Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium

We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium. Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification (TAP) tag. The TAP tag consists of a protein A tag and a calmodulin binding peptide (CBP) and has been successfully used for purification of native protein complexes from yeast and animal cells. Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green- or yellow fluorescent protein (GFP or YFP) tags for fusion at either a C- or N-terminal location. Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34, one of the members of the well characterized Dictyostelium Arp2/3 complex, as bait. After denaturation and SDS–PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified.
Dictyostelium discoideum is a genetically tractable model system for studying remodeling of the actin cytoskeleton during cell locomotion, cell division, phagocytosis and vesicle trafficking and for elucidation of signaling pathways that control chemotaxis and development. Several metazoan-type cytoskeletal components were identified for the first time in Dictyostelium
[1–3], and the process of pleckstrin homology domain mediated recruitment that plays such crucial roles in leukocyte chemotaxis and phagocytosis was also first demonstrated in the Dictyostelids [4]. The Dictyostelium genome is now completely sequenced and each gene is therefore available for functional analysis by gene disruption, overexpression and mutagenesis [5]. Genetic approaches to create tagged mutations, such as REMI (restriction enzyme mediated integration), have identified numerous genes with crucial roles in developmental or cellular processes [6]. However in only a few cases has it been possible to use genetic approaches, such as screens for suppressor mutations, to determine epistatic relationships between genes [7]. Or, in other words, to determine in what order their cognate proteins act on each other in a pathway. In other genetically tractable systems, such as the fruit fly and the nematode, epistatic relationships are usually determined by crossing in loss- and gain-of-function mutants of genes in the pathway. The poor accessibility of the Dictyostelid sexual stage, the macrocyst, has prohibited this approach.
Other genetic screens, such as the yeast two-hybrid system or the split-ubiquitin method are being successfully used to identify interacting proteins in a pathway [8,9]. However, these methods depend on successful heterologous expression of the proteins in yeast, which is not possible for many proteins. The TAP tag was developed about seven years ago to purify native protein complexes from yeast [10,11] and was used for systematic identification of protein complexes in yeast [12–15] and metazoans [16,17]. For C-terminal tagging, the TAP tag contains in tandem, a calmodulin binding peptide tag, a TEV protease cleavage site and a protein A tag. For N-terminal tagging the order of these units is reversed. We have constructed a series of vectors for C- and N-terminal tagging of proteins expressed in Dictyostelium. Protein expression is driven by the strong constitutive actin15 promoter. Additional cloning sites were introduced to allow replacement of this promoter by an inducible promoter. A third green- or yellow fluorescent protein tag was introduced for C- or N-terminal expression in some of the vectors for rapid assessment of the levels of protein expression and the cellular location of the expressed proteins.
Materials and methods
Construction of plasmid vectors
Gene fragments were amplified by polymerase chain reaction (PCR)2 with a 4:1 mixture of the Taq and Pfu DNA polymerases (Promega, Madison, WI). All oligonucleotide primers used in this study are listed in Table 1. The integrating Dictyostelium vector EXP4(+) [18] was used as starting material. An XbaI and a HindIII site upstream of the actin15 promoter were first successively destroyed by filling in the overhanging sites after digestion with the Klenow fragment of polymerase I, followed by religation, resulting in vector EXP4(-XH). This also generated a unique Nhe1 site replacing the HindIII site.
A long multiple cloning site (MCS) was generated by deleting the existing polylinker by BglII/XhoI digestion of the vector and by ligating a duplex of primers MCS1 and MCS2 that carries compatible sticky ends into the BglII and XhoI sites. The resulting vector, EXP5(+) was used as the backbone for all subsequent constructs containing TAP and/or EGFP/EYFP fragments (Table 2).
The N-terminal TAP fragment (NTAP) was amplified from the plasmid pBS1761 [11] using primers NTAP1 and NTAP2 and ligated into the BamHI and HindIII sites of EXP5(+), yielding pDV-NTAP. The C-terminal TAP fragment (CTAP) was amplified from plasmid pBS1479 [10] using primers CTAP1 and CTAP2 and ligated into the ClaI and XhoI sites of EXP5(+), yielding vector pDV-CTAP. Enhanced yellow- and green fluorescent protein genes (YFP) and (GFP) were amplified from the vectors pEYFP-N1 and pEGFP-C3 (Clontech, Mountain View, CA), respectively. For N-terminal location of YFP, primers tapYFP1 and tapYFP2 were used and insertion occurred in the HindIII and SpeI sites of EXP5(+), yielding pDV-NYFP. For C-terminal location of YFP or GFP, primers tapYFP3 and tapYFP4 were used and insertion occurred into the EcoRI and ClaI sites of EXP5(+), yielding pDV-CYFP or pDV-CGFP. All constructs were validated by sequencing across the entire tag and MCS and are listed in Table 2.
Generation and transformation of construct p34-ArcCTAP
The Dictyostelium p34-Arc protein is encoded by the ArpE gene [5,19]. The 1251 bp ArpE coding region was amplified by PCR from genomic DNA isolated from wild-type AX2 cells, using primers p34Arc1 and p34Arc2 (Table 1). The BamHI/XbaI digested PCR product was ligated into the BamHI and XbaI sites of vector pDV-CTAP, yielding vector p34-ArcCTAP. The p34-ArcCTAP, pDV-CGFP-CTAP and pDV-CTAP constructs were transformed by electroporation into D. discoideum strain AX2 and transformants were selected by growth in the presence of 20 μg/ml G418.
Purification of TAP-tagged protein complexes
TAP purification was performed as described previously [10,11] with some modifications. Briefly, 2 × 109 exponentially growing AX2 cells expressing TAP constructs were harvested, washed twice in 10 mM Na/K-phosphate buffer, pH 6.5 and resuspended in 10 ml IPP (150 mM NaCl in 10 mM Tris, pH 8.0), containing one tablet of Complete Protease inhibitor cocktail (Roche, Lewes, UK) per 50 ml. Cells were lysed by addition of Triton X-100 to 1% (v/v) and incubated for 30 min on ice. Lysates were cleared by centrifugation for 20 min at 20000g and 4 °C. The supernatant was rotated for 1 h at 4 °C with 0.1% (v/v) Nonidet P-40 (NP-40) and 200 μl bed volume of IgG-agarose beads (Sigma, St Louis, MO) in Polyprep columns (Bio-rad, Hercules, CA). The beads were washed three times with IPPN (0.1% NP-40 in IPP), and once with 10 ml TEV cleavage buffer (1 mM DTT and 0.5 mM EDTA in IPPN). Bound complexes were liberated by rotating the column for 2 h at 16 °C with 100 units of TEV protease (Invitrogen, Carlsbad, CA) in 1 ml TEV cleavage buffer and recovered by elution.
Subsequently 3 μl of 1 M CaCl2, 3 ml CaM binding buffer (1 mM imidazole, 1 mM Mg-acetate, 10 mM β-mercaptoethanol and 2 mM CaCl2 in IPPN) and 200 μl of bed volume Calmodulin Affinity Resin (Stratagene, La Jolla, CA) were added per milliliter of eluate. The mixture was rotated for 1 h at 4 °C in a second Polyprep column. Beads were washed 3 times with 10 ml CaM binding buffer and bound material was eluted in 5 fractions of 200 μl each with CaM elution buffer (1 mM imidazole, 1 mM Mg-acetate, 10 mM β-mercaptoethanol and 2 mM EGTA in IPPN). Proteins were separated by SDS–PAGE on 4–12% Bis–Tris gels (Invitrogen, Carlsbad, CA) and visualized with a Colloidal Blue Staining kit (Invitrogen, Carlsbad, CA).
Protein mass fingerprinting
Protein mass fingerprint data was obtained by MALDI-TOF-TOF (MS/MS) analysis performed at the University of Dundee ‘Fingerprints’ Proteomics Facility using an Applied Biosystems 4700 Proteomic Analyser. Protein bands were excised, in-gel reductively alkylated and digested in 20 mM NH4HCO3 containing 0.1% N-octylglucoside and 12.5 μg/ml trypsin [20]. One-tenth of each digest was applied to a 192 well MALDI sample plate (Applied Biosystems (AB)), allowed to air dry and mixed with 0.5 μl of a solution of 5 mg/ml α-cyano-4-hydroxy-trans-cinnamic acid matrix (Sigma, StLouis, USA), 50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid in 10 mM NH4H2PO4, and then allowed to air dry prior to analysis.
