/Tc-Antp and Cx/Tc-Scr, we characterized its mutant lesion(s). We found that ptlD60 is associated with an inversion of about 6.6 Mb, with breakpoints in ptl/Tc-Antp and a distant region of LG2 (Fig. 3b). In addition, there are small deletions at each end of the inversion (approximately 3.1 kb of the ptl/Tc-Antp transcription unit including all of exon 2 and approximately 1.5 kb at the other end). Consistent with this conclusion, we find that ptlD60 can act as a crossover suppressor for LG2, reducing recombination between Reindeer (a mutation near one end of LG2) and the HOMC from its normal value of 35–40 cM (Beeman et al. 1996) to approximately 20 cM. These results suggest that breakpoints within the ptl/Tc-Antp gene affect the function of both ptl/Tc-Antp and Cx/Tc-Scr, probably by disrupting the function of Cx/Tc-Scr regulatory elements (see “Discussion”).
Additional evidence for the presence of Cx/Tc-Scr regulatory elements in the vicinity of ptl/Tc-Antp comes from a piggyBac-insertion line recovered during an insertional mutagenesis project. The KT076 line carries a homozygous lethal insertion in the last intron of ptl (Fig. 5). Crosses between KT076 heterozygotes produce a class of embryos (putative homozygotes) with the T1 and T2 legs partially transformed toward antennae (Fig. 4d), a phenotype consistent with partial loss of Ptl/Tc-Antp function. However, individuals carrying the insertion display an embryonic enhancer trap expression pattern (Fig. 4f) very similar to the expression pattern of Cx/Tc-Scr (Curtis et al. 2001; Fig. 4g), despite the fact that the insertion site is about 87 kb upstream of Cx/Tc-Scr. KT076 larvae and pupae also show weak enhancer trap patterns consistent with predicted Cx/Tc-Scr domains (Fig. 4h–i). As expected from the phenotype of homozygotes, KT076 fails to complement ptlD60 as assayed by adult viability, and crosses of KT076 to ptlD60 heterozygotes produce embryos with a phenotype similar to, but slightly weaker than, that of ptlD60 homozygotes (Fig. 4e). In contrast, KT076 fully complements both the embryonic phenotype and the adult viability of Cx61, a null allele of Cx/Tc-Scr (Shippy et al. 2006), indicating that Cx/Tc-Scr function is not compromised by the insertion (data not shown). These results suggest that KT076 is a hypomorphic allele of ptl/Tc-Antp, and it will, hereafter, be referred to as ptlKT76. Together with the data from the mxpDch-3 and ptlD60 mutations, the Cx/Tc-Scr-like enhancer trap phenotype of ptlKT76 provides strong evidence that Cx/Tc-Scr regulatory elements are located near, and probably within, ptl/Tc-Antp. Although additional experiments will be necessary to pinpoint the location of regulatory elements and verify this conclusion, it is intriguing to think that overlap of regulatory elements of one Hox gene with the transcription unit of another Hox gene might be an important mechanism of Hox cluster constraint.
Fig. 5Overlap of Cx/Tc-Scr regulatory elements with the ptl/Tc-Antp locus. The gene structure of ptl/Tc-Antp (coding sequence is shaded gray) and the positions of mutant lesions are shown in the diagram. The inferred positions of Cx/Tc-Scr regulatory elements in the ptl/Tc-Antp region are indicated below the diagram
Discussion
The Hox clusters of several insects have now been completely sequenced. While breaks in the cluster seem to have occurred several times in the Drosophila lineage, the clusters of Apis, Anopheles, and Tribolium are intact. This suggests that many insect clusters are still subject to constraints to maintain their organization. Of the insects with intact clusters, Tribolium is, by far, the most genetically tractable and has a strong history of Hox gene studies, thus offering the best system for understanding the constraints acting on an intact insect Hox cluster. Below, we discuss insights provided by our analysis of the Tribolium HOMC into mechanisms that might be responsible for Hox cluster integrity.
Temporal colinearity
Among organisms for which both Hox cluster sequence and expression data are available, the presence of an intact cluster appears to be correlated with temporal colinearity of Hox gene expression, while disrupted clusters are associated with lack of temporal colinearity (Monteiro and Ferrier 2006). This observation has pushed temporal colinearity to the forefront of discussions about Hox cluster maintenance. However, several questions remain to be answered. Does temporal colinearity really require an intact Hox cluster? Is temporal colinearity required for proper Hox gene function in organisms with intact clusters? If temporal colinearity is a key constraint on Tribolium Hox cluster integrity, we might expect rearrangements to affect the function of most or all Hox genes. However, our analysis of Tribolium Hox cluster mutations provides no evidence for such global effects. The ptlD60 inversion splits the complex into two parts but all of the Hox genes except ptl/Tc-Antp and Cx/Tc-Scr appear to function normally. In addition, the mxpDch-3 rearrangement results in the translocation of several HOMC genes (Tc-zen, Tc-Dfd, Cx/Tc-Scr, and Tc-ftz) to a new chromosomal location. At least one of these genes, Tc-Dfd, is functional because mxpDch-3 fully complements a Tc-Dfd null allele. Likewise, the genes remaining in the HOMC (with the exception of mxp/Tc-pb) apparently function normally. These limited effects of Hox cluster rearrangements are similar to what has been reported for Drosophila (e.g., Abbott and Kaufman 1986; Pultz et al 1988). Although additional experiments will be required to determine whether Tribolium Hox genes exhibit temporal colinearity, our results suggest that constraints on the Tribolium HOMC are more likely to act locally.
Regulatory elements
The reported breaks and transposition sites in the Hox clusters of Drosophila species are all located in intergenic regions near the 3′ end of a gene (Negre et al. 2005; Negre and Ruiz 2007) and, thus, are presumably less likely to separate a gene from its regulatory elements, which are predominantly located 5′ of each Drosophila Hox gene. Interestingly, the one exception to this rule is the region between abd-A and Abd-B, which contains regulatory regions for both genes and is not split in any of the Drosophila species examined so far (Negre and Ruiz 2007). These observations led to the conclusion that Drosophila Hox clusters have a modular organization (with each gene and its regulatory elements representing a separate module) and that Drosophila Hox genes are still partially clustered simply because the regulatory regions are so large that there are relatively few positions where breaks can occur without disturbing a module. That is, most of the remaining linkage in Drosophila Hox clusters (with the possible exception of abd-A and Abd-B) is due to “phylogenetic inertia,” and, given enough time, the clusters will completely disperse (Negre and Ruiz 2007).
The question naturally arises whether phylogenetic inertia could also be the reason for the intact Hox clusters of insects like Tribolium. Lewis et al. (2003) suggested that unusual features of recombination (Ranz et al. 2001) may make drosophilids more tolerant of Hox cluster rearrangements than are most insects. If this is the case, intact clusters may just be the consequence of slower rates of chromosomal rearrangement (Negre and Ruiz 2007). Alternatively, constraints on Hox cluster maintenance may still be functional in Tribolium.
