Proteus mirabilis (PM) is a Gram-negative, rod-shaped bacterium that causes catheter-associated urinary tract infections (CAUTIs). The specific roles of bacterial surface components (BSCs) in PM pathogenicity and CAUTIs remain unknown. To address this knowledge gap, we utilized relevant in vitro adhesion/invasion models and a well-established murine model of CAUTI to assess the ability of wildtype (WT) and seven mutant strains (MSs) of PM with deficiencies in various genes encoding BSCs to undergo the infectious process (including adhesion to catheters) in both model systems. Overall, MSs adhesion to catheters and the different cell types tested was significantly reduced compared to WT, while no invasion of cells was evident at 24 h. In vivo, WT showed a greater number of planktonic (urine) bacteria, bacteria adherent to catheters, and bacteria adherent to/invading bladder tissue when compared to the MSs. Bacterial counts in urine for PMI3191 and waaE mutants were lower than that for WT and other MSs. The complementation of mutated BSC genes resulting in the biggest defects restored the invasion phenotype both in vitro and in vivo. BSCs play a critical role at various steps in the pathogenicity of PM including adhesion to indwelling medical devices and adhesion/invasion of urinary tissue in vivo. Proteus mirabilis (PM) is a Gram-negative bacterium first described by Gustav Hauser in 1885. He named this genus of Proteobacteria after the Greek god Proteus due to the bacteria’s changeability in form as it alternates between an immobile and a mobile, so-called “swarming” condition [PM possesses the enzyme urease that splits urea in urine into carbon dioxide (CO2) and ammonia (NH3), which results in a rise in urinary pH [3PO4) and apatite (CaPO4) and eventual catheter encrustation and struvite stone formation [PM-induced catheter-associated urinary tract infections (CAUTIs), are financially and physically burdensome outcomes of urinary catheter use [PM pathogenicity in the urinary tract is colonization, as the microorganism can attach to epithelial cells with various fimbriae and adhesins and potentially internalize within these cells [PM have been described: fimbriae are outer-membrane pili that extend from the bacterial surface and usually facilitate adherence to surfaces, of which the chaperone-usher fimbriae are linear, unbranching, outer-membrane pili produced by Gram-negative bacteria that mediate their form of secretion and assembly [PM HI4320 encodes 17 chaperone-usher fimbrial operons, and these fimbriae are regarded as key factors in adhesion and bacterial aggregation for PM in vivo [PM [PM cell surface components, generated through previous studies, to identify PM HI4320 fitness factors, to assess their role in PM pathogenicity with specific emphasis on key adhesion and invasion processes [ucaJ, fim14C, PMI3191, arnA, pbpC, and waaE.ioxide CO and ammommonia NH, which rassembly ,7. PM HI in vivo . To datevivo [PM . To begirocesses . These iE. coli [PM. Overexpression of MrpJ decreases flagellum-mediated motility in PM in order to regulate the opposing processes of motility and adhesion. UcaJ is one of 14 paralogues of MrpJ found in PM and functions as an inhibitor of motility when attachment is indicated [Fim14C (PMI2998) is the major pilin subunit of a putative chaperone usher fimbrial operon, and fim8F (PMI1464) is part of another putative chaperone usher fimbrial operon [PMI3191 encodes for a glycosyltransferase that has been shown to contribute to PM lipopolysaccharide (LPS) [ArnA (PMI1045) is the name of a gene that encodes for the bifunctional polymyxin resistance protein ArnA that physiologically catalyzes the oxidative decarboxylation of UDP-glucuronic acid to UDP-4-keto-arabinose, which is important in LPS formation [pbpC (PMI1850) encodes for a protein that is involved in the pathway peptidoglycan biosynthesis with regard to cell wall formation. The penicillin-binding protein 1C (pbpC) is one of several in PM HI4320 and represents a conserved feature across Gram-negative bacteria [waaE encodes for an LPS core biosynthesis glycosyl transferase, an enzyme required for LPS biosynthesis. This outer membrane component of Gram-negative bacteria is critical for antimicrobial peptide resistance, as well as swarming motility [Urothelial cell adhesin (UCA) frimbriae are adhesion proteins that have been observed to play a critical role in the attachment to the urothelium and are related to the K99 fimbriae of enterotoxigenic E. coli . MrpJ isndicated . Fim14C l operon . PMI3191de (LPS) . ArnA , [waaE (PMI3136) was generated for this study via the TargeTron method: a kanamycin resistance cassette was inserted into the gene of interest, following the Sigma TargeTron group II intron protocol, as previously described [cPMI3191 and cwaaE) were generated. Briefly, complementation vectors were constructed by amplifying the indicated gene along with approximately 500 bp of flanking sequence and ligating the amplified sequences into a linearized pGEN-MCS vector. Successful complementation was verified by selection on ampicillin plates and by PCR.The studies were carried out using wildtype 0 days), ,23 along0 days), . Brieflyescribed . In addiFor bacterial culture, bacterial strains were separately cultured from freezer stocks (−80 °C) in fresh Luria broth (LB) media  ± kanamycin or ampicillin. Strains were incubated in a shaking incubator at 225 RPM and 37 °C overnight. The following day, all cultures were sub-cultured in fresh media  and left for a subsequent overnight subculture under the same conditions prior to use for experiments.PM WT and mutants to interact with the catheter surface and form biofilm over time, an adhesion assay using 24G polyurethane (PU) catheters to be used for eventual in vivo analyses was performed in artificial urine. PM wildtype and the 9 mutant and complemented strains were separately cultured from freezer stocks in fresh LB media. Strains were incubated at 37 °C overnight. The following day, all cultures were sub-cultured in fresh LB media and left overnight in the same conditions. Bacterial stock solutions of approximately 1 × 108 CFU/mL were prepared in artificial urine. The artificial urine was prepared as described by Brooks and Keevil [To assess the potential for d Keevil .PM strains, and tubes were placed on a rotator and placed inside an incubator for either 4 or 24 h at 37 °C. At each timepoint, catheters were removed and washed gently in sterile PBS to remove any loosely adhered bacteria. Catheters from each strain were transferred to tubes of fresh PBS, sonicated, vortexed, serially diluted, and plated on LB agar for CFU counts. Agar plates were incubated overnight at 37 °C until bacterial colonies were visible, and distinguishable CFU counts between 2 to 50 colonies were documented.Sterile catheter pieces were placed inside sterile Eppendorf tubes containing artificial urine and one of the 10 4–1 × 105 cells were seeded in each well of a 24-well tissue culture plate. Cells were incubated at 37 °C and 5% CO2 and allowed to grow until 80% confluent. On the day of the experiments, two of the wells were trypsinized to quantify the number of cells per well and the average cell number used to determine the number of bacteria to be added to each well. Cell media were removed, and all cells were gently washed thrice with sterile PBS. Next, each of the bacterial strains to be tested was added at a multiplicity of infection (MOI) of 1:100. The plates were gently centrifuged at 500× g for 5 min to bring bacteria in close proximity to the epithelial cells without forcing them to directly interact. Plates were then incubated at 37 °C and 5% CO2 for either 2 h or 24 h.For adhesion and invasion assays, three different human cell lines were used, including the kidney epithelial cell lines A498 and HEK293 and the human bladder epithelial cells line T24. Cells were cultured from liquid nitrogen freezer stocks. A498 and HEK293 cells were grown in Dulbecco’s Modified Eagle Medium  and T24 cells with Roswell Park Memorial Institute  1640 media, all supplemented with 10% fetal bovine serum (FBS). Once cells had stabilized in their growing patterns and were 80% confluent, they were detached using Trypsin/EDTA. Cells were counted using an automatic cell counter and 1 × 10To assess bacterial adhesion, bacterial cell media were removed after 2 h of incubation, and the monolayers were washed thrice gently with sterile PBS to remove any loosely adherent bacteria. Cells were then lysed using 0.1% Triton-X solution for 15 min at room temperature. The resultant cell lysates were then serially diluted in sterile PBS and plated on LB agar plates followed by overnight incubation at 37 °C and CFU counting the following day. Counts between 2 and 50 visible colonies were recorded and used for bacterial quantification.2 to kill adherent bacteria. Next, cells were washed with PBS and lysed with 0.1% Triton-X at room temperature for 15 min. Internalized bacteria were quantified via CFU counts as described above.To assess bacterial invasion, the media were removed and cells were washed thrice with sterile PBS at the 2 h and 24 h timepoints. Cells were then incubated in fresh media containing 1% Penicillin-Streptomycin for an additional 4 h at 37 °C and 5% COPM WT and mutants were grown in 5 mL LB and incubated at 37 °C overnight. The culture was diluted 1:10 with LB, and a 1.5 ml suspension was created to an OD600 of 0.5. The bacteria were centrifuged at 10,600× g for 10 min, the supernatant was removed, and the LPS extraction was started immediately. Bacterial LPS was extracted via hot aqueous-phenol extraction according to Davies et al. [To verify the complementation of genes affecting LPS synthesis, we extracted bacterial LPS via hot aqueous-phenol extraction followed by separation by SDS-PAGE (15%) . PM WT as et al. . LPS wasThe study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Animal Care Committee of The University of British Columbia (Protocol A17-0297).