Outcome of video-assisted thoracoscopic treatment of idiopathic chylothorax in 15 cats.

OBJECTIVE: To evaluate the outcomes and complications of video-assisted thoracoscopic (VATS) treatment of chylothorax in cats. STUDY DESIGN: Multi-institutional retrospective study. ANIMALS: Fifteen client-owned cats. METHODS: The medical records of cats undergoing thoracoscopic thoracic duct ligation (TDL) for treatment of idiopathic chylothorax were reviewed. Cats undergoing additional procedures including thoracoscopic pericardectomy and/or laparoscopic cisterna chyli ablation (CCA)_were included. Follow up was obtained through communication with the referring veterinarian or owner. RESULTS: All cats underwent thoracoscopic TDL. Thirteen cats underwent simultaneous pericardectomy and two cats underwent laparoscopic CCA without pericardectomy. Conversion from a thoracoscopic to open approach was necessary in 2/15 (13%) of thoracic duct ligations and 1/11 (9%) of pericardectomies. The most common postoperative complication was persistent pleural effusion in five cats (33%). Four of 15 cats (27%) died or were euthanized prior to hospital discharge following surgery. Recurrence of effusion occurred in 1/7 (14%) of cats that sustained resolution of the effusion at the time of surgery with a median follow up of 8 months. The overall mortality attributed to chylothorax was 47%. CONCLUSION: Thoracoscopic treatment of idiopathic chylothorax resulted in a low incidence of intraoperative complications or conversion in the study population; however, mortality related to feline idiopathic chylothorax remained high. CLINICAL SIGNIFICANCE: While VATS treatment of idiopathic chylothorax is technically feasible, further consideration of the underlying pathology and current treatment algorithm is needed to improve outcomes as this remains a frustrating disease to treat in the feline population.

Various repair events following CRISPR/Cas9-based mutational correction of an infertility-related mutation in mouse embryos.

PURPOSE: Unpredictable genetic modifications and chromosomal aberrations following CRISPR/Cas9 administration hamper the efficacy of germline editing. Repair events triggered by double-strand DNA breaks (DSBs) besides non-homologous end joining and repair template-driven homology-directed repair have been insufficiently investigated in mouse. In this work, we are the first to investigate the precise repair mechanisms triggered by parental-specific DSB induction in mouse for paternal mutational correction in the context of an infertility-related mutation. METHODS: We aimed to correct a paternal 22-nucleotide deletion in Plcz1, associated with lack of fertilisation in vitro, by administrating CRISPR/Cas9 components during intracytoplasmic injection of Plcz1-null sperm in wild-type oocytes combined with assisted oocyte activation. Through targeted next-generation sequencing, 77 injected embryos and 26 blastomeres from seven injected embryos were investigated. In addition, low-pass whole genome sequencing was successfully performed on 17 injected embryo samples. RESULTS: Repair mechanisms induced by two different CRISPR/Cas9 guide RNA (gRNA) designs were investigated. In 13.73% (7/51; gRNA 1) and 19.05% (4/21; gRNA 2) of the targeted embryos, only the wild-type allele was observed, of which the majority (85.71%; 6/7) showed integrity of the targeted chromosome. Remarkably, for both designs, only in one of these embryos (1/7; gRNA 1 and 1/4; gRNA2) could repair template use be detected. This suggests that alternative repair events have occurred. Next, various genetic events within the same embryo were detected after single-cell analysis of four embryos. CONCLUSION: Our results suggest the occurrence of mosaicism and complex repair events after CRISPR/Cas9 DSB induction where chromosomal integrity is predominantly contained.

Risk factors for complicated perioperative recovery in dogs undergoing staphylectomy or folded flap palatoplasty: Seventy-six cases (2018-2022).

OBJECTIVE: To analyze risk factors for complicated perioperative recovery of dogs undergoing either staphylectomy or folded flap palatoplasty. STUDY DESIGN: Retrospective study. ANIMALS: Seventy-six client-owned dogs. METHODS: Medical records of dogs that underwent either staphylectomy or folded flap palatoplasty were reviewed for signalment, brachycephalic risk (BRisk) score, history of gastrointestinal signs, laryngeal collapse grade, presence of preoperative aspiration pneumonia, intraoperative respiratory and cardiovascular complications, length of general anesthesia, number of corrected brachycephalic obstructive airway syndrome (BOAS) components, and gastrointestinal and respiratory postoperative complications. Complicated recovery was defined as requirement for prolonged oxygen treatment and/or tracheostomy or perioperative death. Penalized logistic regression was used to identify risk factors. RESULTS: Seventy-six dogs were enrolled in the study. Multivariate penalized logistic regression identified four risk factors for complicated recovery. These include surgery type (p = .0002), age (p = .0113), laryngeal collapse grade >2 (p < .0001) and length of general anesthesia (p = .0051). CONCLUSIONS: In this population, dogs that had staphylectomy, increasing age, laryngeal collapse grade >2 and increasing length of general anesthesia were at increased risk for perioperative complicated recovery. CLINICAL SIGNIFICANCE: The results of this study identified risk factors for perioperative complicated recovery in dogs undergoing elongated soft palate correction and may assist in surgical planning and early prediction of complications.

