Robotic Precisely Oocyte Blind Enucleation Method

Oocyte enucleation is a critical procedure for somatic cell nuclear transfer. Yet, the main steps of oocyte enucleation are still manually operated, which presents several drawbacks such as low precision, high repetition error, and long training time for operators. For improving the operation efficiency and success rate, a robotic precise oocyte blind enucleation method is presented in this paper. The proposed method involves the following key techniques: oocyte translation control, oocyte immobilization and penetration control, and enucleation volume control based on the adaptive slide mode. Compared with the manual blind enucleation method, the proposed robotic blind enucleation method reduced the operation time by 44.5% (manual method: 62 s vs. proposed method: 34.4 s), increased the accuracy of enucleation by 83.1% (manual method: 30.7 vs. proposed method: 5.2), increased the success rate from 80% to 93.3%, and increased the cleavage rate from 41.7% to 63.3%.

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Prognostic Factors for the Outcome of Salivary Gland Holmium: YAG Laser Intraductal Lithotripsy

Ear Nose & Throat Journal ◽

10.1177/01455613211010075 ◽

2021 ◽

pp. 014556132110100

Author(s):

Shuo-Jen Wang ◽

Lung-Che Chen ◽

Yi-Chih Lin ◽

Yen-Chun Chen ◽

Luong Huu Dang ◽

...

Keyword(s):

Logistic Regression ◽

Salivary Gland ◽

Success Rate ◽

Operative Time ◽

Tertiary Care ◽

Operation Time ◽

Laser Lithotripsy ◽

Successful Outcome ◽

Care Hospital ◽

Yag Laser

Objectives: Holmium: YAG laser has gained its popularity throughout the years and is used to treat sialolithiasis, which helps to overcome the limitations of traditional sialendoscopic lithotripsy for larger-sized salivary stones. However, little information is available regarding factors predicting the success rate of Holmium: YAG laser intraductal lithotripsy. The purpose of this study is to investigate the factors affecting the success rates of Holmium: YAG laser lithotripsy for salivary stones treatment in a tertiary care hospital. Methods: A retrospective study conducted in patients receiving sialolithiasis surgery under sialendoscopy from May 2013 to March 2015 at Mackay Memorial Hospital, Taiwan. Data on various factors, including patients’ age, gender, glands, size of largest stone, multiple stones (≥2 stones), location of the stone (distal duct, middle duct, proximal duct, and hilum), and operative time. The success of the surgery defined as patients without any complaints such as swelling or tenderness. Logistic regression and Fisher exact tests were employed to examine these factors on the success rate. Results: Fifty-four patients who received sialendoscopy surgery with a mean age of 35.74 years old recruited. Logistic regression identified the operation time exceeding 210 minutes showed 23.497 folds higher odd ratio of having a result of operation failure ( P < .05). Conclusion: The prolonged operation time is the sole independent factor affecting the successful outcome for salivary gland intraductal laser lithotripsy. We recommend operative time be no more than 210 minutes to increase the success rate in salivary gland Holmium: YAG laser intraductal lithotripsy.

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64COMPARISON OF DEVELOPMENTAL POTENTIAL OF IN VIVO AND IN VITRO RECIPIENT OOCYTES AFTER NUCLEAR TRANSFER IN GOAT

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab64 ◽

2004 ◽

Vol 16 (2) ◽

pp. 154

Author(s):

H.S. Park ◽

M.Y. Lee ◽

S.P. Hong ◽

J.I. Jin ◽

J.K. Park ◽

...

Keyword(s):