Our analysis of mutant breakpoints in the ptl/Tc-Antp region indicates that, in at least one case, Tribolium Hox genes are not modular. That is, at least some of the regulatory elements controlling Cx/Tc-Scr expression are apparently located within the pt/Tc-Antp gene (Fig. 5). Most embryonic enhancers of Cx/Tc-Scr are predicted to lie between the mxpDch-3 and ptlD60 breakpoints because the mxpDch-3 allele appears to lack all Cx/Tc-Scr function, while ptlD60 has almost normal embryonic Cx/Tc-Scr function. This conclusion is further supported by the expression of mxp/Tc-pb in a Cx/Tc-Scr-like pattern in mxpDch-3 mutants, presumably due to juxtaposition of the mxp/Tc-pb transcription unit with sequence between Tc-ftz and ptl/Tc-Antp. Adult Cx/Tc-Scr regulatory elements are likely located within, or 5′ of, ptl/Tc-Antp because the ptlD60 rearrangement seems to have a stronger effect on the adult functions of Cx/Tc-Scr. The presence of Cx/Tc-Scr regulatory elements near, or within, ptl/Tc-Antp is supported by the observation that a piggyBac insertion within the ptl/Tc-Antp transcription unit (ptlK76) shows an enhancer trap phenotype that appears to be driven by Cx/Tc-Scr regulatory elements.
In Drosophila, elements which drive Scr-like expression patterns have been found in the region between ftz and Antp (Gorman and Kaufman 1995). However, these elements are apparently redundant because breakpoints in this region only slightly reduce Scr expression levels within its normal domain. Moreover, a deficiency which removes most, if not all, of the Antp transcription unit has no effect on embryonic Scr expression. Thus, it is possible that the modularity of the Drosophila Hox cluster is a recent innovation resulting from changes in the position of cis-regulatory elements. This newly acquired modularity might have allowed breaks in the Hox cluster in the Drosophila lineage. In contrast, overlap of regulatory elements with neighboring genes might still act as a constraint on the integrity of the Hox cluster in Tribolium.
Noncoding transcripts
Although noncoding transcripts within Hox clusters have been recognized for many years (Cumberledge et al. 1990; Lipshitz et al. 1987; Sanchez-Herrero and Akam 1989), there has recently been renewed interest in these enigmatic RNAs. At least two of these ncRNAs, miR-196 and miR-iab-4, can have homeotic function and attenuate the actions of protein-coding Hox genes (Hornstein et al. 2005; Ronshaugen et al. 2005).
In addition to the miRNAs, numerous long noncoding RNAs have been identified within the Hox clusters of both flies and mammals (Bae et al. 2002; Rinn et al. 2007). ncRNAs are implicated in a vast array of processes, including regulation of transcription, translation, epigenetic control of chromatin, mono-allelic expression, dosage compensation, and silencing (reviewed in Mattick and Makunin 2006). In the Drosophila BXC, these transcripts have been implicated in the control of Hox gene expression, although there is some controversy as to whether they promote (Sanchez-Elsner et al. 2006) or repress (Petruk et al. 2006) Hox gene transcription. Mainguy et al. (2007) found evidence for extensive noncoding transcription in the mammalian Hox clusters and proposed that polycistronic and antisense transcription might play a role in keeping Hox genes clustered. Our transcriptional profiling data demonstrates that Tribolium also shows considerable noncoding transcription in the Hox complex. While the tiling array has provided a revealing snapshot of transcription levels during embryonic development, much additional work will be necessary to characterize the actual transcripts. For example, the high levels of transcription in the ptl/Tc-Antp and Utx/Tc-Ubx regions could be the result of several individual transcripts or one long transcript. Interestingly, dicistronic transcripts spanning the Antp and Ubx orthologs have been reported in crustaceans (Shiga et al. 2006) and centipedes (Brena et al. 2006). If such transcripts have an important function, they might constrain linkage in at least some parts of the Hox cluster.
The data available thus far indicate that noncoding transcripts are prevalent in both intact and broken Hox clusters. However, it is not clear whether these transcripts perform the same functions in all organisms. Perhaps, noncoding RNAs play a different or more critical role in organisms with intact clusters. Future studies in this area are likely to provide important insights into Hox gene function and possibly into Hox cluster conservation.
Conclusions
The results presented here provide a foundation for further studies of the constraints acting on Hox clusters. While it is important to keep in mind that multiple factors may have contributed to the maintenance of Hox clusters during evolution (Kmita and Duboule, 2003), the intact structure of the Tribolium Hox cluster and the suite of tools now available for this insect makes it an ideal candidate for such research.

tribolium
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tiling


Description of the attachment geometry of the anteromedial and posterolateral bundles of the ACL from arthroscopic perspective for anatomical tunnel placement

The anterior cruciate ligament (ACL) consists of an anteromedial bundle (AMB) and a posterolateral bundle (PLB). A reconstruction restoring the functional two-bundled nature should be able to approximate normal ACL function better than the most commonly used single-bundle reconstructions. Accurate tunnel positioning is important, but difficult. The purpose of this study was to provide a geometric description of the centre of the attachments relative to arthroscopically visible landmarks. The AMB and PLB attachment sites in 35 dissected cadaver knees were measured with a 3D system, as were anatomical landmarks of femur and tibia. At the femur, the mean ACL centre is positioned 7.9 ± 1.4 mm (mean ± 1 SD) shallow, along the notch roof, from the most lateral over-the-top position at the posterior edge of the intercondylar notch and from that point 4.0 ± 1.3 mm from the notch roof, low on the surface of the lateral condyle wall. The mean AMB centre is at 7.2 ± 1.8 and 1.4 ± 1.7 mm, and the mean PLB centre at 8.8 ± 1.6 and 6.7 ± 2.0 mm. At the tibia, the mean ACL centre is positioned 5.1 ± 1.7 mm lateral of the medial tibial spine and from that point 9.8 ± 2.1 mm anterior. The mean AMB centre is at 3.0 ± 1.6 and 9.4 ± 2.2 mm, and the mean PLB centre at 7.2 ± 1.8 and 10.1 ± 2.1 mm. The ACL attachment geometry is well defined relative to arthroscopically visible landmarks with respect to the AMB and PLB. With simple guidelines for the surgeon, the attachments centres can be found during arthroscopic single-bundle or double-bundle reconstructions.