Laser-Assisted Retropharyngeal Rigid Nasopharyngoscopy in Dogs.

This article reviews minimally invasive approaches to the nasopharynx. It also introduces a novel approach, transbuccal retrograde rigid pharyngoscopy, and describes three cases in which this approach was used.

New Training Options for Minimally Invasive Surgery Skills.

Veterinary minimally invasive surgery (MIS) training options are becoming more available. This article reviews new developments in this area and the current evidence for manual skills and cognitive training of MIS.

Bilateral neoureterocystostomy with distal ureteral tapering in a dog with severe bilateral hydroureteronephrosis.

A 3.5-year-old intact male Labrador retriever was seen for hematuria. The results of clinical pathology tests were unremarkable. However, urinalysis revealed dark, cloudy, alkalotic, and isosthenuric urine containing red and white blood cells, epithelial cells, and struvite crystals. Severe bilateral enlargement of ureters and markedly enlarged kidneys were identified on abdominal radiographs. Computerized tomography revealed extensive bilateral hydroureteronephrosis with no definitive cause of obstruction. The dog underwent bilateral ureteral tapering with bilateral neoureterocystostomy and placement of temporary bilateral ureteral stents and a cystostomy tube. The dog was monitored in the intensive care unit for 7 d after surgery and was discharged 9 d after surgery and after the stent and cystostomy tube were removed. The dog remained clinically normal and was reported to have been euthanized at 11 y of age (2021) due to unspecified causes. Key clinical message: There are several potential causes of severe bilateral hydroureteronephrosis in animals. Based on this case report, dogs with severe bilateral hydroureteronephrosis that are clinically asymptomatic may have favorable outcomes following bilateral ureteral reconstruction and neoureterocystostomy, even if a definitive cause is not identified.

Néourétérocystostomie bilatérale avec effilement urétéral chez un chien avec hydrourétéronéphrose bilatérale sévère. Un labrador mâle non-castré âgé de 3,5 ans a été vu pour hématurie. Les résultats des tests de pathologie clinique ne présentaient pas d'anomalie. Toutefois, l'analyse d'urine a mis en évidence une urine foncée, trouble, alcaline et isosthénurique, contenant des globules rouges et blancs, des cellules épithéliales et des cristaux de struvite. Une augmentation bilatérale sévère de la taille des urètres ainsi qu'une augmentation de la taille des reins ont été notées lors des radiographies abdominales. Un examen par tomodensitométrie a révélé une hydrourétéronéphrose bilatérale marquée sans cause identifiable d'obstruction. On procéda à un effilement urétéral bilatéral avec néourétérocystostomie bilatérale et mise en place de stents urétéraux bilatéraux temporaires et un tube à cystotomie. Le chien a été sous surveillance à l'unité des soins intensifs pendant 7 j après la chirurgie et a obtenu son congé 9 j après la chirurgie, après que les stents et le tube à cystotomie aient été retirés. Le chien est demeuré cliniquement normal jusqu'à son euthanasie à l'âge de 11 ans (2021) pour des raisons non-spécifiées.Message clinique clé :Il y a plusieurs causes potentielles d'hydrourétéronéphrose bilatérale chez les animaux. Sur la base de ce rapport de cas, les chiens avec hydrourétéronéphrose bilatérale sévère qui sont cliniquement asymptomatique un résultat favorable peut être obtenu à la suite d'une reconstruction urétérale bilatérale et une néourétérocystostomie même si une cause définitive n'est pas identifiée.(Traduit par Dr Serge Messier).

Receiver operating characteristics of computed tomography (CT) compared to cystoscopy in diagnosis of canine ectopic ureters: Thirty-five cases.