The mass spectrometer was internally calibrated using the AB 4700 Proteomic Analyser Calibration Mix. Using the 4000 series Explorer Software (AB), MS spectral data were acquired from the samples and an MS/MS list was automatically generated for further analysis based on the top 5 most intense ions present (trypsin and major keratin ions were excluded). The MS and MS/MS spectral data obtained were exported from the 4700 using the Global Proteome Server (GPS) Explorer Software (AB). The data were then submitted to a local MASCOT search engine (Matrixscience, London, UK) for protein database searching against the Uniprot database (http://www.ebi.ac.uk/swissprot/) for identification.
Results and discussion
Cloning strategies
The integrating Dictyostelium vector EXP4(+) was chosen as the backbone for all constructs. This vector is derived from pATSP [21], itself a derivative of the standard cloning vector pAT153. EXP4(+) carries the Act6NeoR cassette for G418 selection in Dictyostelium, and a cassette for protein expression consisting of the constitutive actin 15 (A15) promoter, the 2H3 terminator and a small polylinker with 6 restriction sites [18]. Because some of the cloning steps used to make this vector could not be retraced, we sequenced about 900 bp each around the points of fusion of the 2H3 terminator with the actin6 promoter, and the NeoR terminal region with the original pAT153 vector backbone. This revealed that the 2H3 terminator was preceded by 257 bp of the 2H3 coding region. However, since this coding sequence is downstream of the stopcodon in EXP4(+) and all derived vectors, it is not incorporated in the expressed tagged proteins. The complete sequence of EXP(4+) was reconstructed and a vector map is presented in Fig. 1.
To obtain a larger multiple cloning region, we first successively destroyed the XbaI and HindIII sites upstream of the A15 promoter, creating a novel NheI site. In addition to the existing SalI site, this site can be used to exchange promoters or to insert genes with their own promoter. The existing polylinker was removed by BglII and XhoI digestion and replaced by an adaptor duplex of oligonucleotides MCS1 and MCS2 (Table 1), yielding vector EXP5(+) (Fig. 1). The new multiple cloning site (MCS) contains an ATG start codon, preceded by 5 adenine residues to generate the Dictyostelium Kozak sequence and followed by 7 unique restriction sites:
The MCS was subsequently used to clone in the TAP tags and enhanced YFP or GFP tags that were amplified by PCR. This is possible in any configuration i.e. TAP alone, YFP(GFP) alone or combinations of TAP and YFP(GFP) at the same site or at opposing sites. When cloned at the same site, the YFP(GFP) is in between the gene of interest and the TAP sequence as indicated above. Here, NTAP and CTAP stand for N- and C-terminal TAP, respectively, while NYFP and CYFP(GFP) stand for N- and C-terminal GFPs and YFPs. Vectors with single NTAP, CTAP, NYFP or CYFP(GFP) tags were created first and validated by DNA sequencing across the entire tag and MCS. The CTAP fragment contained a stopcodon before the XhoI restriction site. Vectors with TAP + GFP(YFP) tags were then generated by removing a tag from one of these vectors and cloning it in the appropriate position in the other vectors. Some restriction sites cannot be used anymore: BglII will digest both the NTAP and the CTAP tags, while EcoRI will digest the CTAP tag. NheI will digest the CTAP tag, which then leaves only SalI to introduce a different promoter. All vectors prepared in the course of this work are listed in Table 2 and their sequences are deposited in GenBank.
Purification of the Dictyostelium Arp2/3 complex with TAP-tagged p34-Arc
To validate that the TAP tag can be used to purify native protein complexes in Dictyostelium we used a TAP tagged D. discoideum p34-Arc protein as bait for purification of members of the well-characterized Arp2/3 complex. The Arp2/3 complex is a complex of 7 proteins, including the p34-Arc protein, involved in the nucleation of actin polymerization [19]. We prepared a C-terminal fusion of the TAP tag with D. discoideum p34-Arc, as was previously done for yeast and human p34-Arc (named Arc35 and ARP2C, respectively) [14]. The p34-Arc gene was amplified from D. discoideum genomic DNA using the primers p34Arc1 and p34Arc2 (Table 1), which harbour BamH1 and XbaI sites, respectively, and cloned into the BamHI and XbaI digested pCTAP vector. This yielded the vector p34-Arc-CTAP, which was transformed into D. discoideum AX2 cells. AX2 cells transformed with the pDV-CTAP vector and with a pDV-CGFP-CTAP vector served as controls. For each transformed cell line, lysates were prepared by detergent treatment from 2 × 109 cells and adsorbed to IgG agarose. After extensive washing, the 4.8 kD CBP moiety of the TAP tag with fused “bait” and complexed “prey” were released from IgG agarose by TEV protease treatment. The proteins or protein complexes were further purified over calmodulin resin, eluted with EGTA, denatured and size-fractionated on SDS–PAGE gels. Fig. 2 shows that a few faint bands were visible in preparations from cells that contained the empty pDV-CTAP vector. Extracts from pDV-CGFP-CTAP transformed cells showed a strong band at 32 kD, the expected size of the CGFP–CBP fusion (26.8 + 4.8 kD) after successful cleavage. Extracts of cells transformed with p34-Arc-CTAP displayed a pattern of 8 bands, similar to that observed after purification of the Dictyostelium Arp2/3 complex by traditional chromatography [19]. Bands 1–8 were cut out from the gel, proteins were digested in gel with trypsin and sequences of the extracted peptides were determined by MALDI-TOF/TOF mass spectrometry. This yielded 12–18 different peptide sequences for each of the 4 larger bands and 7–11 different peptide sequences for each of the four smaller bands. Query of the Uniprot protein database with the peptide sequences yielded all members of the D. discoideum Arp2/3 complex for bands 1–4 and 6–8 (Fig. 2). Band 5, which is also present in the pDV-CTAP and pDV-CGFP-CTAP extracts was identified as discoidin B, a highly abundant Dictyostelium lectin, which is most likely a non-specific contaminant.
Conclusions
We have constructed eleven integrating plasmid vectors for protein expression in Dictyostelium that carry the TAP tag for tandem affinity purification of native proteins or protein complexes. In addition to the TAP tag for N-terminal or C-terminal fusion, a subset of vectors carry an additional enhanced GFP or enhanced YFP tag at either the same or opposite location as the TAP tag. The vectors are constructed in such a manner that the existing constitutive actin15 promoter can easily be swapped for an inducible promoter. Alternatively the pDV-CTAP vectors also allow cloning of a gene under its own promoter. In addition, the neomycin selection cassette can be exchanged for a different selection marker e.g. blasticidin resistance, which requires only a single copy of the bsr gene.
Preferentially, the “bait” gene of choice should be expressed under its own promoter from a single copy vector in a “bait” null mutant. This will avoid artefacts due to ectopic expression and competition of the endogenous “bait” with the tagged “bait” for binding to cellular proteins. Moreover, rescue of the null mutant phenotype by the tagged “bait” will ensure that the tags are not interfering with protein function. However, it may often be desirable to overexpress the “bait” to obtain a sufficient yield of protein complexes, although this also increases the probability of pulling down unrelated proteins.
We show that for the pDV-CGFP-CTAP vector, the highly fluorescent GFP protein is efficiently released and purified by the two affinity steps and the TEV protease treatment. We also show that by using a p34-Arc-CTAP fusion protein as bait, the 6 associated members of the Arp2/3 complex could be isolated with negligible contamination. We therefore expect that these vectors will prove to be useful tools for identification of native protein complexes in Dictyostelium.

tandem affinity purification
expression vector
native protein complexes
yellow fluorescent protein
dictyostelium discoideum


Laparoscopic Pelvic Autonomic Nerve-Preserving Surgery for Sigmoid Colon Cancer

Background To test the feasibility of laparoscopic approach in performing the simultaneous pelvic autonomic nerve preservation during standard anterior resection of sigmoid colon cancer.