Introduction
The anterior cruciate ligament (ACL) consists of two functional bundles [2, 4, 16]. The anteromedial bundle (AMB) originates anteroproximal in the intercondylar notch, close to the over-the-top position at the posterior edge of the notch, from the deep high part of the femoral attachment area and inserts anteromedial on the anterior intercondylar area of the tibia. The posterolateral bundle (PLB) originates more posteriorly and distally in the notch, from the shallow low part of the femoral attachment area and inserts posterolateral on the anterior intercondylar area of the tibia. The ACL reconstruction aims at restoring normal knee function. Most ACL replacements are performed with the isometric single-bundle technique. Isometric positioning of a single-bundle graft results in replacement of the AMB only. Although tensioned over the complete range of motion, the fibres are mostly tight in flexion. The AMB is the major constraint for anterior tibial displacement of the flexed knee [30], but cannot restore normal knee laxity and kinematics near extension [4, 28, 41]. In an effort to improve knee mechanics, double-bundle anatomic ACL reconstructions are now developed with reconstruction of both AMB and PLB [9, 10, 13, 19, 28, 35, 40, 46, 49, 50, 52, 53]. As presented in previous studies, a reconstructed PLB is able to restore stability in knee angles where an isometrically placed graft fails [28, 41, 50]. Additional restraint against anterior displacement in 15° of flexion [48] as well as prevention of the pivot shift is demonstrated [28, 50]. Therefore, a reconstruction with two bundles should be able to approximate normal ACL function over the complete range of motion [39, 41, 51].
Tunnel positioning is an important factor for clinical success of ACL reconstructions. Incorrect tibial [23] and femoral [29, 55] tunnel placements result in abnormal knee mechanics. Anatomical placement restores normal knee function better than isometric placement [36, 55]. However, accurate tunnel placement seems difficult. Misplacement between 25 and 65% of the tibial and femoral tunnels is reported [8, 27, 47]. Double-bundle ACL reconstructions require an anatomical placement of the bone tunnels. It is difficult to identify the ACL remnants in chronic ACL-injured knees. Therefore, detailed information about the approximate native position is essential to determine proper anatomic tunnel placement for the two bundles during arthroscopy [17]. The anatomical position has been the subject of many studies [3, 5, 12, 14–16, 20, 26, 33, 34, 37, 38, 44]. Only a few recent studies have described the anatomic positions of the AM and PL bundles [11, 32, 45, 52]. Due to the two-dimensional and limited view on the arthroscopic monitor, the landmarks and descriptions used in the above-mentioned studies seem not sufficient for correct positioning of the two separate bundles in all planes during arthroscopic surgery.
This study is aimed at acquiring quantitative geometric data of the ACL attachments on tibia and femur, such that these data can be used in an arthroscopically guided procedure for reconstruction of the ACL. We hypothesized that reliable guidelines to find the centres of the AMB, PLB and ACL relative to arthroscopically visible landmarks can be established. For the purpose of an anatomically accurate reconstruction of the ACL, the variations should be equal to or less than reported in other studies. As regard to the dimensions of drilled tunnel holes, normally 10 up to 12 mm, 95% (mean ± 2SD) of the attachment centres should be within this range. Therefore the a priori set assumption is that the maximally acceptable SD is 2.5 up to 3 mm.
Methods
Dissection
Thirty-five intact human cadaveric knee joints of elderly donors preserved in formalin, without signs of gross bony deformity, previous fracture or degenerative disease and with intact knee ligaments were dissected. Because of local post-dissection handling procedures, no exact data on gender and age of the donors were available, but they were older than 60 years. The muscles and anterior capsule were removed. The ligaments were left intact in order to preserve controlled motion of the knee. The femur was fixated in a clamp, the tibia was moved, resulting in flexion and extension of the knee joint. During this repeated passive movement, the two functional components of the ACL were identified, based on a visually detectable difference in their tensioning patterns as described by Girgis et al. [16]. In 90° of flexion, an anterior load was manually applied. This caused tension in the fibres of the medial tibial attachment site, the AMB. The fibres of the PLB remained slack. This procedure enabled a separation of the two bundles at the tibial attachment from ventral. The femur was turned in the clamp to enable a posterior approach and the posterior cruciate ligament (PCL) was removed. In this position the initial division of the ACL at the tibial attachment was visible and used to extend the division towards the femoral origin. The tibia was moved towards extension. The PLB-fibres, inserting at the lateral femoral ACL attachment site, tightened, enabling to complete the separation as far as the femoral attachment. The outline contours of both AMB and PLB attachment areas were marked, with a waterproof felt pen.
Anatomical position
To quantify the position of the centres of the attachment sites relative to arthroscopically visible landmarks, three-dimensional (3D) measurements were made with a 3Space Fastrak electromagnetic tracking system (Polhemus Navigation Sciences, Colchester, VT, USA). The x, y and z co-ordinates of each measured point were recorded with an accuracy of 0.35 mm [42]. On the femur and tibia, the attachment sites of the AMB, PLB and the entire ACL were digitized by means of a collection of points placed at equal distances on the marked outlines. The 3D position of the centres of the ACL, AMB and PLB attachments were calculated by the geometric mean of all points on the outlines. The 3D distances between the centres of the two bundles were calculated. On both femur and tibia, an arthroscopically visible landmark was digitized that served as the origin of a local coordinate system. The absolute two-dimensional (2D) positions of the centres relative to these landmarks were calculated. The position in the third dimension was determined by the surface geometry of the femoral condyle and the intercondylar tibial area, respectively.
Absolute positions can depend on different knee sizes and the dimensions of the femoral notch and the intercondylar tibial area. To correct for this, the relative centre positions were also calculated. Additional reference points near the attachment area on femur and tibia were digitized. Between these points two reference lines were defined to create a 2D coordinate system. The distances defined the dimensions of the femoral notch and intercondylar tibial area. Absolute centre positions were transformed in positions relative to the reference lines (%) within femur and tibia. To detect whether the absolute position of the attachment centres was actually influenced by knee size, statistical analyses were performed. The relation between absolute position and knee size can be demonstrated with a correlation coefficient. The Pearson’s correlations between the distances of the absolute positions and the length of the reference lines, representing knee size, were calculated. Correlations with a Pearson’s correlation coefficient r > 0.6 and a significance level P < 0.05, for a 95% alpha level were considered relevant and confirmed the relation between knee size and absolute centre position.
Femur
In the anatomical nomenclature the femoral ACL attachment uses anterior/posterior and proximal/distal positions, relative to the extended knee [17] (Fig. 1a). Since this study aimed at describing the positions of the AMB and PLB of the femoral attachment from an arthroscopic perspective, the arthroscopic nomenclature was used as recommended by the ‘‘ESSKA Scientific Workshops’’ in 1998 [4]. The definitions, shallow/deep and high/low, refer to the position along the wall and from the roof of the intercondylar notch in a 90° flexed knee (Fig. 1b).