® Polyurethane I.V. Catheter 24 Gauge x 3/4”, Cat. No. SRFF2419, Terumo, Vaughn, Canada) were temporarily removed from the needles, and a 4 mm piece was cut off using a sterile blade. The angiocatheter and small piece were then re-assembled back onto the needle tip. Prior to being used for the in vivo experiments, all catheters were gas-sterilized with ethylene oxide to further ensure sterility and avoid any contamination.Under aseptic conditions, 24G angiocatheters (Terumo Surflash®) at 12 weeks of age. Mice underwent inhalational anesthesia with 3% isoflurane. Under ultrasound guidance,  a 4 mm piece of a modified 24G catheter was implanted into the mouse bladder [5 CFU/mL in 50 μL PBS of one of the strains to be tested was percutaneously injected into the mouse bladder lumen using a 30G needle. Following bacterial injection, all mice were maintained under anesthesia at 1% isoflurane for an additional hour to promote bacterial attachment to the catheter and epithelium and to avoid premature micturition. On day 3, animals were anesthetized as above and urine was collected using a 30G needle under ultrasound guidance for subsequent quantification of planktonic bacteria via CFU counts, as described above. Subsequently, all mice were euthanized on day 4. All catheters were removed from the mouse bladders and aseptically transferred to tubes containing sterile PBS. After gentle washing with PBS, catheter samples were sonicated at 50/60 Hz for 10 min in an ultrasonic water bath  followed by vortexing for 30 s at high speed to promote detachment of bacteria from the catheter. The PBS-bacterial solutions were then serially diluted and plated on LB agar plates for CFU counts, as outlined above.Mice used in these experiments were male C57Bl/6 mice  software .Significance was assessed using one-way analysis of variance (ANOVA), a nonparametric Kruskal–Wallis test, and the Wilcoxon signed-rank test. PM mutants showed similarly reduced adhesion to the catheter surface at the 4 h timepoint  in the adhesion assay at 24 h. CFU counts for the complemented strains (cPMI3191 and cwaaE) were significantly higher than WT counts. When quantifying invasion assay results at both 2 h and 24 h, WT invasion was 3.7 × 106 CFU/mL, and there was no quantifiable invasion of the PM mutant strains at either timepoint (cPMI3191 and cwaaE), no invasion was observed at either timepoint.We first tested adhesion and invasion in A498 kidney epithelial cells. At 2 h p.i., ed to WT A. Howeveed to WT B. Many oimepoint C,D. Addi6 CFU/mL, while the number of adhered mutant strains ranged from 8.2 × 105 CFU/mL to 1.6 × 106 CFU/mL. There was no statistically significant difference between WT and the mutant strains with regard to their adhesion capabilities  showed more adherence to HEK293 cells at both 2 h and 24 h.Next, we tested adhesion and invasion in HEK293 kidney epithelial cells: PM WT at 2 h p.i. was quantified at 1.2 × 10bilities A. At 24 as found B. The co4 CFU/mL, and although not statistically significant, PMI3191 and waaE displayed very low levels of invasion at 1.7 × 103 CFU/mL and 6.0 × 103 CFU/mL, respectively (3 CFU/mL), and none of the mutants invaded the cells  to T24 cells exceeded adhesion levels of WT at 24 h , a bacterial solution containing 5 × 105 CFU/mL in 50 μL PBS of one of the strains to be tested was percutaneously injected into the mouse bladder lumen using a 30G needle. On day 4 (48 h post infection), animals were anesthetized urine aseptically collected using a 30G needle under ultrasound guidance for subsequent quantification of planktonic bacteria via CFU counts as described above. Subsequently, all mice were euthanized.To further investigate the in vitro findings we tested PMI3139 and waaE mutants, with the complementation of these genes resulting in adhesion levels returning to levels similar to WT. All control mice, injected with sterile PBS, yielded 0 CFU counts.On day 4 of the experiment (48 h p.i.), the highest number of bacteria in urine were found for WT, while those for the MSs were significantly lower A. The loPM was found to have the greatest adhesion compared to the mutant strains. Both PMI3139 and waaE mutants adhered at the lowest levels to catheters. The complementation of these genes resulted in adhesion levels similar to that of the WT strain. Catheters from the control did not have any adherent bacteria.In terms of bacterial adhesion to catheters B, WT PM PMI3139 and waaE resulted in a trend towards increased adhesion, which did not reach WT levels.In terms of interaction with the urothelium C, the WTPMI3139 and waaE did rescue the loss of invasiveness, albeit at levels lower than the WT strain.In terms of the number of bacteria that invaded the urothelium D, internProteus mirabilis is a notoriously strong biofilm former and is therefore a frequent cause of urinary tract infections in individuals with indwelling foreign bodies within the urinary tract, such as bladder catheters, ureteral stents or nephrostomy tubes, and often leads to the encrustation of devices and/or infection stones [PM pathogenicity, especially the adherence to indwelling devices leading to biofilm formation and subsequent adhesion and invasion of uroepithelial cells. To address this gap in knowledge, the present work investigates the role of different bacterial components in mediating these key processes involved in PM pathogenicity using both in vitro and in vivo settings.n stones . To dateP. mirabilis has been shown to prefer a higher pH to undergo adhesion processes. Furthermore, bacteria within a biofilm remain alive for at least several days in a higher-pH environment [PMI3191 and waaE mutants, which adhered to a lesser extent than the other mutants. Interestingly, lower numbers were also recovered from the urines of animals infected with these two mutants despite similar inoculation numbers, suggesting that lower adhesion may be the result of fewer bacteria being retained in the urinary tracts of these animals. That being said, reduced adhesiveness and bacterial numbers of these mutants in urine did not translate to the bladder tissue, as both adhesion and invasion levels were similar to the remaining mutants. This may suggest that despite lower numbers being retained in the urine, these mutants were more efficient at adhering to the bladder tissue. The fact that complementation of PMI3191 and waaE resulted in adhesion and invasion levels similar to the WT strain suggests that changes in these key pathogenic steps were driven by changes in LPS structure, as these genes express LPS biosynthesis genes, and at least knocking out waaE resulted in significant changes in LPS banding patterns that were resolved by complementation.All mutants were found to have decreased adhesion to PU catheters. Considering that these experiments were performed in artificial urine, which has limited proteins, adhesion in this setting is mainly driven by charge and hydrophobicity characteristics rather than the direct interaction of adhesins with proteins deposited on the material surface . Interesironment . A similPMI3191 and waaE resulted in adhesion levels that were greater than the corresponding mutant and, in some cases, similar to WT. The fact that complementation of PMI3191 and waaE resulted in the rescue of the invasion phenotype for both HEK293 and T24 cells suggests that the LPS structure plays a role in mediating the invasion of uroepithelial cells by PM. Changes in LPS glycosylation such as those expected from the loss of a glycosyltransferase  have previously been shown to affect the invasiveness of Shigella flexneri, with strains with increased glycosylation showing enhanced invasion [E. coli, which may explain the loss of invasion of the waaE mutant [PM3191 and waaE did not rescue the invasiveness in this cell type, suggesting that a role for PM LPS in invasion may be cell-dependent. This certainly requires further investigation.None of the mutants were found invading the bladder tissue when the animals were sacrificed 48 h post-infection. To better understand the adhesion and invasion patterns of WT and mutant strains, these key processes were assessed in three different cell lines representing the bladder and kidney. Overall, we found adhesion and invasion patterns to differ between the different cell types. While adhesion was somewhat affected for some mutants at the 2 h timepoint for all cell types, no differences were observed at 24 h post-infection. In contrast, none of the mutants were found to invade A498 cells at either timepoint, while some initial invasion was evident for HEK293 cells that were lost by 24 h. A similar pattern was also observed for T24 bladder cells, which suggests that a lack of invasion in the mice may be due to an inability of the mutants to invade bladder cells. In terms of the role for LPS structure in these processes, complementation of E mutant . That saPM virulence, and their crucial role in infection pathogenicity by aiding adhesion and aggregation in vivo has been demonstrated [ucaJ, fim8F, and fim14c mutants showed substantially lower CFU counts in the adhesion assays at both timepoints in the majority of experiments. UcaJ is a uroepithelial cell adhesin (UCA) mutant, and UCA fimbriae have been identified to play a critical role in the attachment of PM to epithelial cells of the urinary tract [ucaA mutant demonstrated diminished adhesion to the urothelium, compared to wildtype [PM may be a crucial factor in kidney colonization, as well as the onset and propagation of CAUTI.Fimbriae have been the subject of continued investigation in nstrated . Fimbrianstrated . These fry tract . Pellegrwildtype . Their rE. coli and Pseudomonas aeruginosa [arnA gene in a PM CAUTI mouse model specifically indicated that it leads to a decrease in bacterial fitness in the urinary tract, as well as the spleen, which may be indicative of bacteremia [arnA mutant bacteria that adhered to kidney epithelial cells were comparable to those of WT, but there were substantially lower CFU counts in the bladder cells when compared to WT, indicating that arnA plays a role in the interaction of PM with the uroepithelium.ArnA is a bifunctional polymyxin resistance protein that is required for the modification of lipid A in LPS, and this modification leads to bacterial resistance to cationic antimicrobial peptides of the host immune system, as well as resistance to certain antibiotics, such as polymyxin . These pruginosa . Anothercteremia . CFU couPM has been identified to possess PBPs, and the pbpC mutant strain used in this study lacks enzymatic abilities associated with peptidoglycan synthesis [pbpC mutant showed the same patterns in the adhesion and invasion assays as mutants in fimbriae, suggesting that changes in peptidoglycan structure play a role in these important processes.Penicillin-binding proteins (PBPs) are high-molecular-weight proteins that are attributed to crucial steps in peptidoglycan synthesis of Gram-negative bacteria . PM has ynthesis . InteresPM, including the adhesion to indwelling medical devices, as well as the adhesion and invasion of urinary tissue cells and the tissue itself. These data suggest a role for all surface components studied in these key processes. Considering that no mutant resulted in the complete loss of tissue adhesion and invasion indicates that these processes likely involve the dynamic interplay between multiple components. Future experiments should focus on utilizing PM strains with mutations on multiple key components to determine whether targeting more than one results in the loss of infection. To that end, the data presented herein represent a critical first step towards the development of targeted therapies to block key steps in the pathogenicity of PM and prevent associated morbidity and mortality.Collectively, the present work provides a broad overview of bacterial surface structures critical for different critical steps in the pathogenicity and the overall infectious process of