Preservation of pelvic autonomic nerves to retain genitourinary function without compromise of oncologic clearance of tumor has been extensively studied in rectal cancer surgery.1–12 However, there is still not much information on sexual and bladder function after the curative resection of sigmoid colon cancer. Theoretically, the pelvic autonomic nerve-preserving surgery is technically much easier during a sigmoid colon resection, as apposed to rectal surgery, because the pelvic deep-seated neural structures, such as pelvic plexus (inferior hypogastric plexus), lateral ligament, neurovascular bundles of Walsh, and periprostate plexus, were not within the boundary of dissection.13–17 It is therefore our opinion that during the anterior resection of sigmoid colon cancer, preservation of autonomic nerves is a priority—that is, autonomic nerve preservation is appropriate unless there are oncologic reasons for the resection of nerves, e.g., the nerves were directly involved by cancer, in terms of better postoperative genitourinary function for the patients. Recently, more and more sigmoid colon cancers were resected laparoscopically.18–20 Therefore, we conducted the present study to test the feasibility of laparoscopic approach in performing the total pelvic autonomic nerve preservation during the standard oncologic resection of sigmoid colon cancer. We hypothesized that given a well-illuminated magnified view by laparoscopy, the autonomic nerves can be well protected from inadvertent surgical damage.
PATIENTS AND METHODS
Patients meeting the following eligibility criteria were recruited for the present study. The inclusion criteria were: (1) curative and elective surgery; (2) sigmoid adenocarcinoma with tumor location generally 15 to 40 cm above anal verge; (3) American Society of Anesthesiology function class I to class III patients; (4) age between 18 to 65 years. The exclusion criteria were: (1) emergency or urgent surgery; (2) evidence of tumor invasion to adjacent organs or distant metastasis; (3) previous major abdominal or pelvic surgery; (4) abnormal preoperative baseline genitourinary function on the basis of validated questionnaire-based interview.10,21,22 Before entering the present study, all patients were well explained regarding the surgical procedures, and informed consents were obtained. The present study was approved by the Institutional Review board of National Taiwan University Hospital and was designated as NTUH 93S-024.
Surgical Strategy
The related neuroanatomy for the nerve-preserving surgery of sigmoid colon cancer is shown in Fig. 1A. The preservation of autonomic nerves was focused on the following three anatomic sites: (1) inferior mesenteric plexus (Fig. 1B); (2) superior hypogastric plexus (Fig. 1C); and (3) the paired hypogastric nerves (Fig. 1D). The details of surgical procedures were well recorded and are shown in the associated video.
Fig. 1(A) Surgical anatomy of pelvic anatomic nerves related to the nerve-preserving surgery of sigmoid colon cancer. (B) The inferior mesenteric plexus (yellow arrow) encircles the root of inferior mesenteric artery (IMA). (C) The superior hypogastric plexus overlying the interiliac trigone area. (D) The right hypogastric nerve travels across the presacral areolar tissue and then adheres to the proper rectal fascia.
To ensure the visualization and preservation of the nerves at all three critical points, we used a specific point-by-point checklist during the operations. The dissection commences at the medial side of sigmoid mesentery at the level of pelvic promontory. By gentle tenting of the sigmoid mesocolon ventrally, the superior hypogastric plexus was identified and preserved at the interiliac trigone just below the peritoneum. Subsequently, the dissection was continued upward along the ventral side of abdominal aorta. The preaortic plexus were then preserved by sparing the preaortic connective tissue. Thereafter, the medial-to-lateral laparoscopic dissection along the areolar tissue between sigmoid mesocolon and Gerota fascia was performed. The medial approach is well adapted for laparoscopy because it provides the working space and demands the least handling of sigmoid colon.23
During the mobilization of sigmoid mesocolon, we did not identify the ureter because the loose connective tissue between Gerota fascia and sigmoid mesocolon is a natural dissection plane. Normally, the ureter and gonadal vessels are covered by the Gerota fascia, and therefore, if the ureter is clearly or intentionally identified, it means that the Gerota fascia is opened and the dissection plane may be imprecise. By simultaneous upward and medial-to-lateral dissection, we subsequently reach the junction of inferior mesenteric artery (IMA) and abdominal aorta. The IMA was divided 1 to 2 cm distal to its origin, or just distal to the left colic branch. This is because the sympathetic fibers encircle the IMA at the level of IMA’s origin from abdominal aorta. If the nerves are not adequately cleared from “behind” the IMA, they can be easily damaged at this point (Fig. 1B). Subsequently, the “holy plane” at the transition of the mesosigmoid to the mesorectum was meticulously dissected to progressively displace the hypogastric nerves dorsally and laterally (Fig. 1D), and thus preserve them.
Assessment of Urinary Function
Urinary function was evaluated on the basis of questionnaire-based interview of patients preoperatively and 3 months after surgery. In patients with major surgical complications, for example, anastomotic leakage, the urinary function was evaluated 3 months after recovery from surgical morbidity, i.e., 3 months after the closure of defunctioning ileostoma. The preoperative functional questionnaire was based on the recollection of function before the development of symptoms from sigmoid colon cancer. Routinely, the urine Foley catheter was inserted for the patients immediately before the operation. In the postoperative period, the date for urine removal of the Foley catheter and the nature of any voiding problems, such as those requiring recatheterization, were recorded. The duration of indwelling urine Foley catheter was counted from the time of insertion to the time of its removal. The questionnaire used was based on International Prostate Symptom Score (IPSS), the parameters of which included incomplete bladder emptying, frequency, intermittency, urgency, weak stream, straining, and nocturia. Further subdivisions were added to the seven items.10 The scoring system for voiding function described above was based on a 0 to 5 scale, as follows: 0, not at all; 1, less than one time in five; 2, less than half the time; 3, approximately half the time; 4, more than half the time; 5, almost always. IPSS was calculated by adding the item scores. We ranked the urinary function as good (IPSS, 0–7); fair (IPSS, 8–14), and poor (IPSS, 15–35). Any voiding problems recovered within 3 months after surgery was considered to be transient bladder voiding dysfunction; otherwise, the voiding problems were deemed persistent.
Assessment of Sexual Function
Sexual function was evaluated preoperatively and 6 months after operation or recovery from surgical complications, if present.
In male patients, the sexual function was evaluated on the basis of potency and ejaculation. The ejaculatory function was graded as good, fair (decrease in ejaculatory amounts up to 50%), and poor (retrograde ejaculation, failure to ejaculate). The grade of potency disorder was measured on the basis of the International Index of Erectile Function (IIEF), which contains five index domains (erectile function, orgasm function, sexual desire, intercourse satisfaction, and overall satisfaction) and 15 items.21 Each item of the IIEF is scored on a 5- or 6-point Likert score. Score for each domain are variable, and the total score range is 5 to 75. We classified the male potency function as good (IIEF, 60–75), fair (IIEF, 44–59), and poor (IIEF, 5–43).
In female patients, sexual function was evaluated on the basis of the Female Sexual Function Index (FSFI), which is a 19-item validated questionnaire that was designed to evaluate female sexual dysfunction, especially female sexual arousal disorder.22 The scale is divided into six domains: desire, arousal, lubrication, orgasm, satisfaction, and pain. Each item is scored with a 5- or 6- point Likert scale. The scales for each domain are variable, and the total score range is 4 to 95. For the present study, we classified the female sexual function as good (FSFI, 76–95), fair (FSFI, 58–75), and poor (FSFI, 4–57). Furthermore, the specific sexual problems in women such as libido, arousal, lubrication, orgasm, and dyspareunia were investigated.
Data Management
Data on patients undergoing laparoscopic autonomic nerve-preserving surgery for sigmoid colon cancer between January 2004 and December 2006 were entered into a prospective database of the present study. Until June 2007, the mean time of follow-up for the patients was 18 months, ranging from 6 to 36 months. The genitourinary function of the patients was analyzed by the intention-to-treat principle. Patients were first contacted by telephone by a research assistant who described the nature of the project. Those who consented to the present study were given a questionnaire-based interview by the research assistant. The data were managed by a statistician. To prevent bias of evaluation, the research assistant was not told which surgical procedure was performed for each individual patient.