Fig. 1The orientation in the femoral notch, used in this study, is based on the recommendations of the ESKKA 1998 [4]. The notch depth (ND) is directed from shallow to deep in anatomic distal–proximal direction. The notch height (NH) is directed from low to high in anatomic dorsal–ventral direction. The notch wall is located at the medial side of the lateral condyle. The notch roof is the connection between the two condyles
The digitized points on the femur are represented in Fig. 2. The main femoral landmark was derived from the over-the-top position, located at the posterior edge of the intercondylar notch. The most proximal high deep point (D) on the lateral condyle was found at the 10.30 o’clock position in the arc of the femoral notch, in a right knee (at 1.30 in a left knee) [17] (Fig. 2). The circumferences of the ACL attachments were digitized, as was the additional reference point, the most distal high shallow point (S). This point is positioned on the distal cartilage edge of the lateral condyle in the anterior notch outlet (Fig. 2a). The femoral coordinate system was defined with two reference lines (Fig. 3). The first-defined femoral reference line (from D to S) divided the notch roof from the notch wall and defined the length of the lateral notch depth (ND). The lowest, dorsal point (L) on the posterior joint cartilage edge was found using a line parallel to the line DS. The second reference line (from L to H) was defined from point L perpendicular to crossing point H, high in the notch at the line DS. It indicated the magnitude of the notch height (NH). After creating this coordinate system, the calculated attachment centres (C) of the ACL, AMB and PLB were projected on the line DS at point P. The absolute centre position (mm) was composed of the distance between the main femoral landmark, the high deep point D and the point P and the distance between the point P and the centre C. The relative (%) position was calculated by dividing the distance DP by DS (notch depth) and distance CP by LH (notch height). Finally the individual results were displayed in a diagram to define the distribution of the centres relative to the means and the advised tunnel position.
Fig. 2a Distal view at a left femur. The high shallow and high deep points of the cartilage border were determined by placing the stylus at the point of an imaginary rectangular corner, indicating the separation between wall and roof, this corresponds with the 1.30 o’clock position in a left knee (10.30 o’clock in a right knee). b View at the medial side of a left lateral femoral condyle. The points that were digitized: AMB (white rounds) and PLB (black rounds) attachments; the cartilage border (grey asterisks), with the most distal, high shallow point (black asterisk) and most proximal, high deep point (white asterisk) indicating the separation between the notch wall on the lateral condyle and the notch roofFig. 3A schematic arthroscopic femoral view through the anteromedial portal. The femoral coordinate system is indicated. The first reference line, from the high deep point (D) to the high shallow point (S) on the cartilage edge separated the notch wall on the lateral condyle from the notch roof and defined the notch depth (ND). The second reference line, a perpendicular line, from the lowest point on the posterior cartilage edge (L) to the crossing point on the line DS (H) and defined the notch height (NH). The calculated attachment centre of the ACL (C) was projected on line DS (P). The absolute distances DP and CP were calculated, as were the distances relative to the reference lines (DP/DS and CP/LH). This was also done for the AMB and PLB, for the sake of clearness, only the centre of the entire ACL is depicted
Tibia
The digitized points on the tibia are represented in Fig. 4. The medial tibial spine (M) was determined as the main tibial landmark. The circumferences of the ACL attachments were digitized, as were the additional reference points. Those were, besides the lateral tibial spine (L), the most anterior points on the margin of the articular surfaces of the medial tibial condyle (MA) and lateral tibial condyle (LA). The tibial coordinate system was defined as follows (Fig. 5). The first tibial reference line (from M to L) was defined between the medial and the lateral tibial spine representing the interspinal distance (ID). A line connecting the anterior points MA and LA indicated the anterior margin of the articular surface of the medial and lateral condyles of the tibia. The second reference line (from M to Q) connected the medial spine (M) perpendicular with the anterior margin at crossing point Q. The length of the second reference line represents the length of the anterior intercondylar area (AL). After creating this coordinate system, the calculated attachment centres of the ACL, AMB and PLB were projected on the line ML at point P. The absolute centre position (mm) was composed of the distance between the major tibial landmark, the medial tibial spine M and the point P, and the distance between the point P and the centre C. The relative position (%) was calculated by dividing the distance MP by ML (interspinal distance) and distance CP by MQ (anterior length). Finally the individual results were displayed in a diagram to define the distribution of the centres relative to the means and the advised tunnel position.
Fig. 4Proximal view at the articular surfaces of the tibial condyles and the anterior intercondylar area. The points that were digitized: AMB (white rounds) and PLB (black rounds) attachments; the lateral tibial spine (light grey oval) and medial tibial spine (dark grey oval) and the most anterior points on the margin of the articular surface of the medial and lateral tibial condyles (white asterisks)Fig. 5A schematic arthroscopic tibial view. The tibial coordinate system is indicated. The first reference line, from the medial spine (M) to the lateral spine (L), defined the interspinal distance (ID). The second reference line, from the medial spine (M) perpendicular to the crossing point (Q) at the anterior line between the most anterior points on the margin of the articular surface of the medial and lateral tibial condyles defined the anterior intercondylar length (AL). The calculated attachment centre (C) was projected on line ML (P). The absolute distances MP and CP were calculated, as were the distances relative to the reference lines (MP/ML and CP/MQ). This was also done for the AMB and PLB, for the sake of clearness, only the centre of the entire ACL is depicted
Surface
To determine the surface dimensions of the bony attachment areas, a line was fitted through the digitized points on the outlines of both the AMB and PLB attachments on femur and on the tibia. The enclosed surface of all areas was calculated (in mm2), with the aid of a Delaunay triangulation based on the Qhull algorithm as provided by Matlab® (version 7 The MathWorks, Inc, more details provided on http://www.mathworks.com). The surface of the entire ACL attachment was calculated as the sum of AMB and PLB. Also the percentage of AMB and PLB surfaces was calculated relative to the ACL attachment surface. Statistical analyses to detect differences in size of the attachment surfaces were performed. Femoral AMB and femoral PLB, tibial AMB and tibial PLB and finally femoral ACL and tibial ACL were compared. A 2-tailed Student’s t test for paired data was used. Statistical significance was defined as P < 0.05, for a 95% alpha level.
Results
Femur
The oval-shaped attachment of the anterior cruciate ligament was situated on the medial surface of the lateral femoral condyle. It was positioned deep in the notch, covering most of the proximal half of the wall. The fibres of the deep low border attached to the edge of the joint cartilage, following the contour of this edge posteriorly on the condyle. In only 4 of the 35 specimens, the attachment site was completely limited to the medial wall of the lateral condyle and had no footprint in the notch roof. In 31 femurs, a small part of the deep high AMB attachment extended into the intercondylar notch roof.
The notch depth (DS = 31.8 ± 2.6 mm) was the largest distance of the femoral dimensions. On average it was 2¼ times the length of the notch height (LH = 14.3 ± 1.5 mm). In shallow–deep direction measured along the notch, relative to the notch depth, the centres of the two bundles were more close to each other, than in high–low direction measured from the roof, relative to the notch height. The mean position of all AMB centres along the roofline of the notch was 7.2 mm shallow from the high deep corner at ¼ of the ND-line (Table 1; Fig. 6). In 9 of the 35 femurs the centre of the AMB was not situated on the condyle wall, but above the transition line on the notch roof. Therefore, the average centre was high in the notch at 1/10 of the NH-line, 1.4 mm from the roof. The centre of the PLB was positioned slightly more shallow at less than 2 mm from the AMB (Table 1; Fig. 6). However, it was situated clearly lower, about 5 mm, on the femoral condyle wall, approximately halfway the notch height. The centre position of the entire ACL was in the middle between the AMB and the PLB at ¼ of both reference lines. Approximately 96% of the mean centres were inside a 12 mm drill hole, if positioned at the mean centre of the ACL attachment. The Pearson’s correlation coefficients between the absolute positions and the reference lines were smaller than 0.3 and not significant (P > 0.05).