Intraoperative goal-directed fluid therapy (GDFT) has been reported to reduce postoperative complications of patients undergoing major abdominal surgery. The clinical benefits of pleth variability index (PVI)-directed fluid management for gastrointestinal (GI) surgical patients remain unclear. Therefore, this study aimed to evaluate the impact of PVI-directed GDFT on GI surgical outcomes in elderly patients.n = 110 each). The primary outcome was a composite of complications within 30 postoperative days. The secondary outcomes were cardiopulmonary complications, time to first flatus, postoperative nausea and vomiting, and postoperative length of stay.This randomised controlled trial was conducted in two university teaching hospitals from November 2017 to December 2020. In total, 220 older adults undergoing GI surgery were randomised to the GDFT or conventional fluid therapy (CFT) group (P = 0.008). In intention-to-treat analysis, there was no difference in overall complications between the CFT group (41.3%) and GDFT group (43.0%) . The proportion of cardiopulmonary complications was higher in the CFT group than in the GDFT group . No other differences were identified between the two groups.The total volumes of fluid administered were less in the GDFT group than in the CFT group  on 1 August 2017. The number of elderly patients undergoing surgical procedures is growing drastically with the ageing of society. This population is at a greater risk of mortality and morbidity after gastrointestinal (GI) surgical procedures than younger patients. Despite advances in surgical and perioperative care, postoperative complications occur in approximately 45% of patients, with an in-hospital mortality rate of approximately 3% in elderly patients undergoing GI surgical procedures , time to first flatus, and postoperative length of stay (PLOS).This dual-centre clinical RCT was conducted in two university teaching hospitals from November 2017 to December 2020  The study analysed whether perioperative GDFT based on PVI reduced composite postoperative complications in elderly patients who underwent major GI surgery compared to conventional fluid management practice It also evaluated cardiopulmonary major postoperative complications, PONV, time to first flatus, and PLOS. This study was approved by the Clinical Research Ethics Committee of Peking University Shenzhen Hospital (IRB 2017-001-2), and written informed consent was obtained from all participants in the trial. Prior to patient enrolment, this trial was registered with the Chinese Clinical Trial Registry . This manuscript adheres to the applicable guidelines of the Consolidated Standards of Reporting Trials (CONSORT).Potential participants were screened on the day before surgery (or on the preceding Friday for those who underwent surgery on a Monday). Adult patients aged ≥ 65 who underwent elective major GI surgery  were considered eligible.2.Patients were excluded if they met any of the following criteria: (1) history of severe cardiac disease, including severe arrhythmia, myocardial infarction, and cardiac insufficiency; (2) presence of hepatorenal dysfunction; (3) presence of cardiac failure (New York Heart Association class III or IV); (4) history of asthma; (5) presence of pulmonary infection; and (6) body mass index ≥ 30 kg/mDetailed information, including baseline demographic data, preoperative medical history, diagnosis at the time of admission, illness severity and perioperative variables were obtained after recruitment. After obtaining written informed consent, baseline data  were collected.A biostatistician not involved in the data management and statistical analyses generated random numbers  using SAS software version 9.2 , with a block size of 4. Results of this randomisation were sealed in sequentially numbered envelopes and kept until the end of the study by a study coordinator (J.Z.) who was not involved in data collection, perioperative care, or postoperative follow-up. During the study period, consecutively recruited patients received either intraoperative GDFT or conventional haemodynamic management, according to the random number allocation by the study coordinator (J.Z.). Intraoperative data of each recruited patient were collected by anaesthesiologists . Anaesthesiologists and investigators did not communicate with each other regarding the patient data collected. Both patients and postoperative investigators were blinded to the study group assignment. Unblinding was conducted after the trial was closed and data of all patients were collected. Older adults undergoing GI surgery were randomly assigned to the GDFT or conventional fluid therapy (CFT) group.2 between 35 and 45 mmHg; and pulse oxygen saturation > 96%. In all cases, the anaesthetic procedure was chosen by the attending anaesthesiologist. Packed red blood cells were administered at the anaesthesiologist’s discretion . Lactate concentrations at the beginning of surgery and at discharge from the post-anaesthesia care unit (PACU) were analysed.No premedication was administered, and solid food and clear fluid intakewere allowed until 8 h and 2 h before surgery, respectively. Patients’ electrocardiographic activity, arterial blood pressure, oxygen saturation, and body temperature were continuously monitored. General anaesthesia was induced with etomidate or propofol and sufentanil; atracurium or rocuronium was used for neuromuscular block. A 20-gauge radial artery catheter and central venous access catheter were inserted at the end of the induction phase. Anaesthesia was maintained with sevoflurane or propofol and remifentanil. The PVI sensor  was placed on the patient’s index finger to monitor the PVI continuously.. The use of additional regional anaesthesia, choice of anaesthetic drugs, and operative pain management were at the discretion of the attending anaesthesiologist. The investigators indicated that the ventilator patterns should be restricted as follows: controlled ventilation with a tidal volume of 8 mL/kg of ideal body weight; an initial respiratory rate of 12 breaths/min adjusted to achieve end-tidal CO−1.h−1. When MAP is < 65 mmHg, the vasoactive drugs) were promptly administered to maintain a mean arterial blood pressure of ≥ 65 mmHg regardless of PVI. When MAP is ≥ 65 mmHg, we judge fluid responsiveness is present via PVI > 13%. Whenever the PVI was > 13% for 5 min, we administered a 250-mL bolus of colloid . The dose was repeated every 5 min until the PVI was < 13% . A bolus of 250-mL colloids was administered whenever acute blood loss was > 50 mL, the mean arterial blood pressure decreased to < 65 mmHg, or the central venous pressure decreased to < 5 mmHg. A repeat bolus was administered after waiting for 5 min if any one of these criteria was met. Whenever the mean arterial blood pressure decreased to < 65 mmHg and remained unresponsive to fluids, vasoactive drugs  were administered to maintain a mean arterial blood pressure of > 65 mmHg.In the CFT group, 500 mL of crystalloid was infused during induction, followed by a continuous infusion of crystalloids . Data were obtained from the clinical files completed by the surgeons responsible for the patient, but who were blinded to the study. Data validation  were performed by the principal investigator (X-H.W.).Based on previous published data .Unless otherwise indicated, all results are expressed as mean and standard deviation or as the median and interquartile range (IQR). Normally distributed continuous data were compared using the independent-samples From 5 November 2017 to 29 October 2020, 537 patients were eligible for enrolment, and 220 patients from two hospitals  were enrolled and randomised. The final follow-up of the last randomised participants was performed in December 2020. Of 220 patients, 9 were excluded after randomisation based on the protocol-defined exclusion criteria: 6 patients withdrew from the study, and 3 patients were excluded because of a change in the surgical procedure. All evaluable patients were followed up for 30 days postoperatively, and none were lost to follow-up. A total of 211 patients were included in the intention-to-treat analyses  . The duration of surgery and anaesthesia was not significantly different between the two groups . The total volumes of fluid administered and crystalloid infusion were lesser in the GDFT group than in the CFT group . Lactate concentrations at the beginning of the surgery  and at discharge  from the PACU were similar between the CFT and GDFT groups . The proportion of cardiopulmonary complication was higher in the CFT group than in the GDFT group . No significant between-group differences in PONV, the time to first flatus, and PLOS were identified  (Table The number of patients with one or more complications at 30 days was similar between the CFT group (43 [41.3%]) and GDFT group (46 [43.0%]) . The mechanism of the beneficial effect of GDFT could not be determined in our study. It is possible that elderly patients receiving GDFT benefit from fluid optimisation and avoid excessive fluid intake and, therefore, have a decreased risk of overload of the heart and tissue oedema, which, compared to CFT, could have potentially lowered the risk of postoperative complications such as pulmonary oedema (1.9% vs. 4.8%), arrhythmia (1.9 vs. 4.8%), pneumonia (1.9% vs. 5.8%), surgical site infection (2.8% vs. 5.8%), and anastomotic leakage (0.9% vs. 4.8%). A recent meta-analysis of GDFT trials concluded that GDFT during general anaesthesia might reduce pneumonia, surgical site infection, and anastomotic leakage, and this result reached moderate certainty in the evidence (Jessen et al. Recovery of GI function is a significant determinant of in-hospital recovery after GI surgery (Augestad and Delaney This study has some limitations. First, GDFT was performed only intraoperatively. Therefore, it is unclear if the intraoperative assessment of fluid administration is adequate to produce a considerable difference in the outcome compared to the perioperative assessment. The use of GDFT throughout the perioperative period may be more effective in improving patient outcomes. Second, the cut-off value for the PVI was a limitation. The threshold value of the PVI in our interventional group was set at 13% based on that reported in previous literature (Cannesson et al. Among elderly patients undergoing GI surgery, intraoperative GDFT based on the PVI showed no superiority to the conventional fluid management regimen in terms of overall complications, PONV, and PLOS, but was associated with a lower cardiopulmonary complication rate than usual fluid management.