Nuclear Transfer ◽

Electric Pulse ◽

Serum Starvation ◽

Cleavage Rate ◽

Developmental Potential ◽

In Vitro Development ◽

Significant Difference ◽

Vitro Development

Recent techniques in somatic cell nuclear transfer (SCNT) have been widely used for animal research. In addition, SCNT techniques may allow for the rescue of endangered species. Despite efforts for wildlife preservation, however, some threatened or endangered wild animal species will likely become extinct. As a preliminary experiment of a series in wildlife research, we tried to identify an improved method for the production of more transferable NT embryos in goats. Mature donor animals of Korean native goats (20–25kg) were synchronized with a CIDR (type G; InterAg, New Zealand) vaginal implant for 10 days followed by a total of 8 twice daily injections of 70mg of FSH (Folltropine, London, Ontario, Canada) and 400IU of hCG (Chorulon, Intervet, Moxmeer, The Netherlands). Oocytes were then collected surgically by retograde oviduct flush or direct aspiration from ovarian follicles in vivo at 29–34h after hCG. Oocytes collected from follicles were matured in TCM-199 containing 10% FBS and hormones. Prepared ear skin cells from the goat were cultured in TCM-199 containing 10% FBS at 39°C, 5% CO2 in air, and confluent monolayers were obtained. Oocytes were enucleated and donor cells from serum starvation (0.5%) culture were fused through a single electric pulse (DC 2.36kvcm−1, 17μs), and then activated by a single electric pulse (AC 5vmm−1, 5s+DC 1.56kvcm−1, 30μs) or chemical treatment (5μgmL−1 ionomycin 5min−1, 1.9mM 6-DMAP/4h). Reconstructed oocytes were cultured in M16 medium with 10% goat serum (GS) for 6–7 days. Data were analyzed by chi-square test. In in vitro development, significantly (P<0.05) more oocytes were cleaved (24/30, 80.0%) and developed (7/24, 29.2%) to morula or blastocyst stage, respectively, in NT oocytes activated by Iono + DMAP compared to electric stimulated oocytes (2/21, 40.0%; 0/2, 0%). There was a significant difference in in vitro development of NT embryos by the method of oocyte collection. Cleavage rate was higher (P<0.05) in NT embryos from in vivo oocytes (23/28, 82.1%) than in in vitro matured oocytes (19/35, 54.3%), and further development to morula or blastocyst was also significantly (P<0.05%) higher in NT embryos from in vivo oocytes (7/23, 30.4%) than in NT embryos from in vitro matured oocytes (0/19, 0%). When we compared NT embryos to parthenotes, developmental rate was not significantly different between NT embryos and parthenotes. These results strongly suggest that the in vivo oocytes will have superior developmental potential to oocytes matured in vitro. Table 1 Effect of different oocyte source on in vitro development following caprine SCNT

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Regrowth of the adenoids after adenoidectomy down to the pharyngobasilar fascial surface

The Journal of Laryngology & Otology ◽

10.1017/s0022215115001437 ◽

2015 ◽

Vol 129 (7) ◽

pp. 662-665 ◽

Cited By ~ 5

Author(s):

L Wei ◽

M Wang ◽

N Hua ◽

K Tong ◽

L Zhai ◽

...

Keyword(s):

Surgical Technique ◽

Blood Loss ◽

Success Rate ◽

Operation Time ◽

Early Complications ◽

Estimated Blood Loss ◽

Endoscopic Guidance ◽

Pharyngobasilar Fascia

AbstractObjectives:This study aimed to explore adenoid regrowth after transoral power-assisted adenoidectomy down to the pharyngobasilar fascial surface.Methods:Transoral adenoidectomy down to the pharyngobasilar fascia surface was performed on 39 patients under endoscopic guidance, using a power-assisted system. The operation time, amount of blood loss and iatrogenic injury, presence of complications, and success and regrowth rates were recorded to assess the feasibility, safety and effectiveness of our surgical technique.Results:In this adenoidectomy procedure, the pharyngobasilar fascia was left intact. The estimated blood loss was 5–50 ml (mean 15 ml), and the success rate was 97.3 per cent. Early complications occurred in 2.3 per cent of patients, while no long-term complications occurred in the cohort. No regrowth was found in the follow-up assessments, which were performed for 18–36 months after surgery.Conclusion:Adenoid regrowth was rare after adenoidectomy down to the pharyngobasilar fascial surface. The pharyngobasilar fascia can therefore be considered a surgical boundary for adenoidectomy.

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Effects of cycloheximide treatment and interval between fusion and activation on in vitro development of pig nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd01114 ◽

2002 ◽

Vol 14 (4) ◽

pp. 191 ◽

Cited By ~ 10

Author(s):

M. A. Martinez-Diaz ◽

K. Ikeda ◽

Y. Takahashi

Keyword(s):