Table 1The positions of the ligament centres in the femoral notch Absolute distances (mm) mean (SD)Relative distances (%) mean (SD)DPCPDP/DSCP/LHAMB7.2 (1.8)1.4 (1.7)23 (6)10 (12)PLB8.8 (1.6)6.7 (2.0)28 (6)47 (13)ACL7.9 (1.4)4.0 (1.3)25 (5)28 (9)DP The absolute distance from the high deep point D until point P, the projection of the mean ligament centre at the DS-line, in the notch depth direction; CP the absolute distance from the mean ligament centre until point P at the LH-line, in notch height direction; DP/DS The relative position of the centre to the notch depth; CP/LH The relative position of the centre to the notch heightFig. 6a A two-dimensional graph of the medial side of a right lateral femoral condyle with the individual centres, and the mean centres with the 95% Confidence Interval of the AMB (red squares), PLB (blue diamonds) and ACL (green dots). The position of a 12 mm drill hole at the ACL centre is also displayed. b A schematic arthroscopic view through the anteromedial portal with the mean centres and the 95% Confidence Interval areas of the AMB (red square with line), PLB (blue diamond with line) and ACL (green dot with line)
The mean centres of the AMB and PLB were situated 6.2 ± 1.2 mm from each other. The AMB attachment area, 45% of the total ACL, was significantly (P = 0.005) smaller than the PLB attachment area (Table 2).
Table 2The surface dimensions of the ACL and the two bundles at femur and tibiaAbsolute surface area (mm) mean (SD)Relative surface to total ACL (%) mean (SD)ACLAMBPLBAMBPLBFemur184 (52)81 (27)103 (39)45 (11)55 (11)Tibia229 (53)136 (37)93 (33)59 (9)41 (9)
Tibia
The tibial attachment area of the ACL was situated between the medial and lateral tibial condyle covering the medial part of the anterior intercondylar area. It was stretched out from the region between the tibial spines to anterior with various extensions, more or less shaped as a footprint. The fibres of the AMB inserted medially along the cartilage edge of the articular surface of the medial tibial condyle. The PLB covered the lateral side of the attachment area and was bounded by the attachment of the anterior horn of the lateral meniscus.
The interspinal distance (ML = 12.9 ± 1.6 mm) between the medial and the lateral spine was on the average half times the length of the anterior intercondylar area (MQ = 24.8 ± 2.7 mm). The average centre of the AMB was situated closest to the medial spine, lateral at ¼ of the ID. The PLB was 4 mm more lateral, approximately halfway between the medial and lateral spine (Table 3; Fig. 7). The ACL was in between at 2/5 of the ID. In anterior direction the AMB, ACL and PLB were close to each other, just over 1/3 of the anterior length. Approximately 94% of the mean centres were inside a 10 mm drill hole, if positioned at the mean centre of the ACL attachment (Table 3; Fig. 7). The Pearson’s correlations coefficients between the absolute positions and the interspinal distance (reference line ML) were smaller than 0.3 and not significant (P > 0.05). The Pearson’s correlations coefficients between the absolute positions and the anterior length (reference line MQ) were significant (P < 0.05), however smaller than 0.6.
Table 3The positions of the ligament centres at the anterior intercondylar tibial areaAbsolute distances (mm) mean (SD)Relative distances (%) mean (SD)MPCPMP/MLCP/MQAMB3.0 (1.6)9.4 (2.2)23 (12)37 (9)PLB7.2 (1.8)10.1 (2.1)55 (13)38 (9)ACL5.1 (1.7)9.8 (2.1)39 (12)38 (8)MP The absolute distance from the medial spine (M) until the point P, the projection of the mean ligament centre at the line ML in lateral direction; CP the absolute distance from the mean ligament centre until point P on the line ML, in anterior direction; MP/ML The relative position of the centre to the interspinal distance; CP/MQ The relative position of the centre to the anterior lengthFig. 7a A two-dimensional graph of a right anterior intercondylar tibial area with the individual centres, and the mean centres with the 95% Confidence Interval of the AMB (red squares), PLB (blue diamonds) and ACL (green dots). The position of a 10 mm drill hole at the ACL centre is also displayed. b A schematic arthroscopic view with the mean centres and the 95% Confidence Interval areas of the AMB (red square with line), PLB (blue diamond with line) and ACL (green dot with line)
The mean centres of the AMB and PLB were situated 4.5 ± 0.1 mm from each other. The tibial AMB attachment area of 59% was significantly larger than the PLB attachment area (P < 0.001). The tibial attachment area of the ACL and of the AMB was significantly larger than on the femur (P < 0.001) (Table 2).
Discussion
Incorrect tunnel placement, tibial [22, 25] as well as femoral [43], is seen as one of the most important causes of clinical failure in single-bundle ACL reconstructions [1]. In double-bundle ACL reconstruction, exact anatomic tunnel placement seems to be even more essential. Two bundles must be accurately placed relative to the surrounding structures and relative to each other. Although exact tunnel positioning is important, it seems difficult, even for experienced surgeons [8, 27, 47]. A clear description of the anatomic centres with guidelines to determine the correct tunnel position during arthroscopic procedures can improve the accuracy.
The femoral positions of the two distinct bundles found in this study, the AMB deep high and the PLB shallow low in the notch, are broadly in line with literature [11, 20, 32, 45, 52]. Compared to others, the landmarks that are used in the present study are more easy to locate during arthroscopy. Harner et al. [20] quantified the cross-sectional shape and area of the femoral and tibial attachments of both components in 10 knees without describing the positions relative to landmarks. Yasuda et al. [52] limit their study to the femoral attachment, using five specimens. They describe the centre of the PLB 5 to 8 mm anterior to the edge of the joint cartilage, on the vertical line through the contact point between the femoral condyle and the tibial plateau in a 90° flexed knee. Because this point depends on the position of the knee, it can be difficult to locate accurately during an arthroscopic procedure. Colombet et al. [11] examined seven specimens, collecting especially data of the attachment dimensions. The femoral results presented by the studies of Mochizuki et al. [32] and Takahashi et al. [45] are more suitable for practical use during arthroscopy; however, no exactly defined landmarks were used. The tibial results of Takahashi et al. [45] cannot easily be transferred to the arthroscopic situation. Finally, above-mentioned studies did not present the centres of the ACL attachment.