OBJECTIVE: The aim of the study was to determine receiver operating characteristics (ROC) of computed tomographic excretory urography (CTEU) in predicting cystoscopic findings of ureteral anatomy. STUDY DESIGN: Retrospective cohort study. ANIMALS: Thirty-five client-owned dogs. METHODS: The medical records of dogs referred for suspected ectopic ureters were reviewed. Inclusion criteria included CTEU findings reported by board-certified radiologists, followed by rigid cystoscopy with or without ureteral cystoscopic laser ablation (CLA). Data included signalment, urinary incontinence degree, body condition score, weight, degree of colon distension, CTEU and cystoscopy findings. ROC analysis was used to compare CT-predicted ureteral orthotopia/ectopia to cystoscopy findings. Additionally, ROC of CT predicted ureteral orifice locations was analyzed. Regression covariate analysis was performed to identify factors that may have influenced accuracy of diagnosis. RESULTS: The ability of CT to identify a normal and intra-or extramural ectopic ureters conclusively and correctly was 13/26 (50%) and 32/41(78%), respectively. Sensitivity and specificity of identifying extramural versus intramural ureters was 2/7 versus 30/46 (29 vs. 65%) and 61/63 versus 17/24 (97 vs. 71%), respectively. Ectopic orifice determination sensitivity and specificity varied widely depending on location from 0% to 76% and 67% to 97%, respectively. Covariate analysis failed to identify interfering factors. CONCLUSIONS: CT did not accurately predict anatomy of ureters; CT findings may need confirmation by cystoscopy and possibly intraoperative fluoroscopy prior to determining if CLA is indicated or not. CLINICAL SIGNIFICANCE: Our results may be of importance for surgeons interpreting the CTEU findings. CTEU prediction of the location of the ureteral orifice shows low sensitivity especially in or close to the urethral sphincter area.

Canine laparoscopic-assisted ovary-sparing hysterectomy does not increase risk of stump pyometra.

OBJECTIVE: To evaluate incidences of pyometra and orthopedic, behavioral, urinary/reproductive, neoplastic, or atopic disease processes as outcomes for dogs undergoing either a laparoscopic-assisted ovary-sparing spay/hysterectomy (LapOSS) or a laparoscopic ovariectomy (LapOVE). ANIMALS: 33 client-owned dogs. PROCEDURES: Medical records of client-owned dogs presenting between August 2013 and May 2020 for elective LapOSS or LapOVE were reviewed. A multiple-choice client questionnaire was emailed to all clients whose dogs' complete medical records were available. RESULTS: 17 of the 33 dogs were in the LapOSS group, and 16 of 33 dogs were in the LapOVE group. Of the 17 dogs undergoing LapOSS, 5 of 17 (29%) underwent an elective OVE at a later date. The mean follow-up time was 4.2 ± 1.8 years for the LapOSS group and 4.3 ± 2.0 years for the LapOVE group. No dogs developed stump pyometra. One LapOSS dog developed mammary tumor, and 2 others developed nonreproductive malignant neoplasia while 2 of the LapOVE dogs developed malignant neoplasia. One of the LapOSS dogs with malignant neoplasia had an ovariectomy prior to development of disease. CLINICAL RELEVANCE: Laparoscopic-assisted ovary-sparing spay appears to provide a safe and reliable method of sterilization, with no observable increased risk of pyometra with hysterectomy. Owners must be counseled prior to surgery regarding the consequences of gonadal hormone retention and multiple heat cycles.

Characterization of ovarian tissue oocytes from transgender men reveals poor calcium release and embryo development, which might be overcome by spindle transfer.