Pregnancy loss is often caused by chromosomal abnormalities of the conceptus. The prevalence of these abnormalities and the allocation of (ab)normal cells in embryonic and placental lineages during intrauterine development remain elusive. In this study, we analyzed 1,745 spontaneous pregnancy losses and found that roughly half (50.4%) of the products of conception (POCs) were karyotypically abnormal, with maternal and paternal age independently contributing to the increased genomic aberration rate. We applied genome haplarithmisis to a subset of 94 pregnancy losses with normal parental and POC karyotypes. Genotyping of parental DNA as well as POC extra-embryonic mesoderm and chorionic villi DNA, representing embryonic and trophoblastic tissues, enabled characterization of the genomic landscape of both lineages. Of these pregnancy losses, 35.1% had chromosomal aberrations not previously detected by karyotyping, increasing the rate of aberrations of pregnancy losses to 67.8% by extrapolation. In contrast to viable pregnancies where mosaic chromosomal abnormalities are often restricted to chorionic villi, such as confined placental mosaicism, we found a higher degree of mosaic chromosomal imbalances in extra-embryonic mesoderm rather than chorionic villi. Our results stress the importance of scrutinizing the full allelic architecture of genomic abnormalities in pregnancy loss to improve clinical management and basic research of this devastating condition. Genomic analysis of products of conception collected after spontaneous pregnancy loss in the first trimester reveals previously undetected chromosomal aberrations and a higher degree of mosaic chromosomal imbalances. Pregnancy loss primarily occurs before weeks 8–9 of gestation1, and there is considerable additional loss in earlier stages of pregnancy that may go unnoticed. Overall, 10.8% of women experience at least one pregnancy loss, and 1.9% and 0.7% have two or three pregnancy losses, respectively1. Identifying the cause of pregnancy loss can provide important prognostic, diagnostic and management recommendations to support future viable pregnancies2. Chromosomal abnormalities, in particular aneuploidy, defined as an incorrect number of chromosomes, in the conceptus are the leading causes of pregnancy losses. It has been established that aneuploidies commonly occur during oogenesis5 and in early embryogenesis8. The incidence of chromosomal aneuploidies increases with maternal age, which contributes to age-related infertility8. This is due to the low fidelity of chromosome segregation in meiosis during oogenesis8 and DNA replication stress9 during mitotic cleavage divisions in pre-implantation embryogenesis7. Previously, we and others demonstrated that, although chromosome instability (CIN) is common in early embryogenesis7, it is not present at birth10. This observation indicates that only embryos with sufficient genome integrity can survive to term and that both meiotic aneuploidies in oocytes and post-zygotic chromosome abnormalities in early embryogenesis may lead to implantation failure and pregnancy losses1.Worldwide, 23 million pregnancy losses occur every year, with a high prevalence of 10–15% of all clinically recognized pregnancies11. Self-correction of human embryos may operate via cellular fragmentation and blastomere exclusion of abnormal cells12 or by rescue mechanisms, such as trisomy or monosomy rescue13. Spatiotemporal allocation of abnormal cells or aneuploidy rescue mechanisms can lead to confined placental mosaicism, which is the main biological cause for discordant abnormal non-invasive prenatal testing (NIPT) results, with normal fetus confirmed after invasive fetal testing14. For instance, we previously showed that more than 70% of large (>100 kilobase (kb)) de novo copy number variations (CNVs) are present only in the placental lineage10.CIN leads to mosaic embryos that contain both chromosomally normal and abnormal cells. It has been shown that aneuploid cells in mosaic embryos can be progressively depleted during pre-implantation development15. However, little is known about the genomic landscape of first-trimester spontaneous pregnancy losses. This knowledge is essential to understanding in utero mechanisms of CIN separately for fetal and placental lineages and to developing strategies for the early detection of high-risk pregnancies leading to pregnancy loss. In this study, we profiled the chromosomal landscape of the chorionic villi and the extra-embryonic mesoderm of first-trimester  spontaneous pregnancy losses, which are derived from the embryonic trophectoderm and the inner cell mass, respectively.The genomic landscape of second-trimester and third-trimester pregnancy losses and elective terminations of fetuses with abnormal in utero phenotypes has been characterizedAfter conventional karyotyping of the products of conception (POCs) that were collected from 1,745 women over a course of 35 years (1987–2021), 866 (49.6%) and 879 (50.4%) pregnancy losses were classified as karyotypically normal and abnormal, respectively   Fig. . LogistiP = 0.8, two-sided Welch’s t-test)  values from expected 1maternal:1paternal allelic ratio indicates the degree (%) of abnormal cells—that is, mosaicism19. Here, we applied haplarithmisis on bulk DNA samples  and made use of chorionic villi as a seed to phase the parental genomes, allowing the reconstruction of trio-based parental haplarithms , the rate of genomic aberrations in POC samples reaches 67.8% by extrapolation  Fig. . These shms Fig. . This alhms Fig.  and 2a,bpolation . This isP = 3.6 × 10−2, Fisher’s exact test).To further characterize the genetic effect of the detected aberrations, we divided the 94 pregnancy losses into sporadic pregnancy loss (SPL), defined as one pregnancy loss , and recurrent pregnancy loss (RPL), defined as two or more consecutive pregnancy losses  Fig. . The SPL30. Aberrations on Chr 16 (n = 5) were all of maternal and meiotic in origin, and aberrations on Chr 7 were maternal and mitotic (n = 4), paternal and mitotic (n = 1) and paternal and meiotic (n = 1) in origin 34, indicating that pregnancies with trisomy 16 may have reduced capacity to reach to later gestational ages.Aberrations on Chr 7 and Chr 16 were most common in first-trimester pregnancy loss Fig. , as obsegin Fig. . TrisomyComparing the haplarithm profiles of extra-embryonic mesoderm and chorionic villi allowed us to determine not only if these tissues carry different large CNVs (>100 kb) but also whether the level of mosaicism is different. The analysis of extra-embryonic mesoderm and chorionic villi DNA samples can be used to probe the spatiotemporal allocation of abnormal cells in early embryogenesis and to deduce whether this allocation affects the fate of early prenatal development and the risk of pregnancy loss. To check for accurate dissection of extra-embryonic mesoderm and chorionic villi tissues, we performed methylome-wide analysis of 13 samples that generated good-quality data  showed a combined yield of approximately 53.7% fetal chromosomal abnormalities28  from uncultured samples  normal cells compared to abnormal cells, leading to low diagnostic yieldsaicism >0% from ules Fig.  with higles Fig. 10. Additon7 Fig.  and 7. T35. It was previously suggested that self-correction mechanisms for aneuploidy are active in the inner cell mass during early embryogenesis and that there is a selective bottleneck in early embryogenesis when aneuploid cells are depleted12. Our data are compatible with a selective scenario in which chromosomal aberrations first emerge in the inner cell mass of the blastocyst and persist at least in extra-embryonic mesoderm, conferring a strong detrimental effect on the pregnancy outcome, which may result in pregnancy loss.Profiling the genomic landscape of pregnancy losses by carefully dissecting both the extra-embryonic mesoderm and chorionic villi tissues of the same POC and applying haplarithmisis allowed us to detect and classify 35.1% of karyotypically normal POCs as abnormal. Moreover, POCs with different levels of mosaicism in extra-embryonic mesoderm and chorionic villi were found, with the prevalence of aberrations being higher in extra-embryonic mesoderm compared to chorionic villi. This finding raises intriguing hypotheses about the origins of mosaic mutations in pregnancy loss. Although extra-embryonic mesoderm develops before gastrulation in primates, it develops during gastrulation in rodents. Therefore, extra-embryonic mesoderm cells are most likely derived from transient primary yolk sac, which is of embryoblast, and not trophoblast, origin56. ESHRE’s guidelines from 2022 define RPL as the loss of two or more pregnancies before 24 weeks of gestation56, which was used in the present study.We found that SPL and RPL cohorts have similar prevalence of genomic aberrations. However, these two groups had different types of genomic aberrations, such that the SPL cohort had significantly more segmental aberrations, whereas the RPL cohort carried more aneuploidies Fig. . Althoug58 as well as interpretation of NIPT results with mosaic aberration indications. In nationwide NIPT studies32, the rate of confirmed confined fetal mosaicism is very low compared to confined placental mosaicism, indicating that abnormal cells in the fetus are less tolerated relative to the placenta. For instance, 94% of rare autosomal trisomies in NIPT were found to be most likely due to confined placental mosaicism31. The impact of chromosomal mosaicism is less clear due to current limitations for its detection. According to some rare cytogenetic studies on spontaneous pregnancy losses and confined placental chromosomal mosaicism, confined placental mosaicism is found in approximately 20% of the POCs, which is higher than the reported rate of 1–2% seen in viable pregnancies in chorionic villus sampling61. Additionally, when we compared the nature and prevalence of genomic aberrations along the gestational weeks of the first trimester, we observed that, on average, there may be a higher level of mosaicism in earlier POCs (Supplementary Tables n = 14), the average level of mosaicism in POCs of gestational weeks 4–7 (n = 7) was 67.9% ± 26.4 s.d., whereas, in POCs of gestational weeks 8–13 (n = 7), the average level of mosaicism was 48.9% ± 30.8 s.d.  in human embryos and pregnancies with relevance for the safety of transferring in vitro fertilized (IVF) embryos with mosaic imbalances62. Specifically, in the case of detected copy number changes after NIPT, genome haplarithmisis can distinguish between meiotic and mitotic errors. NIPT with confined placental mosaicism can lead to false-positive test results. These findings require fetal invasive genetic testing that could potentially be avoided by accurate detection of the segregational origin of the mutation as well as linking low abundant cell-free DNA (cfDNA) to the fetal or placental lineage, which is required to exclude the presence of aberrant cells in the fetus. Aberrations of meiotic origin, if occurring via rescue events into mosaic blastocysts, are likely affecting both fetal and placental lineages and predisposing for spontaneous miscarriage, whereas mitotic aberrations may be specific to the placenta due to confined placental mosaicism10 and are compatible with healthy pregnancies. This allows differentiating low-risk pregnancy aneuploidies with aneuploidies of mitotic origin where the fetus may not be affected from higher-risk pregnancies with severe clinical consequences where the fetus is likely affected, as meiotic aberrations are present in both the fetal and placental lineages. Additionally, epigenetic studies of cfDNA have suggested that fetal tissues other than the placenta-derived trophoblasts may also contribute to the cfDNA mixture of maternal blood63. This makes it possible to differentiate confined placental mosaicism, which is apparently safe for pregnancy, from a situation where (mosaic) aberration is present in both placental tissue and the fetus, with a higher risk for pregnancy loss. This underlines the importance of developing technologies that can reliably identify placental and fetal origin when an aberration is found in early pregnancy. Moreover, haplotyping-based NIPT methods enable a generic approach for detection of monogenic disorders64.This study has practical implications, contributing to the emerging studies using NIPT in RPL65, the meiotic or mitotic origin of mosaicism has not always been determined in these successful cases. For instance, as in PGT of IVF embryos, the DNA sample is derived from a single