Nuclear Transfer ◽

Kinase Activity ◽

Short Interval ◽

Cumulus Cells ◽

Cleavage Rate ◽

In Vitro Development ◽

M Phase ◽

Cytostatic Factor ◽

Vitro Development

The effects of cycloheximide (CHX) treatment and the interval between fusion and activation on the development of pig nuclear transfer (NT) embryos constructed with enucleated oocytes and serum-starved granulosa/cumulus cells were examined. One group of couplets was fused and activated simultaneously (FAS) by a single electrical pulse (activation pulse). Another three groups of couplets were fused electricaly 1.5, 2.5 or 4.5 h before being subjected to the activation pulse (FBA). Each group was divided into two subgroups and incubated with or without CHX. The NT embryos treated with CHX showed a high and stable cleavage rate, regardless of the interval between fusion and activation; however, development to blastocysts was improved only when the NT embryos were subjected to FAS with CHX. These results indicate that CHX-sensitive events occurring shortly after FAS may be responsible for the development to blastocysts. Fusion pulse rarely activated M II oocytes, but rapidly dropped the p34cdc2 kinase activity in NT embryos. A pronucleus-like structure was observed 2-2.5 h after the activation pulse with CHX in NT embryos of both the FAS and FBA groups. Therefore, successive inactivation of M-phase promoting factor and cytostatic factor at a certain short interval may also play an important role in the development of NT embryos.

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18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab18 ◽

2014 ◽

Vol 26 (1) ◽

pp. 123

Author(s):

Y. Liu ◽

A. Lucas-Hahn ◽

B. Petersen ◽

R. Li ◽

P. Hassel ◽

...

Keyword(s):

In Vitro Culture ◽

Nuclear Transfer ◽

Cell Number ◽

Culture Conditions ◽

Developmental Competence ◽

Cleavage Rate ◽

Cell Numbers ◽

Blastocyst Rate ◽

Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

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44 IN VITRO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS GENERATED WITH BOVINE OOCYTES AND EQUINE SKIN FIBROBLASTS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab44 ◽

2011 ◽

Vol 23 (1) ◽

pp. 128

Author(s):

J. Lee ◽

J. Park ◽

Y. Chun ◽

W. Lee ◽

K. Song

Keyword(s):

Nuclear Transfer ◽

Polar Body ◽

Donor Cell ◽

Skin Fibroblasts ◽

Developmental Competence ◽

Cleavage Rate ◽

Cell Stage ◽

Bovine Oocytes

Study for equine somatic cell nuclear transfer (SCNT) is an attractive field for research, but it has not been a major field of study because it is hard to obtain a sufficient number of ovaries and it takes a lot of time and effort for the recovery of oocytes matured in vivo by ovum pickup. It was reported that the bovine cytoplast could support the remodelling of equine donor cells (Zhou et al. 2007 Reprod. Domest. Anim. 42, 243–247). The objectives of this study are 1) to monitor the early events of equine SCNT by interspecies SCNT (isSCNT) between bovine cytoplast and equine donor cell, and 2) to investigate the developmental competence of isSCNT embryos. Bovine oocytes were recovered from the follicles of slaughtered ovaries, and matured in TCM-199 supplemented with 10 mU mL–1 FSH, 50 ng mL–1 EGF, and 10% FBS at 39°C under 5% CO2 in air for 22 h. Fibroblasts derived from bovine or equine skin tissues were synchronized at G0/G1 stage by contact inhibition for 72 h. After IVM, oocytes with polar body were enucleated and electrically fused with equine or bovine skin fibroblasts (1.0 kV cm–1, 20 μs, 2 pulses). Fused couplets were activated with 5 μM ionomycin for 4 min followed by 5 h culture in 10 μg mL–1 cycloheximide (CHX) and/or 2 mM 6-DMAP, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C under 5% CO2, 5% O2, and 90% N2 for 7 days. All analyses were performed using SAS (version 9.1; SAS Institute, Cary, NC, USA). The cleavage rate of isSCNT embryos derived from equine cell was not different (252/323, 78.7%; P = 0.94) from that of SCNT embryos derived from bovine cell (230/297, 79.2%). However, the rate of isSCNT embryos developed to over 8-cell stage was lower (3.3%; P < 0.0001) than that of bovine SCNT embryos (39.4%), and total cell number of isSCNT embryos developed to over 8-cell stage was lower (17.5, n = 12; P < 0.0001) than that (80.8, n = 110) of bovine SCNT embryos. Also, the rate of blastocyst formation of isSCNT embryos (0/323; 0.0%) was lower (P < 0.0001) than that of bovine SCNT embryos (83/297; 29.3%). Meanwhile, reconstructed oocytes for isSCNT were fixed at 8 h after activation to investigate the formation of pseudo-pronucleus (PPN) after post-activation treatment with CHX or CHX+6-DMAP. The ratio of oocytes with single PPN after treatment with CHX+6-DMAP (26/35; 74.3%) was not different (P = 0.63) from that of oocytes treated with CHX (24/36; 68.1%). Although isSCNT embryos derived from bovine cytoplast and equine donor cell could not develop to more than the 16-cell stage, it is believed that the results of this isSCNT study could be used for the preliminary data regarding the reprogramming of donor cell in equine SCNT.