Because genitourinary function is influenced by preoperative function and numerous other variables, including the emotional and physiological status of the patients, as well as other influences such as chemotherapy, each patient was used as his or her own control in evaluating the change of function before and after surgery. The two sets of measurements on the same patient were compared, and the P value was calculated from paired t-tests. A statistically significant level of difference was set at a P value of ≤.05.
RESULTS
A total of 112 patients were operated on with the intent of total preservation of pelvic autonomic nerves and curative resection of sigmoid colon cancer. The demographics and clinicopathologic data are shown in Table 1. The nerve-preserving surgery was considered as failed in four patients when the videos of the surgical procedures were rescrutinized by coauthor colorectal surgeons and one general surgeon. The nerve-preserving anterior resection of sigmoid colon was performed with acceptable operation time, little blood loss, and low morbidity. The number of dissected lymph node was adequate, and the short-term oncologic result was good.
Table 1Demographics, clinicopathologic data, and surgical outcomes of patients undergoing laparoscopic autonomic nerve-preserving surgery for sigmoid colon cancer (n = 112)ParameterValueaAge (years)55.8 ± 6.4Sex (M/F)58/54Body mass index (weight/height2, kg/m2), mean (range)    Male23.4 (20.3∼31.6)    Female24.8 (22.0∼32.5)ASA class    I62    II46    III4TME stage (pathologic)     I8    II54    III50Operative time (minutes)184.4 ± 44.8Conversion rate .9% (n = 1)Blood loss (mL)84.5 ± 24.0Postoperative ileus (hours)48.0 ± 8.4Hospitalization (days)8.0 ± 2.0Postoperative pain (visual analog scale)3.5 ± .4Postoperative complication    Total7.1% (n = 8)    Wound infection4    Deep vein thrombosis1    Pneumonia 1    Anastomotic leakage2No. of lymph nodes collected14.4 ± 4.0Tumor recurrence rate8.0% (n = 9)ASA, American Society of Anesthesiology; TNM, tumor, node, metastasis system.a Continuous data are presented as mean ± SD.
In patients with successful nerve-preserving surgery (96.4%, n = 108), 104 patients completed the evaluation of urinary function (Table 2). The median duration of the indwelling urine Foley catheter was 3.0 days, ranging from 1.0 to 7.0 days. Preoperatively, the baseline urinary function of all patients was ranked as good (IPSS, 0–7). However, after postoperative removal of the urine Foley catheter, the ranks of IPSS were changed to good in 98 patients (94.2%), fair (IPSS, 8–14) in 5 (4.8%), and poor (IPSS, 15–35) in 1 (1.0%). There was no significant difference of preoperative and postoperative IPSS score (P = .075, paired t-test) in patients with successful nerve-preserving surgery. The six patients with fair or poor bladder function experienced only transient dysfunction and recovered thereafter. Forty-four male and 42 female patients who were sexually active before the operation, i.e., whose ejaculatory function, IIEF, and FSFI score were all ranked as good, responded to the assessment of sexual function. Postoperatively, in male patients (Table 3), the ranks of ejaculatory function were changed to good in 40 (90.9%), fair (decrease in ejaculatory amounts) in 3 (6.8%), and poor (retrograde ejaculation, failure of ejaculation) in 1 (2.3%). The grades of the potency were changed to good (IIEF, 60–75) in 41 (93.2%), fair (IIEF, 44–59) in 2 (4.5%), and poor (IIEF, 5–43) in 1 (2.3%). There was no significant difference of preoperative and postoperative IIEF score (P = .082, paired t-test) in male patients whose pelvic autonomic nerves were successfully preserved during surgery. In female patients (Table 3), the ranks of sexual function were changed to good (FSFI score, 76–95) in 36 (85.7%), fair (FSFI score, 58–75) in 4 (9.5%), and poor (FSFI score, 4–57) in 2 (4.8%). However, there was no significant change of FSFI score (P = .122, paired t-test) for female patients before and after successful nerve-preserving surgery. Remarkably, specific sexual problems in women with autonomic nerve preservation included vaginal lubrication in 14.3% (n = 6), dyspareunia in 9.5% (n = 4), sexual arousal in 4.7% (n = 2), and orgasm in 4.7% (n = 2).
Table 2Changes in urinary function before and after surgery for patients with successful nerve-preserving surgery (n = 104)Urinary function (range of IPSS)Before operationAfter operationP valuea    Good (0–7)10498    Fair (8–14)05    Poor (15–35)01IPSS score (mean ± SD)3.20 ± 1.723.68 ± 2.82.075IPSS, International Prostate Symptom Score.aP value was calculated on the basis of statistics of paired t-test.Table 3Changes in sexual function for male and female patients with successful nerve-preserving surgeryFunctionBefore OperationAfter OperationP valueaMale patients (n = 44)Ejaculatory function    Good4440    Fair03    Poor01Erectile function (IIEF Score)     Good (60–75)4441    Fair (44–59)02    Poor (5–43)01IIEF score (mean ± SD)72.4 ± 4.670.3 ± 8.4.082Female patients (n = 42) Sexual function (FSFI Score)     Good (76–95)4236    Fair (58–75)04    Poor (4–57)02FSFI score (mean ± SD)89.0 ± 9.285.4 ± 16.4.122IIEF, International Index of Erectile Function; FSFI, Female Sexual Function Index.aP value was calculated on the basis of statistics of paired t-test.
Of the four patients (3.6%) who failed the procedures of the nerve-preserving surgery, three were female and one was male (Table 4). In them, the mean duration of indwelling urine Foley catheter was 6 days (range, 4–8 days). Although the genitourinary function for the four patients was normal before surgery, the single male patient’s IPSS and IIEF rank declined to the level of “fair” and he experienced retrograde ejaculation after the operation. The IPSS rank of the three female patients changed to the “fair” level in two, but the other patient remained at a “good” level after operation. The sexual function was adversely affected by the failure of nerve-preserving surgery, with a “fair” rank of FSFI in one patient and a “poor” rank in the remaining two patients. All three female patients experienced specific sexual problems, including vaginal lubrication, dyspareunia, sexual arousal, and orgasm.
Table 4Changes of values in specific domains of the scoring system for evaluation of genitourinary function in patients who failed the procedures of nerve-preserving surgery (n = 4)DomainPatient 1 (male)Patient 2 (female)Patient 3 (female)Patient 4 (female)Before OPAfter OPBefore OPAfter OPBefore OPAfter OPBefore OPAfter OPIPSS (score range)    Total0100209012    1. Incomplete emptying (0–5)02010404    2. Frequency (0–5)02010102    3. Intermittency (0–5)01000202    4. Urgency (0–5)01000001    5. Week stream (0–5)01000000    6. Straining (0–5)02000203    7. Nocturia (0–5)01000000IIEF (score range)     Total7544––––––    1. Erection function (1–30)3018––––––    2. Orgasmic function (0–10)106––––––    3. Sexual desire (2–10)106––––––    4. Intercourse satisfaction (0–15)157––––––    5. Overall satisfaction (2–10)107––––––FSFI    Total––955895279532    1. Desire (2–10)––108104105    2. Arousal (0–20)––2012205206    3. Lubrication (0–20)––2010206206    4. Orgasm (0–15)––159153154    5. Satisfaction (2–15)––159153155    6. Pain (0–15)––1510156156IIEF, International Index of Erectile Function; FSFI, Female Sexual Function Index.