trophectoderm biopsy, and the true extent of mosaicism in all embryo compartments, including the inner cell mass, cannot be determined. However, the determination of the segregational origin of mosaic aberrations would help to avoid transferring meiotic (mosaic) IVF embryos, which confer a high risk for inner cell mass aberrations and later pregnancy loss. In contrast, we suggest that the rules for selecting mitotic mosaic embryos for uterine transfer could be more relaxed.Taken together, the detection of the segregational origin of chromosomal aberrations is of paramount importance for prognosing the successful completion of pregnancy. Although embryos with mosaic abnormalities can lead to the birth of healthy babiesIn conclusion, our study shows that as many as two-thirds of all pregnancy losses may be due to fetal chromosomal abnormalities. Furthermore, the ability to accurately determine the segregational and lineage origins of fetal genomic aberrations may enhance the efficacy of human natural and assisted conception and, thereby, improve reproductive genetic care in general.Embryonic tissues and parental blood samples were obtained from the Biobank of Populations of Northern Eurasia, Research Institute of Medical Genetics, Tomsk National Research Medical Center. All couples signed an appropriate informed consent for the transfer of their samples to the biobank for scientific research. This study was approved by the local ethics committee of the Research Institute of Medical Genetics, Tomsk National Research Medical Center, of the Russian Academy of Sciences (protocol no. 15 February 2021). Permission was given for the retrospective analysis of the anonymized biological samples of the biobank.66. Anembryonic pregnancy (AP) was diagnosed in the absence of an embryo in the gestational sac for a period of more than 7 weeks; in addition, ultrasound criteria for AP were a gestational sac ≥13 mm without a yolk sac or ≥18 mm without an embryo. Missed abortion (МА) was diagnosed for embryos with CRL ≥7 mm without cardiac activity or no cardiac activity upon the initial scan and post-7-days scan for embryos with CRL <7 mm. Spontaneous abortion (SA) was diagnosed as a spontaneously terminated pregnancy without ultrasound examination. The most frequent clinical forms of early pregnancy loss in our study were MAs, followed by APs and SAs  ≥7 mm for embryos with no cardiac activity, the absence of an embryo and its cardiac activity 14 d after the detection of a gestational sac without a yolk sac and the absence of an embryo and its cardiac activity 11 d after the detection of a gestational sac with a yolk sac53. The first involves long-term culture of extra-embryonic tissues; most often, cells that are cultured are derived from the stroma of the chorionic villi47. The second approach exploits spontaneously and rapidly dividing cells of the cytotrophoblast to obtain direct chromosome preparations without culturing68. Usually, the results of both techniques are similar. However, discordant results can be obtained in some cases due to tissue-specific placental mosaicism13. Therefore, when large fragments of fetal sac were present, the internal mesodermal layer of extra-embryonic membrane was used for cell culture; otherwise, both extra-embryonic mesoderm and chorionic villi were used (see the ‘Chorionic villi and extra-embryonic mesoderm dissection of the POCs and DNA extraction’ subsection). The tissues were chopped with scissors into small fragments, and long-term cultures were set up in 25-cm2 flasks with 5 ml of DMEM/F12 (1:1) medium (Gibco) supplemented with 20% FBS (HyClone), 1% MEM NEAA solution (Gibco) and 1% penicillin–streptomycin (Gibco). Tissues were incubated at 37 °C with once-weekly medium renewal. Extra-embryonic fibroblasts were cultivated until sufficient mitotic cells for cytogenetic analysis were obtained. Demecolcine (Sigma-Aldrich) was added 4 h before chromosome harvesting, and the samples were processed using standard techniques of hypotonic treatment with 0.55% sodium citrate and cell fixation with a 1:3 mixture of acetic acid:methanol. In some cases, direct preparations of the chorionic villi were used69. Slide preparations and GTG banding were performed by standard protocols in accordance with guidelines70. Subsequently, frozen tissues were used for DNA extraction and preparation of cell suspensions for interphase FISH.The POCs, usually represented by fragments of the gestational sac, were delivered to the laboratory in sterile saline, thoroughly washed and separated from decidual tissues and blood clots under an inverted microscope. Part of each tissue sample was used for cell culture, and the remaining tissue sample was stored at −70 °C for DNA extraction. Traditionally, cytogenetic studies of POCs have used one of two methods to determine the karyotype−1). The samples were incubated for 16 h at 37 °C. Then, 550 μl of phenol was added, mixed gently and centrifuged for 3 min at 12,000 r.p.m. and room temperature. Next, 300 μl of phenol and 300 μl of a mixture  were added to the supernatant liquid, mixed and centrifuged under the same conditions. Afterwards, 550 μl of the mixture  was added to the supernatant, mixed and centrifuged. The supernatant liquid was taken again; 30 μl of 10 M sodium acetate and 660 μl of ethanol were added; and the tube was inverted until DNA was visualized. The solution was removed; 100 μl of 70% ethanol was added to the DNA; the DNA was washed and centrifuged at 12,000 r.p.m. for 3 min; the alcohol was removed; and the precipitate was dried at 37 °C. DNA was dissolved in 100 µl of Tris/EDTA buffer. Likewise, genomic DNA was extracted from the peripheral blood of parents using the standard phenol‒chloroform method.Genomic DNA was extracted from blood samples of the parents and from two distinct locations in the POC: chorionic villi and extra-embryonic mesoderm. The dissection of chorionic villi and extra-embryonic mesoderm by an experienced pathologist is possible from the fourth week of gestation onwards, whereas, from the sixth week of gestation, the separation of extra-embryonic mesoderm and chorionic villi almost always succeeds . Specifically, after thawing, chorionic villi were carefully scraped off under an Axiovert 200 inverted microscope (Carl Zeiss) from extra-embryonic membranes based on their morphology, and DNA was extracted separately from these tissues of each sample. The main limitation for accurate dissection of chorionic villi and extra-embryonic mesoderm, however, is the way that POCs are acquired after pregnancy termination. This is because different methods after pregnancy termination are used in medical practice, including curettage, vacuum aspiration or using specific drugs. Genomic DNA was extracted using a standard phenol‒chloroform extraction method that allows for the isolation of up to 900 ng of DNA from the tissues. To isolate DNA from tissues, a small fragment of tissue (200–300 mg) was taken. Then, samples of chorionic villi or extra-embryonic mesoderm were placed in Eppendorf tubes, and 467 µl of buffer  was added to them, in addition to 25 µl of 20% SDS and 7.5 µl of proteinase K  and treatment with 70% acetic acid for 3–5 min, followed by three washes of the obtained cell suspension with PBS according to a modified protocol67. The extra-embryonic mesoderm cells were obtained by digesting the extra-embryonic membrane  for 30–60 min at 37 °C (ref. 60). Cell suspensions were fixed and stored in 3:1 methanol:acetic acid at −20 °C. For confirmation studies, interphase FISH was performed separately for thawed chorionic villi and extra-embryonic mesoderm cells using centromere-specific DNA probes for chromosomes 2, 15, X and Y as well as subtelomeric DNA probes for chromosome 16 . From 100 to 400 interphase nuclei were scored for each sample using an Axio Imager Z2 microscope (Carl Zeiss) with Metafer and ISIS software (MetaSystems).Conventional CGH and interphase FISH with centromere enumeration probes were performed as described previouslyane Fig.  with 125n = 1,745 and, on average, 10 metaphases) or additional testing by CGH and FISH. All samples with abnormal karyotypes were excluded from downstream haplarithmisis. Of the karyotypically normal cases, 111 families (114 POCs) were randomly selected for SNP haplotyping, given that fetal (extra-embryonic mesoderm and chorionic villi) and parental blood samples were available and that no genetic predisposition for pregnancy loss had been identified in the couple. Twenty families were excluded due to the DNA of one or more family members being of insufficient quantity or quality, causing low SNP call rates or due to one or both parents not being the biological parent, making haplotyping analysis impossible  code GLP28939), which contains approximately 665,000 SNP markers with a mean probe spacing of ~4.4 kb and a median probe spacing of ~2.3 kb. Genotype calls, SNP BAF values and logR values of all samples were computed using Illumina GenomeStudio software. Illumina genotyping was performed at the Core Facility of Genomics, Institute of Genomics, University of Tartu, Estonia.7. This originally allowed tracing the inheritance of linked disease variants. Previously, we demonstrated that, using Illumina’s Global-Screening Array-24, haplarithmisis can detect low-grade mosaicism (>10%), subchromosomal CNVs (>100 kb), the parental and segregational origin of aberrations and maternal cell contamination in placenta10.Haplarithmisis is a conceptual workflow that enables simultaneous genome-wide haplotyping and copy number typing using genotyping information from offspring and parentsSpecifically, in the present study, the parental genotypes were phased by using the chorionic villi genotype of the POC as a seed for phasing. Subsequently, the BAF values—that is, continuous genotype values Fig. —of the e7, haplarithmisis has two features for detection of degree of mosaicism  in combination with the difference between M1 and M2 BAFs (dmat) is characteristic for specific abnormalities, and their specific pattern correlates to segregational origin. For example, if dpat has a value of 0.67 and dmat has a value of 0.33, and the logR value for this chromosome is raised above 0 (~0.58) as compared to the other chromosomes, this is indicative of a trisomy where the maternal chromosome has an abnormal number of two copies—that is, 1paternal:2maternal allelic ratio. Subsequently, segregational origin can be determined by the position of the M1 and M2 values around the centromere. These values around the centromere change depending on whether the centromere contains three different homologs  or two different homologs . To distinguish between meiosis II and mitotic errors, the M1 and M2 values effected by a recombination event are used. This depends on whether there are three different homologs  or two different homologs  at the recombination event. To determine the degree of mosaicism, the genomic coordinates at the logR distortion from the expected value—that is, logR = 0—were used to extract the BAFs and segmented P1, P2 or M1, M2 BAFs of the location of interest. BAF values were then compared to the reference dataset for calculation of level of mosaicism, as described previously19.As described previously in detailism Fig.  and segrism Fig. . First, 19. Parent-of-origin haplotyping allows for the detection of maternal DNA contribution in fetal tissues, as previously described10. In one DNA sample, complete maternal contamination was detected by genome haplarithmisis and validated by quantitative fluorescence polymerase chain reaction (QF−PCR). This DNA sample was excluded from the study.Haplarithmisis was applied to each quartet DNA sample to delineate the allelic architecture of the fetal tissues. Chorionic villi tissue was used as a reference to phase parental genotypes. Parental haplarithms were used to infer the DNA copy number state, parent of origin and level of mosaicism of fetal tissues. In total, 450 DNA samples were analyzed. Levels of mosaicism were calculated as previously described19. Extrapolation of the total abnormalities was calculated using the formula below:Haplarithms of analyzed tissues were classified based on several factors: types of aberrations detected, size of aberrations , placental or embryonic origin based on tissue biopsy, parental  and segregational  origin and level (%) of mosaicism. Levels of mosaicism were calculated based on BAF values as previously describedn = 1,745) and genome haplarithmisis  concerning parental and gestational age were performed by two-sided Welch’s t-test. For