Renal aluminium handling in the rat: a micropuncture assessment

2004 ◽  
Vol 107 (2) ◽  
pp. 159-165 ◽  
Author(s):  
David G. SHIRLEY ◽  
Mary F. WALTER ◽  
Stephen J. WALTER ◽  
Andrew THEWLES ◽  
Christopher J. LOTE
Keyword(s):  
Glomerular Filtration ◽  
Distal Tubule ◽  
High Dose ◽  
Aluminium Content ◽  
Filtration System ◽  
Significant Difference ◽  
Glomerular Filtrate ◽  
Aluminium Citrate ◽  
Distal Site

Uncertainties exist over the glomerular filtration of aluminium and virtually nothing is known about its segmental handling along the nephron. The present study has used micropuncture, combined with electrothermal atomic absorption spectroscopy, to determine directly the aluminium content of glomerular filtrate and of late PCTs (proximal convoluted tubules) and early distal tubules in anaesthetized Munich–Wistar rats infused with three different doses of aluminium citrate (plasma aluminium concentrations, 2.9±0.1, 5.2±0.4 and 10.0±0.9 μg·ml−1 respectively). Aluminium filtration into Bowman's space was found to be considerably greater than that predicted by an in vitro filtration system: in all three groups it was essentially filtered freely. No significant aluminium reabsorption took place along the PCT, but with every dose the FDAl (fractional delivery of aluminium; tubular fluid:plasma aluminium/inulin concentration ratio) was lower at the early distal site than at the late PCT (P<0.001 in each case), indicating net aluminium reabsorption in the loop of Henle. This reabsorption amounted to 19–26% of the filtered aluminium load. In the low- and medium-dose groups, there was no significant difference between FDAl at the early distal site and that in the final urine; however, in the high-dose group, FDAl in the urine (1.02±0.06) exceeded that at the early distal tubule (0.75±0.04; P<0.001), suggesting aluminium secretion in the distal nephron. The results indicate that aluminium loads, when complexed with citrate, are excreted efficiently owing to a combination of glomerular filtration and minimal reabsorption.

scholarly journals Dose dependence of efficacy but not of safety in sublingual immunotherapy

2016 ◽  
Vol 65 (1) ◽  
Author(s):  
F. Frati ◽  
C. Incorvaia ◽  
F. Marcucci ◽  
L. Sensi ◽  
G. Di Cara ◽  
...  
Keyword(s):  
Sublingual Immunotherapy ◽  
Clinical Symptoms ◽  
Meta Analysis ◽  
Dose Dependence ◽  
Subcutaneous Immunotherapy ◽  
High Dose ◽  
Systemic Reactions ◽  
Significant Difference

Sublingual immunotherapy (SLIT) currently represents, as indicated by meta-analysis of its efficacy and safety, a valid option to the generally used traditional subcutaneous immunotherapy (SCIT) for treating respiratory allergy. Regarding efficacy, recent studies demonstrated that, similar to what has already been observed in SCIT as well as in experimental and clinical studies about the magnitudo of allergen exposure, the effectiveness on both clinical symptoms and immunologic changes depends on the amount of allergen administered during treatment. In addition, in vitro studies addressed with the role of dendritic cells, currently considered to be of pivotal importance in orienting toward tolerance the immune response to allergens, showed that the internalisation of allergen molecules, which is followed by tolerogenic presentation to T cells, depends on the amount of allergen. However, such dose dependence is not apparent concerning the safety. In fact, the comparison of studies respectively conducted with high and low allergen doses did not show differences in the rate of systemic reactions, which in any case never had the presentation of anaphylaxis, and instead a significant difference in the rate of local reactions, following the oral and gastrointestinal contact with the allergen extract, in favour of high dose studies.

A Experimental Study and Clinical Result of Prophylaxis aGVHD with Humanized Anti-CD25 Monoclonal Antibody in Haploidentical BMT.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1251-1251
Author(s):  
Shu-Quan Ji ◽  
Hui-Ren Chen ◽  
Heng-Xiang Wang ◽  
Hong-Min Yan ◽  
Mei Xue ◽  
...  
Keyword(s):  
Median Time ◽  
Conditioning Regimen ◽  
Control Group ◽  
High Dose ◽  
Marrow Graft ◽  
Hla Antigen ◽  
Hematopoietic Stem ◽  
Gvhd Prophylaxis ◽  
Significant Difference