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A Comparative Study of Endoscopic versus Microscopic Cartilage Type I Tympanoplasty

International Archives of Otorhinolaryngology ◽

10.1055/s-0039-1693139 ◽

2019 ◽

Vol 24 (01) ◽

pp. e80-e85 ◽

Cited By ~ 3

Author(s):

Dipesh Shakya ◽

Arun KC ◽

Ajit Nepal

Keyword(s):

Comparative Study ◽

Success Rate ◽

Demographic Data ◽

Operation Time ◽

Type I ◽

Success Rates ◽

Type I Tympanoplasty ◽

Graft Success ◽

Retrospective Comparative Study ◽

Endoscopic Tympanoplasty

Abstract Introduction The use of endoscope is rapidly increasing in otological and neuro-otological surgery in the last 2 decades. Middle ear surgeries, including tympanoplasty, have increasingly utilized endoscopes as an adjunct to or as a replacement for the operative microscope. Superior visualization and transcanal access to diseases normally managed with a transmastoid approach are touted as advantages with the endoscope. Objectives The present study aimed to compare the outcomes of endoscopic and microscopic cartilage tympanoplasty (Type I) Methods This was a retrospective comparative study of 70 patients (25 males and 45 females) who underwent type I tympanoplasty between March 2015 and April 2016. The subjects were classified into 2 groups: endoscopic tympanoplasty (ET, n = 35), and microscopic tympanoplasty (MT, n = 35). Tragal cartilage was used as a graft and technique used was cartilage shield tympanoplasty in both groups. Demographic data, perforation size of the tympanic membrane at the preoperative state, operation time, hearing outcome, and graft success rate were evaluated. Results The epidemiological profiles, the preoperative hearing status, and the perforation size were similar in both groups. The mean operation time of the MT group (52.63 ± 8.68 minutes) was longer than that of the ET group (48.20 ± 10.37 minutes), but the difference was not statistically significant. The graft success rates 12 weeks postoperatively were 91.42% both in the ET and MT groups, that is, 32/35; and these values were not statistically significantly different. There was a statistically significant improvement in hearing within the groups, both pre- and postoperatively, but there was no difference between the groups. Conclusion Endoscopic tympanoplasty is a minimally invasive surgery with similar graft success rate, comparable hearing outcomes and shorter operative time period as compared to microscopic use.

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Somatic cell nuclear transfer using transported in vitro-matured oocytes in cynomolgus monkey

Zygote ◽

10.1017/s0967199406003947 ◽

2007 ◽

Vol 15 (1) ◽

pp. 25-33 ◽

Cited By ~ 4

Author(s):