Tunnel positioning in the femoral notch is often determined by the ‘clock’ method [13, 18, 35, 36, 52, 54]. However, this only determines the high–low position from the roof, in the transversal plane along the arc of the femoral notch [17, 54]. The accuracy in the sagittal plane, deep–shallow along the roof of the notch, seems to affect the functional outcome more [17, 21, 29, 55, 56]. This position is often determined with a femoral guide placed behind the posterior edge of the intercondylar notch, at the over-the-top position, not to be confused with the Resident’s ridge [24]. This seems sufficiently accurate for determining the AMB centre, when a guide with a 7 mm offset is placed in the 10.30 or 1.30 o’clock position. The 7 mm offset of the guide places the tunnel in the sagittal plane close to the AMB centre position, found in this study, i.e. 7.2 mm shallow along the roof. Although we did not translate the measured distance in a clock position, the 10.30 clock position seems close to the measured position in the transversal plane, 1.4 mm low from the roof on the condyle wall. This corresponds with the results of Mochizuki et al. [32] and Yasuda et al. [52]. However, the clock method is sensitive to subjective interpretation of positioning the face of the clock [7]. It is not sufficient to find the correct position for the PLB, especially in the sagittal plane. Therefore Colombet et al. [11] defined an extra guideline to position the PLB tunnel: 8 mm lower and ‘shallower’ relative to the AMB centre, found with the clock method. The present study is more precise: 1.3 mm more shallow along the notch and 5.3 mm lower from the roof on the condyle wall, relative to the above-described position of the AMB centre. These positions can best be approached through an anteromedial arthroscopic portal [6].
Various methods can be used to determine the correct drill hole position at the tibia. Some authors prefer placement of the tibial tunnel based on avoiding graft impingement against the roof of the femoral notch with knee extension [22, 34, 44]. Others prefer guides that use the posterior cruciate ligament (PCL) attachment as reference point [31]. Based on these methods, positioning in anteroposterior direction, the sagittal plane, is defined. However, placement of the tunnel in mediolateral direction, the transversal plane, which is also important [26], is not determined. Some studies describe the mediolateral position, relative to the width of the tibial plateau [23, 45]. However, this guideline cannot be used during an arthroscopic procedure. The results of this study can be used for arthroscopic positioning in both directions.
The division of the AMB and PLB on the tibia in anteroposterior direction is similar to Harner et al. [20], resulting in a medial AMB and lateral PLB. This deviates only marginally from other definitions, where the division is in an anteromedial and a posterolateral part [3, 16, 38]. The position of the centres in anteroposterior direction correspond with the results of Takahashi et al. [45], who used similar reference points to define the anterior margin of the articular surface of the tibial condyles. The centres of the two bundles were more close to each other on the tibia, than on the femur.
The size of the femoral ACL attachment 184 ± 52 mm2 was similar to the result of Odensten and Gillquist (200 mm2) [38]. Other studies found smaller attachment areas, 132 mm2 [45] and 113 mm2 [20]. This difference may be caused by a different measuring method, using the largest projection in a 2D image plane. Our study measured the actual three-dimensional attachment area. The partition of the femoral attachment area was nearly equal with 45%, 55%, respectively, for the AMB and PLB. This was comparable to Harner et al. [20] (AMB = 52%, PLB = 48%) and Takahashi et al. [45] (AMB = 50%, PLB = 50%). The average tibial attachment area found in this study was larger than the femoral attachment. This is in line with the results of Girgis et al. [16] and Fuss et al. [15]. On the other hand, Takahashi et al. [45] found that the femoral attachment was larger than the tibial attachment (132 vs 119 mm2). Harner et al. [20] and Odensten and Gillquist [38] found similar sizes for the tibial and femoral attachment areas. In our study the AMB occupied 59% of the tibial attachment, nearly similar to the 57% of Takahashi et al. [45], slightly more than the 52% Harner et al. [20] found.
There was a large variation in knee sizes and dimensions of the reference lines. However, we did not find a correlation between the absolute distance of the attachment centres and the size of the reference lines, i.e. knee size. The absolute positions of the attachment centres were more or less similar for large and small knees. Therefore both absolute as well as relative data can be used as guidelines to find the anatomical positions. At the arthroscopic view, the absolute positions are more useful than the relative positions.
As regards to the dimensions of drilled tunnel holes (10–12 mm), the standard deviations of the attachment centres within 3 mm are acceptable. The femoral positions had less variations than the tibial positions. The positions for the entire ACL attachment had less variations than the attachment positions for the separate bundles. The results of this study seem to produce accurate guidelines to find the anatomic tunnel position during arthroscopic reconstruction. However, some limitations must be mentioned. The subdivision of the ACL in AMB and PLB is not based on anatomically distinct fibre bundles surrounded with fibrous issue [5, 15, 33, 38]. Fibres of both bundles are twisted around each other [33]. Division of the ACL in separate bundles is not easy [3]. Therefore utmost care was taken to divide the two functional bundles according to a previously described method [11, 20]. The method, based on observed tension variation during passive flexion–extension movement was reported to be consistent [32]. This is expressed in the standard deviations (SD) of the mean attachment centres. The variation of the population is expressed by the SD of the ACL centre. The SD of the mean PLB and mean AMB centres show this variation combined with the division error. For the tibia the SDs of the PLB and AMB attachment centres are equal to that of the ACL. For the femur the SDs of the two bundle centres are slightly larger. However, these results do not point to large errors in the identification of the two bundles. The variations of attachment centre positions are actually rather small and similar to other studies [11, 45].
The age of the cadaveric knee specimens was more than 60 years. However, only knees without severe arthritic signs were evaluated. It seems likely that the data can be transferred to the, mostly younger, population receiving an ACL reconstruction [3, 11, 34, 44]. Perfusion fixation preserves the outer contours of shapes i.e. ligament bundles, so neither the identification nor the measurements of the various bundles can be assumed to have been hampered by the fixation.
Conclusions
This study was performed because no earlier study described the ACL, AMB and PLB attachment centres relative to arthroscopically visible landmarks. This resulted in quantitative data of the positions of the attachment centres of the ACL and its two bundles, relative to bony landmarks on femur and tibia, visible through an arthroscope. Using these results, the surgeon will be able to determine the anatomical position of the ACL and the two functional bundles during arthroscopy without additional images or fluoroscopic support. The results can be applied for anatomic single-bundle or anatomic double-bundle techniques.

posterolateral bundle
tunnel placement
anterior cruciate ligament
anteromedial bundle
anatomic acl reconstruction
double-bundle acl reconstruction
arthroscopic view
acl anatomy


The diagnostic strength of the 24-h pad test for self-reported symptoms of urinary incontinence in pregnancy and after childbirth

The clinical impact of incontinence in pregnancy and after childbirth is growing because some studies report the efficacy of physiotherapy in pregnancy and because obstetric choices are supposed to have significant impact on post-reproductive urinary function (Goldberg et al. in Am J Obstet Gynecol 188:1447–1450, 2003). Thus, the need for objective measurement of urinary incontinence in pregnancy is growing. Data on pad testing in pregnancy are lacking. We assessed the clinical relevance of the 24-h pad test during pregnancy and after childbirth, compared with data on self-reported symptoms of urinary incontinence and visual analogue score. According to the receiver operating characteristic curve, the diagnostic value of pad testing for measuring (severity of) self-reported incontinence during pregnancy is not of clinical relevance. However, for the purposes of research, pad tests, combined with subjective/qualitative considerations, play a critical role in allowing comparisons across studies, quantifying the amount of urine loss and establishing a measure of severity.