Abstract Between February 1999 and March 2004, eighty-seven patients with high risk leukemia, age 3–50 (median 19 year), who needed urgent transplant but no HLA-matched or single HLA-antigen mismatched donors available, received unmanipulated HLA haploidentical BMT. The 87 patients were classified as follows AML 27 (CR1 in 7, CR2 in 15 and 5 in relapse), All 38 (CR1 in 4, CR2 in 30 and 4 in relapse) , CML 22 ( 4 in CP, 12 in AP and 6 in BP). All donors were HLA-haploidentical relatives who had at least two major histocompatibility complex antigen mismatched with the recipients. 87 patients underwent haplo-BMT with G-CSF primed BM as stem cells. All patients received a same conditioning regimen including high dose Ara-C, Cyclophosphamide, antithymocyte globulin and total body irradiation to provide both immunosuppression and myeloablation. GVHD prophylaxis consisted of anti-thymocyte globulin, cyclosporin A, methotrexate and mycofenolate mofitel. 72 patients underwent the transplants with the addition of CD25 mAb (Basiliximab Novartis) for GVHD prophylaxis designated as CD25 mAb group. Basiliximab 20mg each by 30min intravenous infusion on 2 hours before transplantion and day 4 after transplantaion. The other 15 patients without Basiliximab for GVHD prophylaxis were as the control group. The two group of patients were comparable in disease status, HLA-disparity and median age of patients. Immunophenotyping, limited dilution assay and colony forming assays were used to measure the effect of Basiliximab on the subsets of lymphocytes, cytotoxic T lymphocyte precursors (CTLp) and hematopoietic cells. All donors were primed with G-CSF at 3-5ug/kg/d for 7 days and the marrow cells were harvested on the eighth day. G-CSF donor priming significantly increased CD34+ and colony forming progenitors in the marrow grafts. More importantly, it significantly reduced lymphocytes and reversed CD4+/CD8+ lymphocyte ratio in the grafts. Both of group who were treated with and without Basiliximab had similar marrow graft contents. All patients established trilineage engraftments.The median time to achieve an absolute neutrophil count 0.5x109/L was 19 days (range, 13 to 24 days). The median time to achieve platelets above 20x109/L was 22 days (range, 16 to 32 days). Between the two groups were no differences in engraftment. Incidence of grades II–IV acute GVHD were 13.9% with GVHD-related deaths 6.9% in Basiliximab group and 33.3% with 20% GVHD-related deaths in control group. There were a significant difference between the anti-CD25 mAb treated Vs non-treated group.Forty-nine patients who survived over 12 months were eligible for the evaluation of cGVHD. 12 patients developed extensive cGVHD, one in control group and eleven in Basiliximab group. 49 were alive in CR during a median follow-up of 30 months (range3–64 months), 42 in Basiliximab group and 7 in control group. Basiliximab significantly decreased alloreactive CTLp by 10–100 fold in limiting dilution assays. It had no effect on hematopoietic stem and progenitor cells as determined by in vitro colony-forming assays.The addition of basiliximab as aGVHD prophylaxis effectively reduced severe lethal aGVHD in haplo-BMT. It is possible to selectively eliminate or reduce the number of alloreactive T cells with anti CD25 antibody, which results in prevention of or a reduction in the severity of GVHD.

In-Vitro and in-Vivo Synergistic Effect of Melphalan and Dual DNA Repair Inhibition in Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3301-3301
Author(s):  
Pritesh R. Patel ◽  
Annie L. Oh ◽  
Vitalyi Senyuk ◽  
Dolores Mahmud ◽  
Nadim Mahmud ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Combination Index ◽  
Fold Increase ◽  
High Dose ◽  
Myeloma Cells ◽  
Synergistic Cytotoxicity ◽  
Significant Difference