STUDY QUESTION: Can spindle transfer (ST) overcome inferior embryonic development of in vitro matured ovarian tissue oocytes (OTO-IVM) originating from testosterone-treated transgender men? SUMMARY ANSWER: ST shows some potential to overcome the embryo developmental arrest observed in OTO-IVM oocytes from transgender men. WHAT IS KNOWN ALREADY: OTO-IVM is being applied as a complementary approach to increase the number of oocytes/embryos available for fertility preservation during ovarian tissue cryopreservation in cancer patients. OTO-IVM has also been proposed for transgender men, although the potential of their oocytes remains poorly investigated. Currently, only one study has examined the ability of OTO-IVM oocytes originating from transgender men to support embryo development, and that study has shown that they exhibit poor potential. STUDY DESIGN, SIZE, DURATION: Both ovaries from 18 transgender men undergoing oophorectomy were collected for the purposes of this study, from November 2020 to September 2022. The patients did not wish to cryopreserve their tissue for fertility preservation and donated their ovaries for research. All patients were having testosterone treatment at the time of oophorectomy and some of them were also having menses inhibition treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sibling ovaries were collected in either cold or warm medium, to identify the most optimal collection temperature. Cumulus oocyte complexes (COCs) from each condition were isolated from the ovarian tissue and matured in vitro for 48 h. The quality of OTO-IVM oocytes was assessed by calcium pattern releasing ability, embryo developmental competence following ICSI, and staining for mitochondrial membrane potential. In vitro matured metaphase I (MI) oocytes, germinal vesicle (GV) oocytes, and in vivo matured oocytes with aggregates of smooth endoplasmic reticulum (SERa) were donated from ovarian stimulated women undergoing infertility treatment and these served as Control oocytes for the study groups. ST was applied to overcome poor oocyte quality. Specifically, enucleated mature Control oocytes served as cytoplasmic recipients of the OTO-IVM spindles from the transgender men. Embryos derived from the different groups were scored and analysed by shallow whole genome sequencing for copy number variations (CNVs). MAIN RESULTS AND THE ROLE OF CHANCE: In total, 331 COCs were collected in the cold condition (OTO-Cold) and 282 were collected in the warm condition (OTO-Warm) from transgender men. The maturation rate was close to 54% for OTO-Cold and 57% for OTO-Warm oocytes. Control oocytes showed a calcium releasing ability of 2.30 AU (n = 39), significantly higher than OTO-Cold (1.47 AU, P = 0.046) oocytes (n = 33) and OTO-Warm (1.03 AU, P = 0.036) oocytes (n = 31); both values of calcium release were similar between the two collection temperatures. Mitochondrial membrane potential did not reveal major differences between Control, OTO-Warm, and OTO-Cold oocytes (P = 0.417). Following ICSI, 59/70 (84.2%) of Control oocytes were fertilized, which was significantly higher compared to 19/47 (40.4%) of OTO-Cold (P < 0.01) and 24/48 (50%) of OTO-Warm oocytes (P < 0.01). In total, 15/59 (25.4%) blastocysts were formed on Day 5 in the Control group, significantly higher than 0/19 (0%) from the OTO-Cold (P = 0.014) and 1/24 (4.1%) in OTO-Warm oocytes (P = 0.026). Application of ST rescued the poor embryo development, by increasing the Day 5 blastocyst rate from 0% (0/19) to 20.6% (6/29) (P = 0.034), similar to that in the ICSI-Control group (25.4%, 15/59). A normal genetic profile was observed in 72.7% (8/11) of OTO-Cold, 72.7% (8/11) of OTO-Warm and 64.7% (11/17) of Control Day 3-Day 5 embryos. After ST was applied for OTO-IVM oocytes, 41.1% (7/17) of the embryos displayed normal genetic patterns, compared to 57.1% (4/7) among ST-Control Day 3-Day 5 embryos. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Due to the limited access to human oocytes and ovarian tissue, our results should be interpreted with some caution, as only a limited number of human oocytes and embryos could be investigated. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study, clearly indicate that OTO-IVM oocytes originating from transgender patients are of inferior quality, which questions their use for fertility preservation. The poor quality is likely to be related to cytoplasmic factors, supported by the increased blastocyst numbers following application of ST. Future research on OTO-IVM from transgender men should focus on the cytoplasmic content of oocytes or supplementation of media with factors that promote cytoplasmic maturation. A more detailed study on the effect of the length of testosterone treatment is also currently missing for more concrete guidelines and guidance on the fertility options of transgender men. Furthermore, our study suggests a potentially beneficial role of experimental ST in overcoming poor embryo development related to cytoplasmic quality. STUDY FUNDING/COMPETING INTEREST(S): A.C. is a holder of FWO grants (1S80220N and 1S80222N). A.B. is a holder of an FWO grant (1298722N). B.H. and A.V.S. have been awarded with a special BOF (Bijzonder Onderzoeksfonds), GOA (Geconcerteerde onderzoeksacties) and 2018000504 (GOA030-18 BOF) funding. B.H. has additional grants from FWO-Vlaanderen (Flemish Fund for Scientific Research, G051516N and G1507816N) and Ghent University Special Research Fund (Bijzonder Onderzoeksfonds, BOF funding (BOF/STA/202109/005)), and has been receiving unrestricted educational funding from Ferring Pharmaceuticals (Aalst, Belgium). The authors declare that they have no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Assisted oocyte activation does not overcome recurrent embryo developmental problems.

STUDY QUESTION: Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)? SUMMARY ANSWER: AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI. WHAT IS KNOWN ALREADY: The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes. STUDY DESIGN, SIZE, DURATION: This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1. MAIN RESULTS AND THE ROLE OF CHANCE: We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure. LIMITATIONS, REASONS FOR CAUTION: Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined. WIDER IMPLICATIONS OF THE FINDINGS: Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research. TRIAL REGISTRATION NUMBER: NCT03354013.

Training residents in minimally invasive surgery; confirming competence or hoping for the best?

BACKGROUND: Veterinary minimally invasive surgery (MIS) is rapidly developing, and most surgeons are performing MIS in their clinical practice. The technical skills of presented surgical techniques are increasingly complex. Required training of American College of Veterinary Surgeons (ACVS) surgical residents in soft tissue MIS (laparoscopy/thoracoscopy) are limited to traditional apprentice training. Unfortunately, such training has been found insufficient to create competent MIS surgeons. AIM OF THE REVIEW: This review discusses development of MIS training for Doctor of Medicine (M.D.) residents in context of veterinary applicability and investigates comparative evidence for how to best train veterinary residents in soft tissue MIS. CONCLUSIONS: A structured curriculum, with validated tasks and clear training goals have been found imperative for training success. Such a curriculum includes both didactic sessions and manual skills training, with video tutorials and reading material to inform and motivate the residents. IMPLICATIONS OF KEY FINDINGS: ACVS residents and diplomates may benefit if a MIS curriculum was developed and made available to all training programs.