N. Chen ◽

S-L. Liow ◽

R. Bin Abdullah ◽

WK. Khadijah Wan Embong ◽

W-Y. Yip ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Donor Cell ◽

Oocyte Retrieval ◽

Development Rate ◽

Cleavage Rate ◽

Immature Oocytes ◽

Polarized Microscopy

SUMMARYSomatic cell nuclear transfer (SCNT) is not successful so far in non-human primates. The objective of this study was to investigate the effects of stimulation cycles (first and repeat) on oocyte retrieval and in vitro maturation (IVM) and to evaluate the effects of stimulation cycles and donor cell type (cumulus and fetal skin fibroblasts) on efficiency of SCNT with transported IVM oocytes. In this study, 369 immature oocytes were collected laparoscopically at 24 h following human chorionic gonadotrophin (hCG) treatment from 12 cynomolgus macaque (Macaca fascicularis) in 24 stimulation cycles, and shipped in pre-equilibrated IVM medium for a 5 h journey, placed in a dry portable incubator (37 °C) without CO2 supplement. A total of 70.6% (247/350) of immature oocytes reached metaphase II (MII) stage at 36 h after hCG administration, MII spindle could be seen clearly in 80.6% (104/129) of matured IVM oocytes under polarized microscopy. A total of 50.0% (37/74) of reconstructive SCNT embryos cleaved after activation; after cleavage, 37.8% (14/37) developed to the 8-cell stage and 8.1% (3/37) developed to morula, but unfortunately none developed to the blastocyst stage. Many more oocytes could be retrieved per cycle from monkeys in the first cycle than in repeated cycles (19.1 vs. 11.7, p < 0.05). There were no significant differences in the maturation rate (70.0 vs. 71.4%, p > 0.05) and MII spindle rate under polarized microscopy (76.4 vs. 86.0%, p > 0.05) between the first and repeat cycles. There were also no significant differences in the cleavage rate, and the 4-cell, 8-cell and morula development rate of SCNT embryos between the first and repeat cycles. When fibroblast cells and cumulus cells were used as the donor cells for SCNT, first cleavage rate was not significantly different, but 4-cell (50.0 vs. 88.9%, p < 0.05) and 8-cell (0 vs. 51.9%, p < 0.01) development rate were significantly lower for the former. In conclusion, the number of stimulation cycles has a significant effect on oocyte retrieval, but has no effect on maturation and SCNT embryo development; however, different donor cell types (cumulus and fibroblast) resulted in different developmental potentials of SCNT embryos.

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Medical Diagnosis of Upper Urinary Calculi Based on Image Filtering in Ureteroscopic Lithotripsy

Journal of Medical Imaging and Health Informatics ◽

10.1166/jmihi.2019.2615 ◽

2019 ◽

Vol 9 (2) ◽

pp. 376-383

Author(s):

Qiyi Hu ◽

Hui Liang ◽

Zhu Wang ◽

Qiong Deng ◽

Jianwen Zhang ◽

...

Keyword(s):

Success Rate ◽

Clearance Rate ◽

Urinary Calculi ◽

Operation Time ◽

Ureteral Stent ◽

Occurrence Rate ◽

Post Surgery ◽

Flexible Ureteroscope ◽

Group A ◽

Group B

Objective: Based on improved image processing techniques, we investigate the therapeutic effect and safety of a flexible ureteroscope applied in treatment of upper urinary calculi with and without preoperative ureteral stent, and analyzed their outcome based on CT and ultrasound medical imaging technology, as well as other clinical data. Methods: These identified patients were randomly divided into two groups, 89 patients suffering from upper urinary calculi in Group A were treated with flexible ureteroscopy lithotripsy and Holmium laser, without preoperative ureteral stent. On the other hand, another 114 patients in Group B received preoperative ureteral stent. In terms of measurement indicators, the total operation time, hospitalization time, hospital costs, success rate of operation, clearance rate of stone, occurrence rate of complications, and the probability of a second operation were recorded, respectively. In order to verify the effect of the operation, we performed Computed Tomography (CT), Nuclear Magnetic Resonance Imaging (MRI), Ultrasound Examination on the patient before and after the operation, and processed the image by downhill filtering algorithm and adaptive median filtering to ensure the validity and accuracy of the experiment. Results: By analyzing other clinical data such as medical images, it is known when making comparisons between two groups, there were no significant difference in terms of stone size, stone site, and success rate of operation (97.8%, 87/89 for group A; 100%, 114/144 for group B). In further, the operation time of group A was longer than that of group B (65.46 ± 10.17 min vs. 59.58 ± 9.25 min, P < 0 05), and the hospitalization time in group A was much shorter (6.53 ± 0.89 d vs. 10.16 ± 1.50 d, P < 0.01). The clearance rate of stone at 3-day post-surgery in group A was much lower than that of group B (36%, 32/89 vs. 51.7%, 59/114, P < 0.05). In addition, the clearance rate of stone at 1-month post-surgery (84.3%, 75/89 in group A vs. 85.1%, 97/114 in group B), and the occurrence rate of complications (21.3%, 19/89 in group A vs. 20.1%, 23/114 in group B) showed no significantly difference (P > 0.05). Besides, the probabilities of a second operation in group A and B were 14.6% (19/89) and 20.1% (23/114), respectively. And no significantly difference could be drawn according to statistical analysis. Conclusion: The application of flexible ureteroscope applied in treatment of upper urinary calculi without preoperative ureteral stent was economical, safe and effective.

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