Abstract High dose melphalan is commonly used in patients with multiple myeloma (MM). Resistance to melphalan has been linked to the ability to repair DNA damage. To test whether DNA repair inhibitors overcome resistance to melphalan and and also have a direct anti-MM effect, we tested MM cell lines RPMI8226 and U266 in-vitro and in-vivo, using a NOD/SCID/ gamma null (NSG) xenograft model. RPMI8226 and U266 cells were initially treated in-vitro with the PARP inhibitor ABT-888. Using a proliferative assay, myeloma cells appeared sensitive to ABT-888 with low GI50 values (8.7μM for RPMI8226 cells, 49μM for U266 cells) and increased γH2AX foci, which persisted at 24 hours after treatment. This was confirmed in methycellulose colony assay where ABT-888 treatment reduced RPMI8226 colonies by 35% (p=0.002). Next we showed synergistic cytotoxicity between ABT-888 and melphalan. In both RPMI8226 and U266 cells strong synergy was displayed with a combination index (CI) less than 1 in proliferative assays (CI 0.5 and 0.3 at 50% proliferation respectively). Combination ABT-888 and melphalan treated cells underwent accelerated senescence compared to cells treated by melphalan alone (27% versus 51% βGal+ staining at 24 hours, p=0.02). This was confirmed by upregulation of senescence related genes p16 (1.6 fold increase) and p21 (1.5 fold increase). We did not find significant difference in apoptosis by Annexin V/ PI staining. Given that increased non-homologous end joining (NHEJ) activity has been shown to lead to resistance to melphalan, we tested whether an inhibitor of NHEJ could be synergistic with PARP inhibition and melphalan. Treatment with the DNA-PK inhibitor NU7026 at 10μM in addition to ABT-888 at 4μM resulted in 46% reduction in proliferation in RPMI8226 cells and 52% in U266 cells. When used in combination with melphalan chemotherapy, the dual DNA repair inhibitor therapy showed marked synergy in RPMI8226 cells with a combination index of 0.39. Finally we tested the ability of the combination of ABT-888 and melphalan to treat myeloma in-vivo. NSG mice were injected via tail vein with 5x106 RPMI8226 cells. Control (untreated) mice subsequently developed myeloma infiltrating the marrow, spleen and axial skeleton, with hind limb paralysis occurring at a median of 42 days. Treated mice received intraperitoneal injections of ABT-888 (3 times a week), or melphalan (weekly) or a combination of both agents starting on day 28 post-injection of MM cells for a total of 3 weeks. Using ABT-888, melphalan and a combination of both agents, median survival of mice was progressively prolonged (44 vs. 67 vs. 107 days, respectively) (p=0.02). Here we show that PARP and DNA-PK inhibition synergizes with melphalan in myeloma cells lines, providing a rationale for the addition of these agents to conditioning chemotherapy. In addition, we also show a direct anti-myeloma activity of these agents without the use of alkylator chemotherapy. Disclosures No relevant conflicts of interest to declare.

Methotrexate-induced renal impairment: clinical studies and rescue from systemic toxicity with high-dose leucovorin and thymidine.

1983 ◽  
Vol 1 (3) ◽  
pp. 208-216 ◽  
Author(s):  
H T Abelson ◽  
M T Fosburg ◽  
G P Beardsley ◽  
A M Goorin ◽  
C Gorka ◽  
...  
Keyword(s):  
Renal Failure ◽  
Glomerular Filtration Rate ◽  
Glomerular Filtration ◽  
Filtration Rate ◽  
Systemic Toxicity ◽  
High Dose ◽  
Severe Toxicity ◽  
Treatment Comparison ◽  
Dose Methotrexate ◽  
Significant Difference

Four separate groups of patients have been studied: (1) The effect of high-dose methotrexate (MTX) administration on glomerular filtration rate was determined by pre- and posttreatment inulin and creatinine clearances in nine patients. Measurements were made prior to and 24-40 hr after drug administration. Inulin and creatinine clearances both decreased a mean of 43%. No signs of systemic toxicity occurred. (2) Three other patients given high-dose courses of MTX developed MTX toxicity. Their creatinine clearance decreased an average of 61%. (3) In a separate group of five patients undergoing weekly MTX treatment, comparison of serum MTX pharmacokinetics with and without alkalinization of the urine demonstrated no significant difference in peak serum MTX levels or serum MTX decay. (4) Eight additional patients with severe renal dysfunction secondary to MTX were treated with increased doses of leucovorin and a continuous infusion of thymidine (8 g/m2/day) once renal failure was recognized. When high-dose leucovorin and thymidine were begun 48-72 hr after the MTX infusion, severe toxicity in the form of leukopenia, thrombocytopenia, diffuse mucositis, stomatitis, or skin rash was averted. We concluded the following: (1) high-dose MTX causes a subclinical decrease in glomerular filtration rate with each administration, even in nontoxic courses; (2) alkalinization of the urine with sodium bicarbonate does not alter plasma MTX decay, while volume expansion (hydration) is maintained constant; and (3) rigorous monitoring of serum creatinine and serum MTX levels 24-48 hr after MTX administration allows for the institution of rescue measures, including leucovorin and thymidine, which will abort the systemic toxicity that accompanies MTX-induced renal failure.