TEAD4 regulates trophectoderm differentiation upstream of CDX2 in a GATA3-independent manner in the human preimplantation embryo.

STUDY QUESTION: What is the role of transcriptional-enhanced associate (TEA) domain family member 4 (TEAD4) in trophectoderm (TE) differentiation during human embryo preimplantation development in comparison to mouse? SUMMARY ANSWER: TEAD4 regulates TE lineage differentiation in the human preimplantation embryo acting upstream of caudal-type homeobox protein 2 (CDX2), but in contrast to the mouse in a GATA-binding protein 3 (GATA3)-independent manner. WHAT IS KNOWN ALREADY: Tead4 is one of the earliest transcription factors expressed during mouse embryo preimplantation development and is required for the expression of TE-associated genes. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination, have shown that Tead4-targeted embryos have compromised development and expression of the TE-specific Cdx2 and Gata3 is downregulated. Cdx2 and Gata3 act in parallel pathways downstream of Tead4 to induce successful TE differentiation. Downstream loss of Cdx2 expression, compromises TE differentiation and subsequent blastocoel formation and leads to the ectopic expression of inner cell mass (ICM) genes, including POU Class 5 homeobox 1 (Pou5f1) and SRY-box transcription factor (Sox2). Cdx2 is a more potent regulator of TE fate in mouse as loss of Cdx2 expression induces more severe phenotypes compared with loss of Gata3 expression. The role of TEAD4 and its downstream effectors during human preimplantation embryo development has not been investigated yet. STUDY DESIGN, SIZE, DURATION: The clustered regularly interspaced short palindromic repeats-clustered regularly interspaced short palindromic repeats (CRISPR)-associated genes (CRISPR-Cas9) system was first introduced in pronuclei (PN)-stage mouse zygotes aiming to identify a guide RNA (gRNA), yielding high editing efficiency and effective disruption of the Tead4 locus. Three guides were tested (gRNA1-3), each time targeting a distinct region of Exon 2 of Tead4. The effects of targeting on developmental capacity were studied in Tead4-targeted embryos (n = 164-summarized data from gRNA1-3) and were compared with two control groups; sham-injected embryos (n = 26) and non-injected media-control embryos (n = 51). The editing efficiency was determined by next-generation sequencing (NGS). In total, n = 55 (summarized data from gRNA1-3) targeted mouse embryos were analysed by NGS. Immunofluorescence analysis to confirm successful targeting by gRNA1 was performed in Tead4-targeted embryos, and non-injected media-control embryos. The downregulation of secondary TE-associated markers Cdx2 and Gata3 was used as an indirect confirmation of successful Tead4-targeting (previously shown to be expressed downstream of Tead4). Additional groups of gRNA1 Tead4-targeted (n = 45) and media control (n = 36) embryos were cultured for an extended period of 8.5 days, to further assess the developmental capacity of the Tead4-targeted group to develop beyond implantation stages. Following the mouse investigation, human metaphase-II (MII) oocytes obtained by IVM were microinjected with gRNA-Cas9 during ICSI (n = 74) to target TEAD4 or used as media-control (n = 33). The editing efficiency was successfully assessed in n = 25 TEAD4-targeted human embryos. Finally, immunofluorescence analysis for TEAD4, CDX2, GATA3 and the ICM marker SOX2 was performed in TEAD4-targeted (n = 10) and non-injected media-control embryos (n = 29). PARTICIPANTS/MATERIALS, SETTING, METHODS: A ribonucleoprotein complex consisting of a gRNA-Cas9 mixture, designed to target Exon 2 of Tead4/TEAD4, was microinjected in mouse PN stage zygotes or human IVM MII oocytes along with sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. In mouse, an additional group of Tead4-targeted and media-control embryos was cultured in vitro for an extended period of 8.5 days. Embryonic development and morphology were assessed daily, during culture in vitro of mouse and human embryos and was followed by a detailed scoring at late blastocyst stage. Targeting efficiency following gRNA-Cas9 introduction was assessed via immunostaining and NGS analysis. MAIN RESULTS AND THE ROLE OF CHANCE: NGS analysis of the Tead4-targeted locus revealed very high editing efficiencies for all three guides, with 100% of the mouse embryos (55 out of 55) carrying genetic modifications resulting from CRISPR-Cas9 genome editing. More specifically, 65.22% (15 out 23) of the PN zygotes microinjected with gRNA1-Cas9, which exhibited the highest efficiency, carried exclusively mutated alleles. The developmental capacity of targeted embryos was significantly reduced (data from gRNA1), as 44.17% of the embryos arrested at the morula stage (2.5 days post coitum), coincident with the initiation of TE lineage differentiation, compared with 8.51% in control and 12.50% in sham control groups. High-quality blastocyst formation rates (Grade 3) were 8.97% in the gRNA1-targeted group, compared with 87.23% in the media-control and 87.50% in the sham group. Immunofluorescence analysis in targeted embryos confirmed downregulation of Tead4, Cdx2, and Gata3 expression, which resulted from successful targeting of the Tead4 locus. Tead4-targeted mouse embryos stained positive for the ICM markers Pou5f1 and Sox2, indicating that expression of ICM lineage markers is not affected. Tead4-targeted embryos were able to cavitate and form a blastocoel without being able to hatch. Extended embryo culture following zona pellucida removal, revealed that the targeted embryos can attach and form egg-cylinder-like structures in the absence of trophoblast giant cells. In human embryos, Exon 2 of TEAD4 was successfully targeted by CRISPR-Cas9 (n = 74). In total, 25 embryos from various developmental stages were analysed by NGS and 96.00% (24 out of 25) of the embryos carried genetic modifications because of gRNA-Cas9 editing. In the subgroup of the 24 edited embryos, 17 (70.83%) carried only mutant alleles and 11 out of these 17 (64.70%) carried exclusively frameshift mutations. Six out of 11 embryos reached the blastocyst stage. In contrast to mice, human-targeted embryos formed blastocysts at a rate (25.00%) that did not differ significantly from the control group (23.81%). However, blastocyst morphology and TE quality were significantly compromised following TEAD4-targeting, showing grade C TE scores, with TE containing very few cells. Immunofluorescence analysis of TEAD4-targeted embryos (n = 10) confirmed successful editing by the complete absence of TEAD4 and its downstream TE marker CDX2, but the embryos generated retained expression of GATA3, which is in contrast to what we have observed and has previously been reported in mouse. In this regard, our results indicate that GATA3 acts in parallel with TEAD4/CDX2 towards TE differentiation in human. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: CRISPR-Cas9 germline genome editing, in some cases, induces mosaic genotypes. These genotypes are a result of inefficient and delayed editing, and complicate the phenotypic analysis and developmental assessment of the injected embryos. We cannot exclude the possibility that the observed differences between mouse and human are the result of variable effects triggered by the culture conditions, which were however similar for both mouse and human embryos in this study. Furthermore, this study utilized human oocytes obtained by IVM, which may not fully recapitulate the developmental behaviour of in vivo matured oocytes. WIDER IMPLICATIONS OF THE FINDINGS: Elucidation of the evolutionary conservation of molecular mechanisms that regulate the differentiation and formation of the trophoblast lineage can give us fundamental insights into early implantation failure, which accounts for ∼15% of human conceptions. STUDY FUNDING/COMPETING INTEREST(S): The research was funded by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01) and Ghent University (BOF.BAS.2018.0018.01). G.C. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 11L8822N). A.B. is supported by FWO-Vlaanderen (Flemish fund for scientific research, Grant no. 1298722 N). We further thank Ferring Pharmaceuticals (Aalst, Belgium) for their unrestricted educational grant. The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Ability to Perform Laparoscopic Intra- and Extracorporeal Suture Ligations in a Live Canine Ovariectomy Model after Simulation Training.