dichotomous outcomes, the chi-squared test was applied to analyze parental and segregational origin outcomes of SPL and RPL. Due to low sample size (non-parametric) and paired samples (extra-embryonic mesoderm and chorionic villi from a single fetus), the Wilcoxon signed-rank test was applied to assess the difference in mosaicism degree between extra-embryonic mesoderm and chorionic villi. To calculate the required sample size for the Wilcoxon signed-rank test for matched pairs to test the difference in mosaicism between extra-embryonic mesoderm and chorionic villi, we assumed a normal parent distribution, a mean percentage of 43% mosaicism in the chorionic villi group, an s.d. of 30 in both groups and a correlation between the groups of 0.5. A total sample size of 35 participants was required to test a 15% difference between the extra-embryonic mesoderm and chorionic villi groups, performing a two-sided test using an alpha of 0.05 and a power (1-β) of 0.80. The sample size calculation was performed using G*Power 3.1.9.7. In addition, a power calculation was performed for mosaicism dynamics across gestational age bins. A total sample size of 86 samples was required to test a 15% difference between week bins 4–7 (n = 7) and 8–13 (n = 7) for extra-embryonic mesoderm tissues containing aberrations of mitotic origin, performing a two-sided test using an alpha of 0.05 and a power of (1-β) of 0.80, indicating that our study is underpowered concerning mosaicism dynamics across gestational age, with a power of (1-β) of 0.18 due to low sample size  were randomly selected based on the following inclusion criteria: (1) availability of sufficient extracted DNA; (2) female sex ; and (3) absence of chromosomal abnormalities according to haplarithmisis. Extracted DNA from both chorionic villi and extra-embryonic mesoderm tissues was processed as follows: 300 ng of DNA from each sample was bisulfite converted using the EZ DNA Methylation Kit (Zymo Research) according to the manufacturer’s instructions and analyzed using the Illumina Infinium MethylationEPIC version 1.0 BeadChip Kit , thereby allowing us to examine DNA methylation at more than 850,000 CpG sites across the human genome. The Illumina methylation array was performed at the Core Facility of Genomics, Institute of Genomics, University of Tartu, Estonia.73. In brief, the data were normalized with subset-quantile within array normalization (SWAN)74, and poor-quality sites and samples were removed based on the Greedycut algorithm . The following additional sites were removed: (1) sites on the sex chromosomes, (2) sites near SNPs, (3) sites with missing values in more than 10% of samples and (4) sites not in a CpG context. Additional sample quality control—namely, sex prediction and SNP probe analysis—was carried out using the sEst75 package and RnBeads, respectively. Methylation beta values, representing the methylated signal intensity divided by the sum of the methylated and unmethylated signal intensity, were used for all analyses. Cellular deconvolution of the samples was performed using the reference-based Houseman algorithm76 on data pre-processed with the recommended preprocessNoob77 method and implemented using the minfi78 package. For this, the reference site and cell type data for stromal, Hofbauer, endothelial, trophoblast, syncytiotrophoblast and nucleated red blood cells (NRBCs) provided by Yuan et al.79 were used. Welch’s t-tests were applied for statistical comparison of the cellular proportions predicted in extra-embryonic mesoderm and chorionic villi samples. All high-quality samples and CpG sites were used to conduct a PCA in which beta values were centered but not scaled. The significance of the associations between the principal components (PCs) and sample features was tested as follows: (1) permutation tests  for continuous numerical variables  and (2) two-sided Wilcoxon rank tests for binary categorical variables (tissue type).Data pre-processing was carried out using the RnBeads R package as previously describedWDR60 gene, which is located at 7q36.3, were used. The reference gene was HEXB, which encodes the β subunit of hexosaminidase and is located at 5q13. Reference genomic DNA was obtained from the peripheral blood lymphocytes of a healthy donor. The results confirmed the diploid genome in chorionic villi and the mosaic monosomy in extra-embryonic mesoderm  in the chorionic villi. To validate these findings, RT‒qPCR was performed with a diploid control, a hemizygous deletion case, two times diluted DNA from the hemizygous deletion case and extra-embryonic mesoderm and chorionic villi from PL2074. Primers for exon 16 of the MBD5 gene (2q23.1), exon 12 of the ASXL2 gene (2p23.3) and exon 1 of the CHCHD2 gene (7p11.2) were used to confirm tetrasomy for Chr 2 and Chr 7 in DNA from chorionic villi of PL1758. The following calculations and formulas were used to determine the fold change between the copy number of the test loci in the pregnancy loss and reference DNA: average value for three CT; logQT test primer = (CT test DNA − CT reference DNA) / slope test primer; (logQT test primer − log QT control primer); fold change = 10logQT test primer −logQT control primer. Fold change values were used to build a chart  or meiotic II . To validate whether haplotyping with chorionic villi as a reference for extra-embryonic mesoderm would yield accurate parent-of-origin results, additional haplotyping with two different siblings and grandparents from the maternal side as references was performed. The results from PL1758 confirm the segregational origin of the aberrations, even when chorionic villi is used as a reference . Fragment length was determined using internal length standards (Size Standard GeneScan 550) and GeneMapper 4.1 software.To confirm relationships and exclude maternal cell contamination, analysis of short tandem repeats (STRs) was carried out using the COrDIS EXPERT 26 Kit for DNA identification of 26 STR markers (Gordiz). The analysis included identification of 26 loci: Chorionic villi DNA samples from SAs and control male and female DNA samples were labeled by a SureTag Labeling Kit  and purified by SureTag Purification Columns . Labeled DNA samples were hybridized using the GenetiSure Pre-Screen Complete Kit (8 × 60K)  at 67 °C for 24 h, according to the manufacturer’s protocol. Microarray images were obtained using a SureScan Microarray Scanner (Agilent) and analyzed by Agilent Feature Extraction (version 12.2.0.7) and CytoGenomics software (version 5.2).Further information on research design is available in the Any methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41591-023-02645-5.Supplementary Tables 1–6.Reporting SummarySource data for Fig. 1c,d.Source data for Supplementary Table 2.


The placenta of hystricomorph rodents, lagomorphs and some primates includes an unusual structure, termed a subplacenta, which essentially consists of trophoblastic cells located deep to the central implantation site within the area of decidualization. It has been suggested that the subplacenta is functionally important, although considerable controversy remains on the issue. In this context, our objective was to compare the architecture and structure of the subplacentas of different hystricomorph species, to investigate the possibility that it is active in hormone synthesis.Hydrochaeris hydrochaeris), 2 pacas (Agouti paca), 5 agoutis (Dasyprocta leporina), 5 rock cavies (Kerodon rupestris) and 3 guinea pigs (Cavia porcellus) at different stages of pregnancy  were used for gross and microscopic examination. This included the preparation of latex injection casts, immunohistochemistry for steroidogenic enzymes, scanning and transmission electron microscopy. Tissue steroid concentrations were also determined.In total, the placentas of 3 capybaras (The gross morphology and microvascular arrangement of the subplacentas were similar among the hystricomorphs studied including ultra-structural verification of cytotrophoblast and syncytiotrophoblast in all species. In guinea pigs, trophoblast cells exhibited characteristics consistent with intense metabolic and secretory activity in general. However, immuno-histochemical evidence also indicated that subplacental trophoblast expressed key steroidogenic enzymes, mainly in the chorionic villus region, consistent with tissue steroid concentrations.The subplacentas within placentas of hystricomorph rodent species are structurally similar and, in guinea pigs, have potential for steroid hormone secretion from, at least the early stages of pregnancy. The success of viviparity in eutherian mammals is dependent on the establishment of a fully functional chorioallantoic placenta in order to sustain successful embryonic and fetal development. In this context, placental development from trophoblast is mediated in part by interactions between maternal endometrial and extra-embryonic tissues during the implantation process –5. The pSome species within the Hystricomorph and Lagomorph sub-orders of the Rodentia have a specialized structure within their placentas, called a subplacenta –18, thatIt is presumed that the subplacenta plays an important, specialized role during pregnancy in hystricomorph rodents , 24–26. Hydrochaeris hydrochaeris), 2 pacas (Agouti paca), 5 agoutis (Dasyprocta leporina), 5 rock cavies (Kerodon rupestris), and 3 guinea pigs (Cavia porcellus) in different stages of pregnancy . The samples were collected from multiple mothers. The guinea pig pregnancy age estimation was performed based in [In total, placentae were analyzed from 3 capybaras . Different colours were injected in a uterine vein, a uterine artery, an umbilical artery and the umbilical vein. The placentae were then immersion fixed in 10 % formalin, dissected and analyzed.Preparations of histological sections for light microscopy were performed at the University of São Paulo’s School of Veterinary Medicine and Animal Science. Fragments of the placentas collected at different stages of gestation were fixed in 4 % paraformaldehyde solution. After fixation the material was washed in phosphate–buffered saline (PBS), followed by dehydration in a series of increasing concentrations of ethanol (from 70 to 100 %), followed by diaphanization in xylol and paraffin embedding (Histosec) . ParaffiTissues collected for immunohistochemistry were immersion fixed in 4 % paraformaldehyde in 70 mM phosphate buffer for 48 h, and then washed in cold PBS for 72 h. The samples were embedded in paraffin, sectioned to 5 μm in a microtome (Leica RM2265) and transferred to silanized slides . The sections were rehydrated and washed in citrate buffer  for antigen recovery. Endogenous peroxidase was blocked with 3 % hydrogen peroxide diluted in distilled water for 30 min in a darkroom. Tissue sections were blocked with 2 % bovine serum albumin (BSA) diluted in PBS for 30 min. Primary antibodies: cytokeratin , vimentin , 17β-hydroxysteroid dehydrogenase , 3β-hydroxysteroid dehydrogenase , and aromatase cytochrome P450  were incubated overnight in a dark, humid chamber at 4 °C. The reaction was revealed using Dako Advance HRP  and stained with DAB  according to the manufacturer’s instructions, and the slides were counterstained with hematoxylin. Between each step prior to antibody incubation, slides were rinsed in PBS containing 0.2 % BSA. Negative controls included no primary antiserum.Slides were then mounted, viewed, and photographed on the Nikon Eclipse 80I microscope.To study the microvasculature, term placentae were injected with Mercox™ CL-2R resin  . The proFragments of guinea pig placental tissue were collected at different stages of gestation (middle and late). Samples of subplacenta and labyrinthine zones were frozen at -20 °C for hormonal analysis. Steroid hormone concentration were measured using enzyme-immunoassays (EIA), previously validated by . SteroidII). However, a major gross vasculature structure was formed by maternal veins .The gross morphology and microvascular arrangements of the subplacenta of these hystricomorph rodents were observed during pregnancy Fig. A-D. The ins Fig. DII.II). The giant cells of the cytotrophoblastic region showed a positive reaction to cytokeratin, however there was no immuno-staining in the fetal mesenchyme . There was positive actin labeling in the cells of the muscle layer of the fetal vessels, but no evidence of actin immuno-staining in the syncytotrophlast region . Vimentin-positive labeling was observed on vessel walls and giant cells .Fetal and maternal blood vessels were prominent in the labyrinth layer and near the junctional