DISCUSSION
The present study confirmed our hypothesis that by taking advantage of the optic properties of laparoscopy, we can precisely preserve the pelvic autonomic nerve at the following three anatomic sites: inferior mesenteric plexus, superior hypogastric plexus, and paired hypogastric nerves. However, we failed to preserve the autonomic nerve in four (one male and three female) patients of this case series—a finding that needs to be addressed. After the completion of nerve-preserving surgery, we routinely tented the superior hypogastric plexus to check the total preservation of associated nerves. In our scrutiny of the surgical videos, we found that the mechanisms of nerve injury for the three female patients were the same: the nerve injuries were all located at the level of the hypogastric nerves and were bilateral. In our experience, the fibers of pelvic autonomic nerve were generally thinner in female that than in male patients. Furthermore, anatomically, the paired hypogastric nerves run downward, close to or tethering the proper rectal fascia along the pelvic wall (Fig. 1D). At this point, the right-sided hypogastric nerves of the three female patients were subjected to avulsion injury. On the other hand, the left-side hypogastric nerves of these three female patients were mistaken as pelvic fibrotic bands and were cauterized. We thought that some female patients might be predisposed to chronic pelvic inflammation, thus blurring the pelvic dissection plane, and therefore the hypogastric nerves and retroperitoneal fibrotic bands could not be clearly differentiated, even under the well-illuminated and magnified view of laparoscopy. In the only male case of autonomic nerve injury, we found that the patient’s body mass index (BMI) was 32.0 kg/m2. During the surgery, the hypogastric nerve was coated by fatty tissues and was inadvertently severed bilaterally. Because obese patients (BMI > 30 kg/m2) represented only 6 (10.3%) of the 58 male and 4 (7.4%) of 54 female patients in this case series and nerve injury related to obesity occurred in only 1 male patient, we therefore were not sure whether high BMI was a risk factor for nerve injury. Further recruitment of more obese patients for study will be necessary to solve this interesting question.
Remarkably, CaverMap Surgical Aid (CM; Blue Torch Medical Technologies, Inc., Ashland, MA) has been recommended to assist surgeons in intraoperatively localizing the autonomic nerves, ultimately leading to improved preservation of autonomic nerves and sexual function.24 We thought that CaveMap might be useful in case the nerve-preserving procedures were for the treatment of rectal cancer, in which case the neural structures to be preserved were deep-seated in the pelvis. However, for the oncologic resection of sigmoid colon cancer, the autonomic nerves at the above-mentioned three critical anatomic points were usually be clearly identified. In the rare cases when exact identification of the autonomic nerves was difficult, we recommended that the surgeons find the precise dissection plane between sigmoid mesentery and retroperitoneal tissue, and thus a plate of nerve-containing tissue overlying the abdominal aorta and interiliac trigone area can be left intact. On the other hand, the present study showed that laparoscopic nerve-preserving surgery for sigmoid colon cancer was technically feasible and could achieve function-preserving outcomes as good as those of traditional open surgery.10,11 However, the functional results of most reported series to date were based on the patients with rectal cancer undergoing open laparotomy. Therefore, further randomized studies in this respect that focus on sigmoid colon cancer are necessary.
Neurophysiologically, the internal urethral orifice is innervated by sympathetic signals and has dual functions: one to ensure urinary continence, and the other to prevent backflow of semen during ejaculation. The present study provided the insights into the functional significance of the internal urethral orifice by the clinical observation that the female patients manifested some degree of urinary incontinence and the male patients experienced retrograde ejaculation as a result of the failed preservation of bilateral hypogastric nerves. However, the influence of damage of pelvic sympathetic nerves on female sexual function has been ambiguous but generally considered because it would most likely result in impairment of vaginal lubrication. The manifestation of specific sexual problems for the three female patients with inadvertent sympathectomy supported this hypothesis. Remarkably, our previous study on the canine modal showed that when one side of hypogastric nerve was severed, the other side of hypogastric nerve could compensate for the genitourinary functional loss to a normal level.15 To clarify the role of pelvic sympathetics in the human genitourinary function, further recruitment of more patients with iatrogenic sympathetic nerve injury for study is required. Because the intentional severance of sympathetic nerve during the surgical practice is not ethical, we think that further study subjects will be focused on patients with an advanced rectosigmoid cancer in which unilateral or bilateral sympathectomy was a necessity for the radical resection of cancer.
In the present study, 6 patients (5.8%) had transient voiding disorder, 4 (9.1%) of 44 male patients had ejaculatory disorder, and up to 14.3% (6 of 42) female patients had sexual problems after the operation, even though their sympathetic nerves were successfully preserved during surgery. Our explanations for this phenomenon were as follows: first, anatomically, the inferior mesenteric plexus, thoracolumbar sympathetic splanchnic nerves, and superior hypogastric plexus were grossly manifested as a continuous plate of weblike connective tissues underlying the root of IMA and overlying the lower abdominal aortic and the interiliac trigone area. Therefore, some minor partial injury of nerve fibers may not be detected during surgery. Second, the retroperitoneal dissection by electrocauterization sometimes causes side injury and/or ischemia change of nerve fibers and thus compromised the neurophysiologic function. Third, the sexual function is influenced by various psychosocial factors. For example, the alteration of body image and/or bowel function after the operation may also have negative effect on sexual function.
Genitourinary functional disorders are generally subjective symptoms, and their measurement, either by questionnaire-based interviews or laboratory manometric and volumetric studies, are vulnerable to evaluation bias. Therefore, before the translation of technical feasibility of nerve-preserving surgery into the retaining of normal genitourinary function, the methodology used for the evaluation of functional outcomes in the present study needs to be further addressed. First, we used IPSS, IIEF, and FSFI to measure the genitourinary function of patients. These three kinds of validated questionnaires have been generally considered to be highly sensitive, and they have been widely adopted internationally for the study of genitourinary function.21,22 Second, in this study, both the patients and the assessors were informed of the intent to preserve the sympathetic nerves during sigmoidectomy but actually had no idea whether the nerve-preserving procedure was successful. The blinding of both assessors and patients to the surgical procedure can lessen the placebo effect. Third, the genitourinary function varies at different time points of evaluation. In this study, the evaluation of postoperative genitourinary function was conducted at the standardized time points before and after the operation. However, the preoperative baseline data was based on patient recall of genitourinary function before the appearance of bowel symptoms. The evaluation of baseline genitourinary function was therefore inevitably subject to methodology limitations, i.e., the potential recall bias. However, to validate the preoperative baseline data of patients, the spouses or sexual partners of some selected patients were also interviewed. We emphasize that patients were included in this study only when their normal baseline genitourinary function was ensured.
In conclusion, the genitourinary function of patients can be successfully preserved with good technical efficiency during laparoscopic sigmoidectomy. The present study should facilitate further study regarding the impact of preservation of genitourinary function on the postoperative quality of life for patients with sigmoid colon cancer.
Electronic supplementary material
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pelvic autonomic nerve
sigmoid colon cancer
laparoscopic surgery


Meta-regression analysis of high-frequency ventilation vs conventional ventilation in infant respiratory distress syndrome

Objective There is considerable heterogeneity among randomized trials comparing high-frequency ventilation (HFV) with conventional mechanical ventilation (CMV) in premature neonates with respiratory distress syndrome. We investigated what factors explained differences in outcome among these trials.
Introduction
High-frequency ventilation (HFV) has been compared with conventional mechanical ventilation (CMV) since the 1980s. In HFV, patients are ventilated with small tidal volumes, even smaller than the dead space of their airways, at high frequencies, normally between 5 and 10 Hz. Because HFV combines high mean airway pressures with small tidal volumes, this technique of ventilation has been regarded by some to be the most optimal form in patients with infant respiratory distress syndrome (IRDS), adult respiratory distress syndrome (ARDS), and other forms of severe lung disease [1].
The HFV has been extensively investigated in premature neonates with IRDS, a population specifically at risk for chronic lung disease (CLD). Unfortunately, the results of these studies were equivocal [2, 3]; thus, the question remains whether or not HFV better prevents CLD than conventional mechanical ventilation (CMV) in patients with severe lung disease. A significant number of meta-analyses have been performed to answer this question [4, 5, 6, 7, 8]. Pooled estimates of pulmonary outcomes failed to show clinically relevant differences among HFV and CMV [7]; however, significant heterogeneity existed between studies included in these meta-analyses. In a recent cumulative meta-analysis we identified improvements of the conventional treatment of IRDS and ventilation strategies applied in both HFV and CMV as important sources of heterogeneity [4]. These associations could be confounded by other explanatory variables. Although a meta-analysis may pool results from randomized trials, differences among trials will not be randomly or independently distributed. A meta-analysis constitutes an observational study of trials, subjected to bias inherent to observational research. In a meta-regression analysis it is possible to adjust for confounding covariates. A number of alternative hypotheses have been formulated to explain heterogeneity between trials:
The observed regression of the cumulative relative risks to the level of unity was due to publication bias.Use of the Sensormedics ventilator resulted in better results in HFV treated patients.A prolonged ventilation on CMV before initiating HFV treatment could reduce the benefits of HFV.In subgroups of more premature neonates with lower birth weight with a higher susceptibility for CLD, HFV could result in better pulmonary outcome.With outcome rates increasingly representing more severe disease, HFV could have an increasing advantage over CMV [9, 10]; therefore, we used meta-regression analysis to better estimate relative treatment effects through adjustments for factors that could explain trial heterogeneity.