Introduction
Incontinence is reported frequently in pregnancy and after childbirth [2–4]. It has been suggested that urinary incontinence in pregnancy is a predictor of the chance to develop post-partum urinary incontinence [5]. In that respect, prevention such as physiotherapy during pregnancy is advised in women with positive symptoms of urinary incontinence in pregnancy [6–9]. Based on questionnaires on symptoms of post-partum urinary incontinence, Goldberg et al. found a strong protective effect of cesarean delivery against the development of post-partum urinary incontinence and highlighted the impact of obstetric choices on post-reproductive urinary function [1]. When the possible consequences of the fact that a pregnant women reports urinary incontinence grow, such as an advise for preventive physiotherapy or an advice regarding the mode of delivery, there is a greater need for objectivity in diagnosing the problem. Pad testing yields an objective measurement of fluid loss over a certain period. In non-pregnant women, the diagnostic value of pad testing for self-reporting of symptoms of urinary incontinence has been questioned [10, 11]. These data are lacking for pregnant women. Most common used types of pad tests are the 1-h and the 24-h test [12–15]. The 24-h pad test is almost certainly more representative to the patients’ day-to-day experiences and is more likely to correlate with self-reported symptoms [16]. The 24-h pad testing has been studied and described exclusively in non-pregnant women. In this study, we focussed on the diagnostic strength of pad testing to measure (the severity of) urinary incontinence in pregnancy and after childbirth. The aims of this study were (1) to describe pad weight gain as measured by the 24-h pad test in a cohort of pregnant women and (2) to assess the clinical usefulness of the 24-h pad test in pregnancy and after childbirth in terms of the relationship between objective urine loss and the self-reported symptoms of urinary incontinence.
Materials and methods
One hundred and seventeen women who attended the outpatient clinics of the University Hospital Groningen and the Martini Hospital Groningen enrolled for the study, with a mean age of 30 years (range 17–41). All women were of Caucasian origin, except three who were of Mediterranean origin. All women were nulliparous and had no history of incontinence, pelvic operations or neurological disease. Written informed consent was obtained from all participating women. The study was approved by the medical ethical committees of both hospitals. For this study, the women were investigated at 28–32 and at 36–38 weeks of pregnancy and 6 weeks and 6 months post-partum.
At each visit, all women completed a questionnaire and a visual analogue score (VAS) on symptoms of urinary incontinence. Complaints of more than five on the VAS scale 0–10 were defined as severe complaints. Women were asked to classify their incontinence as mainly: (1) stress urinary incontinence, involuntary leakage on effort or exertion or on sneezing or coughing, and (2) urge urinary incontinence, involuntary leakage accompanied by or immediately preceded by a strong desire to void [17]. Three pads were packed, each in a plastic bag, and weighed by the investigators before and after use. The women received a written instruction and were free to wear one, two or three pads. It was emphasised that the bag should be closed carefully every time a pad was changed to prevent evaporation. If the bags were open or less than three pads were returned, the test was excluded from evaluation. Pads were given to all women, to be worn for 24 h preceding their appointment. The outcome of the 24-h pad test was recorded as the weight gain as measured by a verified spring balance. Weighing was done by the first or second author, within 3 days after the pad test was carried out. According to the literature, pads were assigned as wet if the total weight gain per 24 h was ≥9 g [11].
Statistical analysis
Pad test results have a non-parametric distribution. For continuous variables, non-parametric tests are used. Numeric data are analysed by cross tabulation, chi-squared test and risk analysis. Pearson correlation test was used to identify significant relationships between variables. Data are presented as median or numbers.
Results
Pregnancy, pad test At 28 weeks of pregnancy, 115 of 117 patients (98%) returned their pads according to the protocol. The median weight gain was 5 g (range 0–36). At 38 weeks, data were available from 98 women (84%). Two patients had withdrawn from the study because of inconvenience, while 17 pads were not, or were not according to the protocol returned. At this stage of pregnancy, the median weight gain was also 5 g (range 0–22). Distributions of pad test weight gain are given in Fig. 1.
Fig. 1Distribution of 24-h pad test results at 28 and 38 weeks of pregnancyPad test results at 28 and 38 weeks of pregnancy were related (r = 0.452, p < 0.0001). Twenty-seven out of 115 (23%) and 17out of 98 pads (17%) were wet at 28 and 38 weeks of pregnancy, respectively, and again, results at 28 and 38 weeks were related (r = 0.317, p < 0.001).
Pregnancy, questionnaire At 28 weeks of pregnancy, 35 of 117 women (30%) reported incontinence (stress, 28 of 35, and urge, 7 of 35), and at 38 weeks, 40 of 115 (35%) did (stress, 33 of 40 [82%], and urge, 7 of 40 [8%]). Reported incontinence at 28 and 38 weeks of pregnancy was related (r = 0.55, p < 0.001). Severe complaints of incontinence were reported in 17 of 117 (15%) and 22 of 115 cases (19%), which were also related (r = 0.482, p < 0.001).
Pregnancy, pad test and questionnaire Women with self-reported incontinence at 28 and 38 weeks of pregnancy had a median pad weight gain of 6.0 (range 0–36) and 6.0 g (range 0–22), respectively, while women without self-reported incontinence had a pad test result of 4.0 (range 0–22) and 4.0 g (range 0–22), respectively, showing no differences between these groups of women. To evaluate the diagnostic value of the pad test for measuring self-reported incontinence, the sensitivity and specificity for several cutoff levels for the pad test was calculated, graphically known as the receiver operating characteristic (ROC) curve. The ROC curve for pad tests and self-reported incontinence at 28 weeks of pregnancy did not differ from the reference line (area 0.575 vs 0.5, p = 0.207), showing a non-diagnostic test (Fig. 2). At 38 weeks of pregnancy, the ROC curve showed a significant difference from the reference line (area 0.663 vs 0.5, p = 0.008), the optimum cutoff point at 5.5 g with a sensitivity of 0.629 and a specificity of 0.619 (Fig. 2). When stratified for stress incontinence or for severe symptoms of urinary incontinence, the ROC curve did not improve.
Fig. 2ROC curve, the diagnostic value of the pad test at different cutoff levels (g/24 h) for measuring self reported incontinence at 28 (a) and 38 (b) weeks
Puerperium, pad test At 6 weeks post-partum, 80 of 117 patients (68%) returned their pads according to the protocol. The median weight gain was 3 g (range 0–40). At 6 months post-partum, pad data were available from 76 patients (65%). Six patients had withdrawn from the study because of inconvenience, three patients had their delivery preterm, 32 pads were not, or were not according to the protocol returned. These women did not want to participate in the pad study anymore or did not return their pads according to the protocol. The women that withdrew from the pad test study were asked to fill up the questionnaires on symptoms of urinary incontinence to check for bias. The women did not differ for self-reported symptoms on urinary incontinence from the women that continued the study on pads. In the study group, the median weight gain was again 3 g (range 0–40). Distributions of the pad test weight gain are given in Fig. 3. Pad test results at 6 weeks and 6 months post-partum were related (r = 0.792, p < 0.0001). Seven out of 80 (9%) and 3 out of 76 pads (4%) were wet at 6 weeks and 6 months post-partum, respectively, and again, the results at 28 and 38 weeks were related (r = 0.320, p = 0.017).