zone of the capybara subplacenta Fig. A. Giant yme Fig. BI − II. ion Fig. CI − II. lls Fig. DI − II.I). Evidence of expression of 3β-HSD was also evident in the same region but was not as extensive expression . The expression of aromatase (P450) was positive around the peripheral membrane protein of the labyrinth region . No labeling was observed in the absence of the primary antisera, the negative controls , in any of the sections.In guinea pig placentae, there was positive immuno-reactivity consistent with steroidogenesis, mainly in the region of chorionic villi and, in the labyrinth region, only in the blood vessel region. Among the enzymes tested, 17β-HSD expression was especially evident in the trophoblastic cells of the villi Fig. A, AI. EvVascular molds examined by the scanning electron microscopy technique demonstrated evidence of larger branches supplying the subplacenta. The division of these larger branches allows the branching of smaller vessels, which become tortuous with sinus dilations and constrictions in the vessel lumen as they course through the tissue Fig. .Fig. 4SIn capybara the nuclei of the cytotrophoblast cells were large in relation to the amount of cytoplasm and nucleoli were prominent Fig. . The basTo verify the capacity for hormonal production by the subplacenta, samples of guinea pig subplacenta were analyzed for progesterone, testosterone and estradiol content and compared with the labyrinth region. In general, for all steroids, samples from mid-gestation had higher steroid concentrations when compared to samples collected from late gestation. In addition, the subplacenta region showed higher hormone levels than labyrinthine samples Table .Table 2The subplacenta, as a structure found only on hystricomorph rodents, lagomorphs, and some primates, has attracted interest, especially in terms of comparative morphology. However, to the best of our knowledge, no studies have yet established an endocrine function for the subplacenta during conceptus development. The results of early studies investigating the potential for progesterone synthesis by the guinea pig placenta indicated that the spongy trophoblast was a likely source , 35. HowLagostomus maximus) subplacenta [Chinchilla lanigera) and coypu (Myocastor coypus) [Myocastor coypus) [The subplacenta has been described in several species among the Hystricomorph rodents , 36–40. placenta  and was  coypus) . All cav coypus) , which a coypus) , the maj coypus) , 43, 44 Castor fiber) carrying twin versus triplet fetuses. The transcripts identified were consistent with functions related to cellular processes, biological regulation, response to stimuli and metabolism but, compared to other parts of the placenta or even compared to other species, there appeared to be little difference in functional terms [Prior indirect evidence supports a possible endocrine role for the subplacenta. Recent work resulted in the creation of a library of differentially expressed genes (DEGs) from the subplacenta of the beaver  presents a distinct pattern of vascularization, typical of endocrinal glands. Different aspects including the central vasculature of the labyrinth and junctional zones, and subplacental vasculature, were comparable with small capillaries, around larger vessels, where it was possible to identify sinusal dilatations and anastomotic channels, like those described previously . FurtherMore direct evidence for endocrine function was provided by the detection of steroid hormones in subplacental tissue and the expression of steroidogenic enzymes including 17β-HSD and 3β-HSD by immuno-histochemistry which is consistent with the synthesis of progesterone and of testosterone and estradiol. The catalytic activity of 17β-HSD is required for the reduction of DHEA to androstenediol, androstenedione to testosterone and estrone to estradiol. The presence of progesterone in subplacental tissue is also consistent with expression of 3β-HSD and the oxidation of pregnenolone and 17α-hydroxypregnenolone to progesterone and 17α-hydroxyprogesterone, respectively , 52. TheThe subplacenta does not differ greatly in terms of the structural aspects among the hystricomorphs investigated, and it is has potential for hormonal secretion during the early stages of pregnancy based on evidence consistent with steroidogenic enzyme activity.


Current expectations are that surgeons should be technically proficient in minimally invasive low anterior resection (LAR)—both laparoscopic and robotic-assisted surgery. However, methods to effectively train surgeons for both approaches are under-explored. We aimed to compare two different training programs for minimally invasive LAR, focusing on the learning curve and perioperative outcomes of two trainee surgeons.We reviewed 272 consecutive patients undergoing laparoscopic or robotic LAR by surgeons A and B, who were novices in conducting minimally invasive colorectal surgery. Surgeon A was trained by first operating on 80 cases by laparoscopy and then 56 cases by robotic-assisted surgery. Surgeon B was trained by simultaneously performing 80 cases by laparoscopy and 56 by robotic-assisted surgery. The cumulative sum (CUSUM) method was used to evaluate the learning curves of operative time and surgical failure.For laparoscopic surgery, the CUSUM plots showed a longer learning process for surgeon A than surgeon B (47 vs. 32 cases) for operative time, but a similar trend in surgical failure (23 vs. 19 cases). For robotic surgery, the plots of the two surgeons showed similar trends for both operative times (23 vs. 25 cases) and surgical failure (17 vs. 19 cases). Therefore, the learning curves of surgeons A and B were respectively divided into two phases at the 47th and 32nd cases for laparoscopic surgery and at the 23rd and 25th cases for robotic surgery. The clinicopathological outcomes of the two surgeons were similar in each phase of the learning curve for each surgery.For surgeons with rich experience in open colorectal resections, simultaneous training for laparoscopic and robotic-assisted LAR of rectal cancer is safe, effective, and associated with accelerated learning curves. Its technical advantages, including ergonomically designed surgical instruments, can not only translate into clinical benefits for patients, such as lower conversion rate, shorter hospital stay, reduced complications, and faster recovery in urogenital function  involves teaching laparoscopy first, followed by robotics, after achieving proficiency in laparoscopy. However, effective methods for training surgeons to be skilled using both approaches are relatively unexplored. Previous literature reports have shown that prior experience in laparoscopic rectal surgery can facilitate the learning curve for robotic rectal resection , 12 and This retrospective study aimed to compare two different training programs (“standard” and “novel” programs) for learning minimally invasive low anterior resection (LAR) for rectal cancer, focusing on the learning curves and perioperative outcomes of two trainee surgeons, who were novices in minimally invasive colorectal surgery.22.1Data from patients with histologically confirmed rectal adenocarcinoma treated at the Henan Provincial People’s Hospital from May 2018 to May 2022 were retrospectively reviewed. Overall, 302 consecutive patients who underwent minimally invasive LAR by surgeons A and B were initially included. After excluding 12 cases operated by surgeon A  and 18 cases by surgeon B , 136 cases were finally enrolled for each surgeon, including 80 laparoscopic and 56 robotic cases, respectively Figure 1Surgeons A and B performed their first laparoscopic LAR (L-LAR) in May 2018 and April 2019, and then performed their first robotic LAR (R-LAR) in August 2019 and June 2019, respectively. During the study period, surgeon A performed the first 80 laparoscopic cases and then the 56 robotic cases using the da Vinci surgical robot (“standard program”); while surgeon B performed an initial 15 cases using the laparoscope and then the remaining 121 cases using the laparoscope and the da Vinci surgical robot alternately (“novel program”). The two surgeons had similar case volumes (L-LAR and R-LAR) per year.Analyzed data included patient demographics, preoperative assessment, intra- and postoperative outcomes, and pathological parameters. Rigid colonoscopy with biopsy and pelvic magnetic resonance imaging (MRI) were performed as routine examinations before the operation. Preoperative chemoradiotherapy (CRT) was recommended for patients with locally advanced rectal cancer (cT3–4 and/or N+) based on MRI examination. Final decisions were made in multidisciplinary treatment meetings. Operative time (OT) was from skin incision to closure. Surgeon console time was the time the surgeon used the robot. Docking time was the time to position the robot and secure the arms to the ports. Postoperative complications included only anastomosis-related complications, such as anastomotic leakage or bleeding. Complications ≥ grade III, according to the Clavien–Dindo classification, were considered severe. Circumferential resection margin (CRM) and distal resection margin (DRM) were considered involved if malignant cells were found within ≤ 1 mm from the CRM or DRM. Surgical failure was defined as the presence of one or more of the following four parameters: conversion, R1 resection (CRM or DRM involved), severe anastomosis-related complications (≥ Clavien–Dindo grade III), and number of harvested lymph nodes< 12. Otherwise, the case was considered a surgical success.This study was approved by the Ethical Committee of the Henan Provincial People’s Hospital and written informed consent was obtained from all patients.2.2For R-LAR, all cases were performed using the da Vinci Si surgical system. A totally robotic technique was adopted in all cases, and standardized as follows. After pneumoperitoneum was established, the assistant docked the robot. The inferior mesenteric vessels were first ligatured and then, the left colon and sigmoid colon were dissected in a medial-to-lateral approach. The splenic flexure was taken down, if appropriate. Then, the pelvic cavity was dissected along the nonvascular plane between the parietal fascia and the visceral fascia of the pelvis, first by a posterior dissection, followed by lateral and anterior dissections. The robot was undocked when the dissection was completed. The rectum was transected laparoscopically and an intracorporeal end-to-end anastomosis was constructed. The specimen was extracted through a Pfannenstiel incision using a wound protector. An ileostomy was created during the operation on the decision of the surgeon when the following criteria were met: uncertainty of the blood supply of the anastomosed stoma, tension in the anastomosed stoma, and poor nutritional status. L-LAR was performed in the same manner as described above for R-LAR.2.3Both surgeons had approximately 10 years of experience in the Department of Gastrointestinal Surgery. During this period, they had undergone training for open colorectal resections, under the supervision of an experienced surgeon and performed open colorectal resections independently on > 200 cases. Before performing the first L-LAR, they received rigorous dry laboratory training in laparoscopy and then participated in colorectal resections as assistants during fellowship training, with surgeons A and B assisting in 50 and 58 cases, respectively. Then, they performed laparoscopic operations for simple surgeries, followed by more difficult surgeries, such as LAR. Surgeons were similarly trained in the robotic procedure during the fellowship training period: first, dry laboratory training; then, assisting robotic colorectal surgeries ; and finally, using the robot independently. The initial cases conducted by the two surgeons were supervised by the previous surgeon who had rich experience in laparoscopic and robotic colorectal resections. He monitored and advised on surgical tips for the manipulation of the instruments and technical tips for laparoscopic and robotic surgeries. After approximately 20 cases, surgeons A and B could perform the operations independently, without supervision.2.4Learning curves were analyzed using the cumulative sum (CUSUM) method, based on two parameters: OT and surgical failure, with the following equation:Xi is an individual value, and 0X is the mean of the overall value for each surgeon.where OT for the first case was the difference between the OT for the first case and the mean OT of all cases for each surgeon. The CUSUMOT of the second case was