Methods
Trials were included based on a previous meta-analysis that we had conducted [4]. The same search strategy, as well as the same inclusion and exclusion criteria as in our previous meta-analysis, were used for an update, yielding two more studies that could be included for this meta-regression analysis. Validity of studies was assessed by criteria published by Jadad et al. [11]. The validity was generally deemed as high with adequate allocation concealment in all trials. Blinding of treatment was not possible due to the nature of the interventions.
Data extraction was performed as has been reported in our previous meta-analysis. The following outcome measures were used: mortality, chronic lung disease (CLD) as defined by supplemental oxygen need or ventilator dependency at the age of 30–36 weeks post-menstrual. A number of explanatory variables were extracted as well: year of publication; type of ventilator used for HFV (Sensormedics 3100A ventilator versus other); ventilation strategies applied in the HFV and CMV treatment groups were obtained as previously described [4]; time on CMV before study initiation; gestational age and birth weight; and outcome rates in the control population were taken as proxy for baseline disease severity in the source population. The Sensormedics ventilator was singled out because previous research suggested better performance compared with other oscillator ventilators [2, 4].
Statistical analysis
All data were extracted according to the intention-to-treat principle. The number of patients surviving without chronic lung disease was subtracted from the total number of randomized patients in each treatment arm to calculate the composite outcome of death or CLD. To calculate the risk of CLD, the number of surviving patients was put in the denominator. Publication bias was assessed by visual appraisal of symmetry of funnel plots and performing rank tests. Smaller studies could show different results than larger studies which could suggest publication bias, but in fact was caused by systematic differences among studies; therefore, an analysis of publication bias stratified for ventilation strategies was performed to determine whether the observed association between the inverse of the standard error with the risk ratio was confounded by ventilation strategies. Meta-regression analysis was used to evaluate other hypotheses. The dependent variables, RR of CLD and RR of CLD or death, were natural log transformed to linearize the regression models. Individual studies were weighted by inverse variances of relative risks of outcomes of interest so that the more precise studies had more influence in the analysis. Firstly, linear regression analyses were applied to explanatory variables. Secondly, linear regression analyses with continuous covariates were conducted stratified by HLVS, LPVS, and use of surfactant. Finally, multivariable linear regression analyses were performed to calculate adjusted contributions of different explanatory variables of rivalling hypotheses to changes in RR. The relative effects of covariates were evaluated by relative risk ratios (RRR). A relative risk ratio quantifies the relative change in RR that is associated with a specified change of a covariate. For continuous variables the RRR was calculated for the ranges of minimum and maximum values of covariates that were reported in trials. For example, the RRR for year of publication was calculated by using the range between the publication year of the first year and the publication year of the last included trial. The RRR for year of publication thus estimates the relative change in RR due to the difference in years of publication between the first and last trials. All analyses were conducted using SPSS 12.0.1 for Windows software (SPSS, Chicago, Ill.) and Excel (Microsoft, Redmond, Wash.).
Results
For the analyses 15 studies were available that specified either CLD in survivors or death or CLD as outcome measures [2, 3, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24]. In 11 trials a high frequency oscillatory ventilator was used [2, 3, 12, 13, 16, 17, 19, 20, 22, 23, 24], in 7 of these trials this was the SensorMedics ventilator [2, 12, 13, 17, 22, 23]. Two studies used a high-frequency jet ventilator [14, 15] and in two studies a high-frequency flow interrupter ventilator was used [18, 21]. In the HFV group a total of 1141 patients were included for the outcome of CLD with 373 events and a total of 1457 patients with 671 events for the outcome death or CLD. In the CMV group a total 1159 patients were reported for the outcome of CLD with 428 events and a total of 1473 patients with 730 events for the outcome death or CLD. A forest plot of these trials can be found in the Electronic Supplement.
Table 1 summarizes the main characteristics of the studies. The outcome of CLD was available in all studies but one [22]. Time on CMV to start of the study was not reported by Plavka et al. [17] and Craft et al. [21]. In only one study was surfactant not used as concomitant treatment [12]. A high lung volume strategy (HLVS) was used in all but two studies [14, 16]. A ventilation strategy in the CMV-treated patients that could qualify as lung protective (LPVS) was reported in the most recent 9 studies [2, 3, 18, 19, 20, 21, 22, 23, 24]. Studies were published over a range of 13 years. Other reported ranges of covariates were 8.7 h average time on CMV before start of study, 5 weeks average gestational age, and 0.65 kg average birth weight. These ranges were used to calculate RRRs. Two studies dominated the analyses by virtue of the weight they received in the analyses: Johnson et al. [3] and Courtney et al. [2] (together 69% for CLD and 73% for death or CLD as outcome).
Table 1Study characteristicsReferenceYearTime on CMVAgeBirth weightSensorMHLVSLPVSSurfCLD lnRRWeightDeath or CLD lnRRWeight [12]1992 9.0281.100YYNN-1.290.01-0.580.01 [13]1996 3.0311.500YYNY-0.670.04-0.550.02 [14]1996 7.2270.950NNNY 0.020.01-0.230.10 [15]1997 8.0271.020NYNY-0.700.03 0.480.03 [16]1998 1.0281.100NNNY 0.000.00 0.310.00 [17]1999260.850YYNY-1.030.01-0.740.01 [18]1999 0.5270.870NYYY 0.090.06 0.010.04 [19]2001 2.6260.840YYYY-0.980.02-0.590.02 [20]2001 0.3280.990NYYY-0.200.05-0.060.05 [2]2002 2.7260.850YYYY-0.060.16-0.220.13 [3]2002 1.0260.850NYYY-0.010.54-0.020.60 [23]2003 1.0291.200YYYY 0.320.03 0.270.04 [22]200314.0270.980YYYY-0.040.05 [21]2003260.726NYYY 0.100.05 0.090.03 [24]2005 0.3270.880NYYY-1.440.01-1.200.00Year: year of publication. Time CMV: Mean time on CMV before start of the study in hours. Age: Mean gestational age (weeks). Birth weight: mean birth weight (kg). HLVS: high lung volume strategy in the HFV group, LPVS: lung protective ventilation strategy in the CMV group, Surf: use of surfactant in the study. CLD: chronic lung disease, defined as on oxygen at 30–36 weeks postmenstrual age, LnRR: natural log of the relative risk
A funnel plot of the inverse of the standard error against the natural logarithm of the RR for CLD was indicative of publication bias because of asymmetry round the line of the pooled effect (Fig. 1). A rank test showed a p-value of 0.112. A stratified analysis of publication bias is indicated by different colors in Fig. 1. To visually evaluate publication bias within subgroups of ventilation strategy, the distribution of trials round the corresponding colored lines (mean effect size within subgroup) was assessed. Stratification by ventilation strategy (HLVS and LPVS vs either no HLVS and/or no LPVS) showed p-values of 0.456 and 0.851, respectively, indicating less evidence of publication bias. The distribution of stratified studies round the lines of pooled estimates showed less asymmetry (Fig. 1). Publication bias for the composite outcome of death or CLD was less likely with a p-value of 0.329. Stratified analysis showed p-values of 0.677 and 1.000.