Veterinary resident training in minimally invasive surgery is currently inconsistent and depends on innate psychomotor skills. Simulation training has been shown to effectively increase basic skills, but demonstration of simulation training effects on advanced skills in the operating room is sparse. We aimed to determine if simulation-trained novice surgeons were able to perform laparoscopic suture ligation in live dogs. Three novice laparoscopic surgeons underwent a 12-session simulation training program with subsequent laparoscopic skills testing to demonstrate competency. The median skills scores of trainees and of one experienced surgeon were 417 and 472, respectively. Eighteen healthy client-owned (shelter) dogs were operated on by four surgeons: one experienced American College of Veterinary Surgeons (ACVS) diplomate, two novice ACVS residents, and one novice ACVS diplomate. Laparoscopic ovariectomy was performed with suture ligation of the ovarian pedicles. Successful surgery was defined as no evidence of ovarian vessel bleeding after transection of the pedicles. Simulation-trained novices performed successful suture-ligated ovariectomies in 11/13 dogs (85%), and the experienced surgeon in 5/5 (100%) dogs. Median total ligation time was 30 minutes (range: 17-57), which was not different among surgeons (p = .118). Median total surgery time was 105 minutes (range: 69-156) for novices and 89 minutes (range: 65-99) for the experienced surgeon (p = .038). Extensive simulation training including suturing may contribute toward surgery residents being able to perform complex laparoscopic procedures. These results need to be confirmed in larger numbers of trainees.

Complications and outcomes associated with laparoscopic-assisted splenectomy in dogs.