the CUSUMOT of the previous case added to the difference between the OT of the second case and the mean OT for all cases for each surgeon. This procedure was repeated for each patient except the last one, which was set as zero. For surgical failure, Xi was an individual attempt and 0X was the observed failure rate of each approach for each surgeon, with Xi assigned a score of 0 for success and 1 for failure.For example, concerning OT, all cases were arranged in chronological order for each surgeon. The CUSUMCUSUM curves ascended when the set value was not reached, which reflected an ongoing learning process; otherwise, the performance was more often on target than expected. Curve analysis involved the identification of the turning point at which the graph adopted a general downward slope; this point represents the end of the initial learning process, after which the target value began to be observed.2.5t test or the Mann–Whitney U test, as appropriate. P< 0.05 was considered statistically significant.All statistical analyses were conducted using SPSS statistical software . Categorical variables were compared using chi-squared or Fisher’s exact tests. Continuous variables are presented as mean ± standard deviation or median (range) and were compared using an independent two-sample 33.1p > 0.05) , American Society of Anesthesiologists score (ASA), tumor location, and CRT ( > 0.05) Table 1.3.2p = 0.085) (p = 0.402). Two conversions occurred for surgeon A and three for surgeon B, due to insufficient experience . There were three severe anastomosis-related complications (two anastomotic leakages and one anastomotic bleeding) for surgeon A and two (one anastomotic leakage and one anastomotic bleeding) for surgeon B (p = 1.000), all of which were treated by endoscopy. Patients operated by surgeon A had significantly shorter hospital stays  and fewer lymph nodes  than those operated by surgeon B. All three patients with positive CRM received postoperative chemoradiotherapy and no local recurrence was observed during follow-up. Surgical failure rates were comparable between the two surgeons .For laparoscopic surgery, OT values were similar between surgeons A and B  Table 1.p > 0.05)  Table 1.3.3rd and 19th cases, respectively, indicating a shorter learning process than that of OT. Therefore, the CUSUMOT was taken as a surrogate to determine the learning curve. Learning curves for surgeon A were determined for phase 1  and phase 2 ; for surgeon B phase 1  and phase 2   and phase 2 (24th–56th cases) for surgeon A; phase 1 (1st–25th cases) and phase 2 (26th–56th cases) for surgeon B . Considering that surgeon B performed 15 laparoscopic cases before undertaking the first robotic cases, we further divided phase 1 into subgroup 1 (the first 15 cases) and subgroup 2 (the remaining cases) at the 15th case. The results showed that OT was significantly longer for surgeon A than for surgeon B in subgroup 2 , while the OT showed no significant difference between surgeons in subgroup 1 , phase 2  (p > 0.05)   Table 2.3.5p = 1.000). For phase 2, 23 patients operated by surgeon A underwent protective ileostomy, compared with 14 operated by surgeon B . Other outcomes were comparable between the two surgeons (p > 0.05)  Table 3.4In most cases, surgeons are trained in laparoscopic and robotic surgery sequentially. However, preliminary experience with laparoscopic LAR is not a necessary pre-requisite for robotic LAR . A few sMost patients in this study had mid-high tumors, and a few had low tumors. This was mainly because the tumor distance from the anal verge reflected the depth of dissection in the pelvis and surgical difficulty . Low tumThe learning curve represents the process of gaining adaptation or becoming skilled in performing a surgical procedure , and is th case. As shown in The most important finding was that training on both approaches simultaneously could produce a crossover effect, which can facilitate the learning curves. This study showed that the laparoscopic learning curve was shorter in the simultaneous training program 32 cases) than that in the sequential training program (47 cases), suggesting that the experience gained in robotic surgery can facilitate the laparoscopic learning curve for rectal cancer surgery. Furthermore, considering that the initial 15 laparoscopic cases conducted by surgeon B were trained differently from the other 65 cases, phase 1 was divided into subgroups 1 and 2 at the 15 cases thThe poor surgical skills of a surgeon during the initial learning period may not compromise surgical quality. Previous reports have suggested that prolonged operative time for colorectal surgery is associated with worse surgical quality , 34. In This study had several limitations. First, the results are limited by its retrospective and non-randomized study design. When applying a new technique with which the surgeon has minimal experience, patients with favorable clinical profiles tend to be selected, leading to a bias in the learning curve and clinical outcome data. A randomized controlled trial is necessary to provide deep insight into this novel training program. Second, the first 15 laparoscopic cases conducted by surgeon B were performed before the first robotic case, impairing the consistency of the novel training system. Another limitation was that, during the learning curve, 30 cases of LAR combined with other operations were excluded from this study. These cases could have biased the learning curve to some extent. In addition, only two surgeons were included in this pilot study, which may result in a high risk of selection bias. Therefore, enrolling more surgeons in a future study will provide more robust and convincing data. Despite these limitations, this study provides preliminary insights into the surgical outcomes and learning curves associated with this novel training program.5For surgeons with rich experience in open colorectal resections, simultaneous training to conduct laparoscopic and robotic-assisted LAR for rectal cancer is safe and effective. This novel training program does not increase the learning load, but does shorten learning curves, which can be attributed to a crossover effect; that is, the skills required for the two approaches are mutually transferrable. Furthermore, surgical outcomes are not compromised. Our results highlight a potential alternative approach for surgeons to rapidly master these two techniques. A randomized controlled trial with more surgeons will provide a more solid methodology for investigating the potential benefits of this novel training program.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Henan Provincial People’s Hospital. The patients/participants provided their written informed consent to participate in this study.JZ, ZW, YW, DW, and CZ performed the experiment conception and design. JZ, ZW, YW, DW, and CZ performed the research. YW, DW, and CZ retrieved the data and performed the data collection. YW and DW analyzed the data and did the paper writing. All authors contributed to the article and approved the submitted version.


Sternorrhyncha in China, although this region hosts massive species distribution data. Here, based on the 15,450 distribution records of Sternorrhyncha species in China, we analyzed patterns in species richness and endemism at 1° × 1° grid size and determined the effects of environmental variables on these patterns using correlations analysis and the model averaging approach. We found that species richness and endemism of Sternorrhyncha species are unevenly distributed, with high values in the eastern and southeastern coastal regions of mainland China, as well as Taiwan Island. Furthermore, the key factors driving species richness and endemism patterns are inconsistent. Species richness patterns were strongly affected by the normalized difference vegetation index, which is closely related to the feeding habits of Sternorrhyncha, whereas endemism patterns were strongly affected by the elevation range. Therefore, our results indicate that the range size of species should be considered to understand the determinants of species diversity patterns.One of the main goals in biogeography and ecology is the study of patterns of species diversity and the driving factors in these patterns. However, such studies have not focused on  Macro-scale patterns of species richness are central to many fundamental questions in ecology and biogeography . SpeciesSternorrhyncha suborder, a tiny sucking phytophagous insect of Hemiptera, consists of approximately 18,700 species worldwide , Dysmicoccusbrevipes  and Phenacoccusmadeirensis Green, 1923, are major agricultural pests and invasive species. However, some species are beneficial to humans in various ways. Scale insects, for example, provide many useful materials such as red dyes, waxes, resins and medicines  for biogeography research  investigate the effects of multiple environmental variables on two types of patterns.This paper aims to (1) analyze the species richness and endemism patterns of Sternorrhyncha species were obtained from previous studies (https://www.gbif.org/). Species richness and endemism patterns were mapped by calculating the species numbers and weight endemism values in 1° × 1° grids, respectively. The weighted endemism of a grid was calculated as the sum of the range-down-weighted species values after each species was down-weighted by the number of grids in which it was found  and annual potential evapotranspiration (PET); 2) water availability: mean annual precipitation (MAP) and annual actual evapotranspiration (AET); 3) climate seasonality: temperature annual range (TAR), temperature seasonality (TS), and precipitation seasonality (PS); 4) habitat heterogeneity: elevation range  and slope (SP); 5) productivity: normalized difference vegetation index (NDVI) and net primary productivity (NPP); and 6) historical climate stability: MAT change and MAP change; these two variables were defined as the absolute value of the difference between the current MAT/MAP and the Last Glacial Maximum MAT/MAP. Because past climate models were uncertain, MAT and MAP in the Last Glacial Maximum were calculated as the mean of the Model for Interdisciplinary Research on Climate Earth system (MIROC-ESM) and Community Climate System Model v.4 (CCSM4) models.Thirteen environmental variables were selected to identify key ecological factors that drive patterns of species richness and endemism and they were grouped into six categories as follows: 1) ambient energy: mean annual temperature . The NDVI and NPP were obtained from the Resource and Environment Data Cloud Platform (http://www.resdc.cn/). The PET and AET were downloaded from the CGIAR-CSI database (http://www.cgiar-csi.org). Elevation data were obtained from CGIAR SRTM (http://srtm.csi.cgiar.org/). The spatial resolutions of NDVI and NPP were 1 km2 and that of MAT and MAP in the Last Glacial Maximum was 2.5 arc min (~5 km). The spatial resolutions of other environmental variables were 30 arc-seconds (~1 km). Environment variable values were obtained in a 1° grid by calculating the mean of all pixels within it using ArcGIS v.10.5 .The reliability of the relationship between species diversity and environmental variables is affected by sampling bias. As a result, assessing sampling bias is an unavoidable step in biogeography. Based on previous studies, a linear regression model was developed using the square-root-transformed number of records and the observed richness in a 1° × 1° grid , 2021. TAIC) was considered the best model to assess the relationship between species diversity and environmental variables. However, AIC is sensitive to the presence of spatial autocorrelation and produces minimum adequate models that are unstable and overfitted .Pearson correlation analysis was used to assess the relationships between species richness/endemism and each environmental variable. Spatial autocorrelation is frequently found in data on species richness, increasing the rate of a type I error in a significance test . To solverfitted . Therefoerfitted . To reduSternorrhyncha in China are shown in Fig. All distribution records of Sternorrhyncha was strongly correlated with NDVI, ER and PET and moderately correlated with MAT, TAR and PS  were used, but NPP played a smaller role than NDVI , because molecular data currently available is insufficient. Thus, once a complete phylogenetic tree using the molecular data was constructed in the future, the importance of evolutionary/historical factors can be determined.In summary, species richness and endemism patterns of