Fig. 1Funnel plot. Selection bias in reporting RR of chronic lung disease (CLD) as suggested by asymmetry of the distribution of studies. x-axis: inverse of the standard error of the RR; y-axis: natural logarithm of the RR. Blue diamonds: studies with either no high lung volume strategy (HLVS) or no lung protective volume strategy (LPVS); pink diamonds: studies with both HLVS and LPVS; dotted line: estimated pooled RR including all studies; dashed colored lines: pooled RR of subgroups of studies. Publication bias was visually appraised by assessing symmetry of distribution of studies around the lines of pooled estimates. CMV conventional mechanical ventilation
Figures 2, 3, 4 show the results of the linear meta-regression analyses for continuous explanatory variables with relative risk of CLD as dependent variable. Two studies dominate these figures, designated by the weight they received in the analyses [2, 3]. Over the years the reported benefit of HFV over CMV seemed to diminish (Fig. 2). A longer time on CMV prior to study initiation and a higher gestational age and increase of birth weight (data not shown) seemed to be positively associated with a relatively better outcome in HFV (Figs. 3, 4). Table 2 shows the results of linear meta-regression analyses, showing significant associations with year of publication (3.1 times higher RR with change of publication year from 1992 to 2005) and whether or not a protective ventilation strategy was applied (1.9 times higher RR with change of protective ventilation from no to yes; Table 2). In the linear regression analyses with death or CLD as composite outcome no significant associations were detected. Whether or not a Sensormedics high-frequency oscillatory ventilator was used and baseline incidence in CMV (0.75 vs 0.08) treated patients displayed the smallest effects on trial outcome (RRR = 0.84 and 0.90 for CLD and RRR = 0.85 and 0.99 for death or CLD, respectively).
Fig. 2Linear regression analyses. Crude and subgroup linear regression analyses of the effect of year of publication, prior time on CMV and gestational age with natural logarithm of RR of CLD as dependent variable. y-axis: natural logarithm of the RR; x-axis: explanatory variables. Blue diamonds: studies with either no HLVS or no LPVS; pink diamonds: studies with both HLVS and LPVS. The size of the diamonds reflects the weights the individual trials contribute to the analyses. Thin blue line: regression line including all studies; thick pink line: regression line including only studies with both HLVS and LPVS. CMV conventional mechanical ventilationFig. 3Same as Fig. 2Fig. 4Same as Fig. 2Table 2Univariable linear regression analysis95% confidence interval95% confidence intervalCrude BSig.LowerUpperRRRLowerUpperboundaryboundaryboundaryboundaryAll studiesCLD Year 0.090.025 0.010.163.131.18 8.27 SensorM-0.170.351-0.550.210.840.58 1.24 (no to yes) TimeCMV-0.090.055-0.190.000.440.19 1.02 Age-0.080.237-0.230.060.660.32 1.36 Weight-0.760.163-1.870.350.540.22 1.33 HLVS-0.110.883-1.741.520.890.17 4.57 LPVS 0.640.009 0.191.101.911.21 3.00 Surf 1.210.168-0.593.003.340.5620.03 CMV-0.180.774-1.531.170.900.42 1.92Death or CLD Year 0.050.096-0.010.122.010.86 4.65 SensorM-0.170.132-0.390.060.850.67 1.06 TimeCMV-0.010.590-0.050.030.920.65 1.29 Age-0.020.733-0.130.100.910.52 1.61 Weight-0.220.611-1.160.710.840.40 1.77 HLVS-0.370.698-2.441.690.690.09 5.45 LPVS 0.190.275-0.180.561.210.84 1.76 Surf 0.520.289-0.511.561.690.60 4.75 CMV-0.020.963-0.910.870.990.60 1.63Studies with surfactant, HLVS, and LPVSCLD Year 0.000.971-0.230.220.960.0517.34 TimeCMV-0.050.698-0.340.250.660.05 8.75 Age 0.040.727-0.220.301.220.33 4.49 Weight 0.410.693-1.992.811.380.20 9.44Death or CLD Year 0.010.846-0.150.171.200.15 9.72 TimeCMV 0.000.819-0.050.040.960.65 1.43 Age 0.060.406-0.100.211.340.61 2.92 Weight 0.550.396-0.891.991.550.49 4.90Simple linear regression analyses were calculated for chronic lung disease (CLD), defined as on oxygen at 30–36 weeks postgestational age, and
death or CLD. The following co-variates were evaluated: Year: Number of years after the first included study; SensorM:
Whether or not a Sensormedics type of HFV was used; TimeCMV: Mean time on CMV before start of the study in hours; Age: Mean gestational age (weeks); Weight: mean birth weight (kg); HLVS:
high lung volume strategy in the HFV group; LPVS: lung protective ventilation strategy in the CMV group; Surf:
use of surfactant in the study; B was the estimated crude coefficient; RRR: relative risk ratio = RRcovariate=1/RRcovariate=0,
for binary variables (SensorM, HLVS and LPVS), for continues
variables the extreme values reported in the studies were used to calculate the ranges, 13 for years, 8.7 for Time on CMV,
5 for Age, 0.8 for Weight and 0.65 for CMV (RRRyears = RRyear=2005/RRyear=1992,
RRRtime on CMV = RRtime=9 h/RRtime=0.3 h, RRRage = RRage=31 weeks/RRyear=26 weeks,
RRRweight = RRweight=1.5 kg/RRyear=0.7 kg, RRRincidence
of CLD in CMV=0.75/RRincidence=0.08)
Figure 5 shows how the incidence of CLD in the CMV treated patients was related to the incidence in HFV treated patients for each of the studies. The diagonal line represents the line of no effect in this figure. A trend line was fitted by weighted linear regression, showing a small effect of change in incidence in CMV on incidence in HFV-treated patients.
Fig. 5Linear regression analysis of incidence of CLD in CMV on incidence of CLD in HFV. y-axis: incidence of CLD in HFV; x-axis: incidence of CLD in CMV. Thin pink line: regression line including all studies
Year of publication was not related to change in relative risk of CLD in the subgroup of studies with HLVS, LPVS and concomitant use of surfactant (RRR = 0.96). There was only a small increase in relative risk for death or CLD (RRR = 1.20; Fig. 2; Table 2). Opposite effects of gestational age (RRR = 1.22 for CLD and 1.38 for death or CLD vs RRR = 0.66 for CLD and 0.91 for death or CLD, respectively) and birth weight were detected in the subgroup analysis (Fig. 4; Table 2). Prior time on CMV exerted less effect on outcome compared with the crude analysis, RRR = 0.66 for CLD and 0.96 for death or CLD and RRR = 0.44 for CLD and 0.92 for death or CLD in the adjusted and crude analyses, respectively (Fig. 3; Table 2).
Multivariable linear regression analyses were conducted to assess the independent contributions to change in RR by explanatory variables (Table 3). The RRRs in Table 3 have the same meaning as in Table 2, only they represented adjusted RRRs. Year of publication was not considered as an independent explanatory variable but rather as proxy for changes in treatment and patient population. Gestational age and birth weight were collinearly related by nature; only gestational age was fitted in the model. One study contributed to the fact that surfactant was not used; therefore, surfactant was not used in the multiple linear regression analyses. Two models were fitted. Model A used Sensormedics, time on CMV, gestational age, HLVS, and LPVS as covariates. The largest estimated effects were caused by ventilation strategies, HLVS, and LPVS, adjusted for use of Sensormedics ventilator, prior time on CMV, and gestational age. These estimations were consistent for the outcomes CLD (RRR = 0.42 and RRR = 2.02 for HLVS and LPVS, respectively) and death or CLD (RRR = 0.42 and RRR = 1.98 fro HLVS and LPVS, respectively). Use of a Sensormedics ventilator seemed to have a much smaller effect on RR for outcome. The RRR of gestational age, comparing 26 weeks with 31 weeks, for CLD and death or CLD were larger (RRR = 1.17 and RRR = 1.47). The effect of a difference in prior time on CMV of 8.7 h on CLD vs death or CLD was not consistent (RRR = 0.85 and RRR = 1.07, respectively).