OBJECTIVE: To report the perioperative characteristics and outcomes of dogs undergoing laparoscopic-assisted splenectomy (LAS). ANIMALS: 136 client-owned dogs. PROCEDURES: Multicentric retrospective study. Medical records of dogs undergoing LAS for treatment of naturally occurring splenic disease from January 1, 2014, to July 31, 2020, were reviewed. History, signalment, physical examination and preoperative diagnostic test results, procedural information, complications, duration of hospitalization, histopathologic diagnosis, and perioperative outcomes were recorded. Perioperative complications were defined using the Veterinary Cooperative Oncology Group - Common Terminology Criteria for Adverse Events (VCOG-CTCAE v2) guidelines. RESULTS: LAS was performed for treatment of a splenic mass (124/136 [91%]), immune-mediated disease (7/136 [5%]), splenomegaly (4/136 [3%]), or immune-mediated disease in conjunction with a splenic mass (1/136 [1%]). Median splenic mass size was 1.3 cm3/kg body weight. Conversion to open laparotomy occurred in 5.9% (8/136) of dogs. Complications occurred in 78 dogs, with all being grade 2 or lower. Median surgical time was 47 minutes, and median postoperative hospital stay was 28 hours. All but 1 dog survived to discharge, the exception being postoperative death due to a suspected portal vein thrombus. CLINICAL RELEVANCE: In the dogs of this report, LAS was associated with low rates of major complications, morbidity, and mortality when performed for a variety of splenic pathologies. Minimally invasive surgeons can consider the LAS technique to perform total splenectomy in dogs without hemoabdomen and with spleens with modest-sized splenic masses up to 55.2 cm3/kg, with minimal rates of complications, morbidity, and mortality.

CRISPR/Cas gene editing in the human germline.

The ease and efficacy of CRISPR/Cas9 germline gene editing in animal models paved the way to human germline gene editing (HGGE), by which permanent changes can be introduced into the embryo. Distinct genes can be knocked out to examine their function during embryonic development. Alternatively, specific sequences can be introduced which can be applied to correct disease-causing mutations. To date, it has been shown that the success of HGGE is dependent on various experimental parameters and that various hurdles (i.e. loss-of-heterozygosity and mosaicism) need to be overcome before clinical applications should be considered. Due to the shortage of human germline material and the ethical constraints concerning HGGE, alternative models such as stem cells have been evaluated as well, in terms of their predictive value on the genetic outcome for HGGE approaches. This review will give an overview of the state of the art of HGGE in oocytes and embryos, and its accompanying challenges.

SPERM FACTORS AND EGG ACTIVATION: Fertilization failure after human ICSI and the clinical potential of PLCZ1.

Two decades have passed since the discovery of phospholipase C zeta (PLCZ1) as the sperm oocyte-activating factor. At present, there is a general consensus that PLCZ1 is responsible for triggering the calcium (Ca2+) oscillations necessary to start the oocyte activation process in mammals. One proof is that abnormal, reduced, or absent PLCZ1 in human spermatozoa leads to fertilization failure (FF) after intracytoplasmic sperm injection (ICSI). ICSI is the most effective assisted reproduction technique and enables overcoming almost all male infertility conditions. Despite fertilization rates of up to 80%, FF does occur in 1-3% of ICSI cycles, which leaves these patients with few options for obtaining genetically related offspring. Assisted oocyte activation (AOA) using Ca+2 ionophores has emerged as a useful treatment option for these patients. While AOA has been proven very beneficial for the treatment of sperm-related FF, some cases of female-related FF cannot be overcome by AOA. Therefore, the development of appropriate diagnostic tests that predict the prognosis of AOA treatment would be advantageous to improve the clinical management of these patients and shorten the time to pregnancy. The aim of this review is to provide an up-to-date overview of the genetic causes of FF after ICSI and to discuss the advantages and disadvantages of using PLCZ1 as a diagnostic marker or therapeutic molecule in comparison with currently available diagnostic tests and treatments.

The ASAS-OMERACT core domain set for axial spondyloarthritis.

BACKGROUND: The current core outcome set for ankylosing spondylitis (AS) has had only minor adaptations since its development 20 years ago. Considering the significant advances in this field during the preceding decades, an update of this core set is necessary. OBJECTIVE: To update the ASAS-OMERACT core outcome set for AS into the ASAS-OMERACT core outcome set for axial spondyloarthritis (axSpA). METHODS: Following OMERACT and COMET guidelines, an international working group representing key stakeholders (patients, rheumatologists, health professionals, pharmaceutical industry and drug regulatory agency representatives) defined the core domain set for axSpA. The development process consisted of: i) Identifying candidate domains using a systematic literature review and qualitative studies; ii) Selection of the most relevant domains for different stakeholders through a 3-round Delphi survey involving axSpA patients and axSpA experts; iii) Consensus and voting by ASAS; iv) Endorsement by OMERACT. Two scenarios are considered based on the type of therapy investigated in the trial: symptom modifying therapies and disease modifying therapies. RESULTS: The updated core outcome set for axSpA includes 7 mandatory domains for all trials (disease activity, pain, morning stiffness, fatigue, physical function, overall functioning and health, and adverse events including death). There are 3 additional domains (extra-musculoskeletal manifestations, peripheral manifestations and structural damage) that are mandatory for disease modifying therapies and important but optional for symptom modifying therapies. Finally, 3 other domains (spinal mobility, sleep, and work and employment) are defined as important but optional domains for all trials. CONCLUSION: The ASAS-OMERACT core domain set for AS has been updated into the ASAS-OMERACT core domain set for axSpA. The next step is the selection of instruments for each domain.