Fig. 3Distribution of 24-h pad test results at 6 weeks and 6 months post-partum
Puerperium, questionnaire At 6 weeks post-partum, 21 of 115 women (18%) reported incontinence (stress, 16 of 21, and urge, 5 of 21), while at 6 months post-partum, 16 of 109 (15%) did (stress, 12 of 16, and urge, 4 of 16). Reported incontinence at 6 weeks and 6 months post-partum is related (r = 0.61, p < 0.001). Severe complaints of incontinence was reported in 15 of 115 (13%) and 11 of 109 cases (10%), which were also related (r = 0.691, p < 0.001).
Puerperium, pad test and questionnaire Women with self-reported incontinence at 6 weeks and 6 months post-partum had a median pad weight gain of 5.5 (range 0–40) and 6.0 g (range 0–40), respectively; women without self-reported incontinence had a pad test result of 3.0 (range 0–16) and 3.0 g (range 0–9), showing a differences between these groups of women (p = 0.01 and p = 0.045, respectively). The ROC curve for pad tests and self-reported incontinence at 6 weeks post differed from the reference line (area 0.767 vs 0.5, p = 0.001), the optimum cutoff point at 4.5 g and the sensitivity of 0.722 and specificity of 0.742 (Fig. 4). At 6 months post-partum, the ROC curve shows also a significant difference from the reference line (area 0.666 vs 0.5, p = 0.047), the optimum cutoff point at 5.5 g and the sensitivity of 0.629 and specificity of 0.619 (Fig. 4). Again, stratifying for stress incontinence or for severe symptoms of urinary incontinence, the ROC curve did not improve.
Fig. 4ROC curve, the diagnostic value of the pad test at different cutoff levels (g/24 h) for measuring self-reported incontinence at 6 weeks (a) and 6 months post-partum (b)
Discussion
In this study, we focussed on the use of pad testing to investigate its prognostic value for objectively measuring (the severity of) self-reported urinary incontinence during pregnancy and after childbirth. The clinical impact of incontinence in pregnancy and after childbirth is growing because some studies report the efficacy of physiotherapy in pregnancy and because obstetric choices are supposed to have a significant impact on post-reproductive urinary function [1]. This growing impact requires objective measurement. In a meta-analysis, the symptom of stress incontinence was 91% sensitive but only 51% specific for detecting genuine stress urinary incontinence as defined by the International Continence Society, based on history and urodynamic testing [18]. Because of pregnancy and because we are interested in an instrument for screening, it is obvious that urodynamic testing cannot be the instrument of choice. Pad testing is an objective, simple and non-invasive instrument capable of measuring fluid loss in a certain period. First of all, we need to define normal values, data that describe the results of pad testing in a cohort of pregnant women without a history of incontinence before pregnancy. Secondly, we need comparison with the criterion standard in pregnancy and the patients’ history.
The median weight gain in the 24-h pad test in pregnancy as reported in our study is in accordance with results reported in a group of non-pregnant, not incontinent, premenopausal women, 2.6–7.0 g, with an upper confidence limit of 5.5–8 g [16, 19]. In post-menopausal continent women, a much lower weight gain of 0.3 g is reported [20]. When compared to the pre-menopausal women, the state of pregnancy does not lead to a higher weight gain in the 24-h pad test nor does the puerperal state.
It is remarkable that the group of controls as referred to had similar pad test results but did not report incontinence, whereas in our pregnant group, 30 (28 weeks of pregnancy) and 35% (38 weeks of pregnancy) of the women did. With the same weight gain in pregnancy, women report more incontinence than in the sample of non-pregnant women. It seems therefore that not the amount of weight gain in the pad test but the pregnancy state itself is more discriminating for the chance that a woman qualifies herself as incontinent.
In our study, during pregnancy, pad test results had only limited diagnostic value for self-reporting of incontinence. In their review article, Ryhammer et al. [21] stated that “incontinence is a complex condition in which differences in the individual patients’ personal characteristics influence the perception of leakage and the identification of the problem. Pregnancy seems to modulate this perception in such a way that it cannot be measured by pad test. As pad testing did not show to have high sensitivity and specificity for self-reported urinary incontinence in pregnancy and after childbirth, there remains confusion about the accurate diagnosis. This becomes important in deciding on management options such as offering preventive physiotherapy in selected cases or strategies that influence the mode of delivery.
After childbirth, the median weight gain is also in accordance with results in non-pregnant continent women. In our study group, 15 to 18% of the women report positive for symptoms of incontinence. Just like in pregnancy, the pad test result has a significant value for testing self-reporting incontinence but again low figures for sensitivity and specificity. Like us, Morkved and Bo [22] reported a discrepancy between self-reported symptoms and stress urinary incontinence assessed by their (short) pad test, 8 weeks after delivery. To assign a women to an intervention, one needs a higher specificity, which according to the curves as shown, will rapidly lead to lower sensitivity. Depending on the chosen intervention, this may or may not be accepted.
The calculation of the diagnostic strength of our 24-h pad test was made with the self-reporting of symptoms of urinary incontinence as the gold standard. At 28 weeks, the pad test failed to capture eight subjects who stated they were wet; at 38 weeks, this was higher. Such results are possibly related to the high threshold for definition of incontinence in the women with some leaking less than 9 g describing some leakage. The rationale for using a high cutoff is established in both men and women, but subjects themselves may perceive this a severe incontinence. It is possible that pregnant and post-delivery women perceive leakage differently than their non-pregnant counterparts.
When adding severity of symptoms to the gold standard, as reported by VAS, the diagnostic strength of the pad test did not improve.
In general practice, questioning about incontinence will provide the clinician with adequate information on the presence, absence or severity of incontinence from a patient perspective, and cumbersome pad tests are unnecessary. In a review article on questionnaires for women with pelvic floor disorders, Barber [23] concludes that measuring symptom severity and quality of life changes in women with pelvic floor disorders is an important part of the evaluation and treatment of women and may be the only practical way to clinically assess symptoms. However, for the purposes of research, pad tests, combined with subjective/qualitative considerations, play a critical role in allowing comparisons across studies, quantifying the amount of urine loss and establishing a measure of severity. Indeed, the International Continence Society standards for research strongly recommend the pad test as one measure in all incontinence research. The fact that the pad test results and patient-reported incontinence were not strongly correlated illustrates the importance of both quantitative and qualitative measures when considered an intervention trial with pelvic floor muscle exercises, for example.
From our study, we conclude that pad testing measures fluid loss over a certain period but does not quantify self-reported symptoms of urinary incontinence. Both measurements are of interest but cannot replace each other. Stressing of the pelvic floor by pregnancy and childbirth modulates the sensation of urinary leakage in such a way that women in this state do report symptoms of urinary incontinence more frequently than nulliparous pre-menopausal women do.

diagnostic strength
pad test
urinary incontinence
pregnancy
childbirth