Table 3Multivariable linear regression analysisAdjusted95% confidence interval95% confidence intervalBSig.Lower boundaryUpper boundaryRRRLower boundaryUpper boundaryModel ACLD (Constant)-0.660.900-13.0311.70 SensorM-0.040.884 -0.75 0.660.960.47 1.94 TimeCMV-0.020.903 -0.38 0.340.850.0419.22 Age 0.030.850 -0.36 0.421.170.16 8.32 HLVS-0.880.306 -2.80 1.040.420.06 2.84 LPVS 0.700.506 -1.73 3.142.020.1823.12Death or CLD (Constant)-1.860.412 -7.22 3.49 SensorM-0.170.309 -0.55 0.210.850.58 1.24 TimeCMV 0.010.722 -0.05 0.061.070.68 1.69 Age 0.080.299 -0.09 0.251.470.62 3.47 HLVS-0.880.407 -3.38 1.620.420.03 5.06 LPVS 0.680.127 -0.28 1.651.980.76 5.19Model BCLD (Constant) 0.070.904 -1.21 1.35 SensorM-0.060.698 -0.38 0.260.940.69 1.30 HLVS-0.810.203 -2.14 0.520.440.12 1.68 LPVS 0.720.011 0.21 1.232.061.23 3.43Death or CLD (Constant) SensorM-0.110.318 -0.33 0.120.900.72 1.13 HLVS-0.790.363 -2.66 1.080.450.07 2.93 LPVS 0.460.089 -0.09 1.011.590.92 2.74Multiple linear regression analyses were calculated for chronic lung disease (CLD),
defined as on oxygen at 30-36 weeks postgestational age, and death or CLD. The following co-variates were evaluated:
SensorM whether or not a Sensormedics type of HFV was used; TimeCMV
mean time on CMV before start of the study (in hours). HLVS high lung volume
strategy in the HFV group, LPVS lung protective ventilation strategy in the
CMV group. B was the adjusted estimated coefficient. RRR relative risk
ratio=RRcovariate=1/RRcovariate=0, for binary variables (SensorM,
HLVS and LPVS), for continues variables the extreme values reported in the studies were used, 8.7 for Time on
CMV (RRRage = RRage=31 weeks/RRyear=26 weeks)
A sensitivity analysis was conducted by fitting a second model (model B) with the most important variables, HLVS and LPVS, combined with whether or not a Sensormedics ventilator was used. The reported RRRs were comparable to those in the first model. Type of ventilator did not have a large effect compared with ventilation strategies (RRR = 0.94 and RRR = 0.90). The HLVS was associated with a decrease of the RRs comparing HFV with CMV (RRR = 0.44 and RRR = 0.45), while LPVS increased the RRs to the line of no effect (RRR = 2.06 and RRR = 1.59).
Discussion
Our meta-regression analysis showed a clear trend of decreasing differences in pulmonary outcome between HFV and CMV in randomized trials conducted in premature neonates with IRDS over the years. The most likely hypothesis for this trend was the application of a LPVS in the most recent studies. Use of surfactant could also have a significant contribution, but only one study did not use surfactant [12]. In previous meta-analyses, subgroup analyses or cumulative methods were used to explore heterogeneity [4, 5, 7]. Subgroup analysis is equivalent to meta-regression with a categorical trial-level covariate. Considering subgroup analysis formally as a meta-regression has advantages, since it focuses on differences between subgroups as is appropriate, rather than the effects in each subgroup separately. Furthermore, it is appropriate to use meta-regression to explore sources of heterogeneity, even if an initial overall test for heterogeneity is non-significant. This test often has low power and therefore a non-significant result does not reliably identify lack of heterogeneity [25].
In this meta-regression analysis we evaluated in a quantitative way a number of hypotheses that were raised to account for different results between randomized trials. A relatively large proportion of well-conducted trials were available for the analyses. For most explanatory variables there were important differences among trials. The effects of the two most important covariates, HLVS and LPVS, were consistent in the different models and were even increased in effect size by adjusting for other covariates. None of the competing hypotheses were more likely to influence results as shown by calculating the RRRs. Common pitfalls in meta-regression analysis can occur, such as multiple or post-hoc analyses, and lead to data dredging and a high probability of false-positive conclusions [25]. We, therefore, restricted our analyses to a limited number of pre-specified explanatory covariates.
Publication bias was considered unlikely as an explanation of the apparent diminishing relative effect of HFV. Publication bias is selection bias. If trials are selectively published either because of their size or because of significant results, this would result in an association between trial size and/or precision and the trial outcome. Strictly speaking, funnel plots probe whether studies with little precision (small studies) give different results from studies with greater precision (larger studies). Asymmetry in the funnel plot may therefore result not from a systematic under-reporting of negative trials but from an essential difference between smaller and larger studies that arises from inherent between-study heterogeneity [26]; thus, if larger studies were also associated with changes in ventilation strategies and these strategies resulted in changes in reported RRs, the assumed publication bias would be, in fact, a real association between ventilation strategy and study outcome; therefore, we conditioned the association between precision and effect size, presumably caused by publication bias, on ventilation strategies. This resulted in a lower p-value for publication bias and more symmetrical distribution of studies in subgroups in the funnel plots; therefore, what appeared to be publication bias could also be explained by differences in ventilation strategies related to both study size and observed relative risks. However, it should be pointed out that the strength of this evidence is difficult to assess because fewer studies in the subgroups automatically resulted in less power to detect publication bias.
Other alternative hypotheses that have been formulated to explain differences among studies were also less compatible with the evidence [9]. The type of ventilator, Sensormedics vs other types of high-frequency ventilators, displayed RRR close to one. In the crude analyses, prior time on CMV before study initiation showed contradictory effects to what was hypothesized [10]. The adjusted analyses showed conflicting results depending on the outcome. Gestational age and birth weight could also influence the magnitude of the effect of HFV compared with CMV. In the adjusted analysis gestational age did not change the RR for CLD but showed an increase of the RR for less premature neonates. Finally, an increased risk of CLD was not accompanied by a greater relative benefit of HFV as compared with CMV.
The observed effects of continuous variables, such as time on CMV or gestational age, could be exaggerated by small studies with outlying results. For the covariate, time on CMV, the two largest studies showed results that were compatible with the hypothesis that this had no important impact on the results of these trials [2, 3]. The same fact applied to the effect of baseline incidence of CLD or death or CLD. Gestational age and weight were comparable between the two largest trials, which made it more difficult to ascertain the relevance of the hypothesis that in smaller and more premature infants HFV performed better than CMV treatment. The observed direction of the effect of gestational age and birth weight, however, was opposite to what the hypothesis predicted. If gestational age was to be interpreted as a higher risk of acquiring CLD, one would expect that an increase in the incidence of CLD was associated with a relatively lower incidence of CLD in HFV treated patients; however, linear regression analysis showed perfectly equal increase in both treatment groups. Still, the possibility remains that the relationship with patient averages, such as gestational age and birth weight, across trials was not the same as the relationship for patients within trials, and therefore an effect of these patient characteristics cannot be excluded but only considered in relation to other covariates [25].
Similar findings of the effects of ventilation strategies have been reported by us and other authors as well [4, 5]; however, meta-analyses are subject to bias when differences among trials are used to explain differences in reported RRs. In this meta-regression analysis we were able to estimate adjusted association measures, thereby diminishing the effects of possible confounders/effect-modifiers. By calculating less biased estimates of the effects of ventilation strategies and the effect of using a Sensormedics ventilator instead of other ventilators on the outcome in the different HFV trials we were able to reinforce the hypothesis that ventilation strategies are more important than type of ventilator to prevent CLD.
The results of this meta-analysis stresses the importance of using appropriate ventilation strategies to prevent ventilator-induced lung damage in a highly vulnerable group of patients; therefore, in clinical practice the question of how to use the ventilator is more important than the question of which ventilator should be used. The major theoretical advantage of HFV to CMV is delivery of smaller tidal volumes to an optimally recruited lung. As this meta-regression analysis did not confirm that subgroups of more premature neonates, avoidance of CMV prior to initiating HFV, or neonates with higher risk of CLD were more likely to benefit form elective HFV in IRDS, future research should be directed at identifying patients in whom HFV does have a benefit over CMV. To improve the robustness of these conclusions and to avoid the limitations of meta-analysis of trials, an individual-patient-data-based meta-regression analysis should be conducted.
Conclusion
In conclusion, confining randomized trails to smaller or more premature children with IRDS did not seem to result in better pulmonary outcomes of HFV compared with CMV. A generally held opinion that a prolonged ventilation time on CMV prior to initiating HFV diminished the benefits of HFV was not in agreement with the current evidence. The most important effects resulting in differences among trials were probably caused by ventilation strategies applied in HFV- and CMV-treated patients.
Electronic supplementary material
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meta-regression analysis
high-frequency ventilation
infant
respiratory distress syndrome
conventional mechanical ventilation