Laparoscopic esophagopexy, fundopexy, and hiatal herniorrhaphy for refractory regurgitation in a racing Alaskan husky sled dog.

A 2-year-old intact female Alaskan husky sled dog was presented with a history of chronic exercise-induced regurgitation refractory to medical management. Previous diagnostics were unremarkable except for an endoscopic examination and histopathologic evaluation of the upper gastrointestinal tract that revealed the presence of Helicobacter spp. and mild non-specific inflammation of the proximal duodenum. A laparoscopic hiatal herniorrhaphy, esophagopexy, fundopexy, and ovariectomy were performed without complications in anesthesia or surgery and clinical improvement was observed with continued follow-up for 8 months after surgery. Key clinical message: Surgical treatment for hiatal hernia may be considered in racing Alaskan sled dogs with regurgitation refractory to gastric protectant therapy.

Oesophagopexie laparoscopique, fundopexie et herniorraphie hiatale pour régurgitation réfractaire chez un chien de course de traîneau husky de l'Alaska. Une chienne de traîneau husky de l'Alaska, âgée de 2 ans, a présenté des antécédents de régurgitation chronique induite par l'effort réfractaire à la prise en charge médicale. Les diagnostics antérieurs n'étaient pas remarquables, sauf pour un examen endoscopique et une évaluation histopathologique du tractus gastro-intestinal supérieur qui a révélé la présence d'Helicobacter spp. et une légère inflammation non spécifique du duodénum proximal. Une herniorraphie hiatale laparoscopique, une oesophagopexie, une fundopexie et une ovariectomie ont été réalisées sans complications sous anesthésie ou en chirurgie et une amélioration clinique a été observée avec un suivi continu pendant 8 mois après la chirurgie.Message clinique clé :Un traitement chirurgical de la hernie hiatale peut être envisagé chez les chiens de traîneau de course de l'Alaska présentant une régurgitation réfractaire au traitement protecteur gastrique.(Traduit par Dr Serge Messier).

Effects of head position on internal and external carotid pressures in standing sedated horses.

The effects of head position on internal carotid artery (ICA) and external carotid artery (ECA) pressures in standing sedated horses were evaluated in this study. The common carotid artery (CCA) was catheterized in 6 horses using an ultrasound-guided technique to facilitate placement of a pressure transducer within the ICA and ECA at the level of the guttural pouch. Transducer position was confirmed by endoscopic visualization. Mean arterial pressure (MAP) was measured with horses in both a head-up and head-down position. The dorsal metatarsal artery was catheterized as a control. Maintaining a head-up position decreased MAP in both the ICA (median: 75.21 mmHg) and ECA (median: 79.43 mmHg), relative to the head-down position (ICA median: 104.65 mmHg; ECA median: 102.26 mmHg). Mean arterial pressure in the dorsal metatarsal artery was not affected by head position. The head-up position resulted in lower arterial pressures in both the ICA and ECA (P = 0.03) compared with the head-down position in standing sedated horses.

Cette étude a évalué les effets de la position de la tête sur la pression artérielle au niveau de l'artère carotide interne (ICA) et de l'artère carotide externe (ECA) chez des chevaux sous sedation debout. L'artère carotide commune (CCA) a été cathétérisée chez six chevaux en utilisant une technique échoguidée pour faciliter le placement d'un transducteur de pression dans l'ICA et l'ECA au niveau de la poche gutturale. La position du transducteur a été confirmée par endoscopie. La pression artérielle moyenne (MAP) a été mesurée chez les chevaux avec la tête en position haute et en position basse. L'artère métatarsienne dorsale a été cathétérisée et a servi comme témoin. Les MAP enregistrées au niveau de l'ICA (médiane: 75,21 mmHg) et de l'ECA (médiane: 79,43 mmHg) lorsque la tête est en position élevée sont plus faibles que celles enregistrées lorsque la tête est en position basse (médiane ICA: 104,65 mmHg; médiane ECA: 102,26 mmHg). La MAP de l'artère métatarsienne dorsale n'a pas été affectée par la position de la tête. En conclusion, chez les chevaux sédatés et debout, la position élevée de la tête produit des pressions artérielles plus faibles au niveau de ICA et ECA (P = 0,03) que celles obtenues lorsque la tête est en position basse.(Traduit par les auteurs).