High-dose dexamethasone shifts the balance of stimulatory and inhibitory Fcγ receptors on monocytes in patients with primary immune thrombocytopenia

Blood ◽  
2011 ◽  
Vol 117 (6) ◽  
pp. 2061-2069 ◽  
Author(s):  
Xin-guang Liu ◽  
Shi-hui Ma ◽  
Jian-zhi Sun ◽  
Juan Ren ◽  
Yan Shi ◽  
...  
Keyword(s):  
Immune Thrombocytopenia ◽  
High Dose ◽  
Fcγ Receptor ◽  
Fcγ Receptors ◽  
Phagocytic Capacity ◽  
High Dose Dexamethasone ◽  
Significant Difference ◽  
Primary Immune Thrombocytopenia ◽  
Before And After

Abstract The human Fcγ receptor (FcγR) system is composed of 2 opposing families, the activating FcγRs (FcγRI, FcγRIIa, and FcγRIII) and the inhibitory FcγR (FcγRIIb). The disturbed balance of the activating and inhibitory FcγRs has been implicated in the pathogenesis of many autoimmune diseases. In this study, the expression of FcγRs on monocytes was determined in 23 patients with primary immune thrombocytopenia (ITP) before and after high-dose dexamethasone (HD-DXM) treatment. The FcγRI expression was significantly higher in ITP patients and decreased after HD-DXM treatment. The ratio of FcγRIIa/IIb mRNA expression on monocytes was significantly higher in untreated patients than in healthy controls. After HD-DXM therapy, the ratio decreased and the increased expression of FcγRIIb mRNA and protein coincided with a remarkable decrease in the expression of FcγRIIa, FcγRI, and monocyte phagocytic capacity. There was no significant difference in FcγRIII expression on monocytes between patients and controls. In vitro cell-culture experiments showed that DXM could induce FcγRIIa and FcγRIIb expression in monocytes from ITP patients, with FcγRIIb at higher amplitudes. These findings suggested that the disturbed FcγR balance might play a role in the pathogenesis of ITP, and that HD-DXM therapy could shift monocyte FcγR balance toward the inhibitory FcγRIIb in patients with ITP.

scholarly journals Daptomycin Is Effective in Treatment of Experimental Endocarditis Due to Methicillin-Resistant and Glycopeptide-Intermediate Staphylococcus aureus

2008 ◽  
Vol 52 (7) ◽  
pp. 2538-2543 ◽  
Author(s):  
Francesc Marco ◽  
Cristina García de la Mària ◽  
Yolanda Armero ◽  
Eurídice Amat ◽  
Dolors Soy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rabbit Model ◽  
Interquartile Range ◽  
High Dose ◽  
Methicillin Resistant ◽  
Significant Difference ◽  
Mrsa Strains ◽  
Lipopeptide Antibiotic

ABSTRACT Daptomycin is a lipopeptide antibiotic with potent in vitro activity against gram-positive cocci, including Staphylococcus aureus. This study evaluated the in vitro and in vivo efficacies of daptomycin against two clinical isolates: methicillin-resistant S. aureus (MRSA) 277 (vancomycin MIC, 2 μg/ml) and glycopeptide-intermediate S. aureus (GISA) ATCC 700788 (vancomycin MIC, 8 μg/ml). Time-kill experiments demonstrated that daptomycin was bactericidal in vitro against these two strains. The in vivo activity of daptomycin (6 mg/kg of body weight every 24 h) was evaluated by using a rabbit model of infective endocarditis and was compared with the activities of a high-dose (HD) vancomycin regimen (1 g intravenously every 6 h), the recommended dose (RD) of vancomycin regimen (1 g intravenously every 12 h) for 48 h, and no treatment (as a control). Daptomycin was significantly more effective than the vancomycin RD in reducing the density of bacteria in the vegetations for the MRSA strains (0 [interquartile range, 0 to 1.5] versus 2 [interquartile range, 0 to 5.6] log CFU/g vegetation; P = 0.02) and GISA strains (2 [interquartile range, 0 to 2] versus 6.6 [interquartile range, 2.0 to 6.9] log CFU/g vegetation; P < 0.01) studied. In addition, daptomycin sterilized more MRSA vegetations than the vancomycin RD (13/18 [72%] versus 7/20 [35%]; P = 0.02) and sterilized more GISA vegetations than either vancomycin regimen (12/19 [63%] versus 4/20 [20%]; P < 0.01). No statistically significant difference between the vancomycin HD and the vancomycin RD for MRSA treatment was noted. These results support the use of daptomycin for the treatment of aortic valve endocarditis caused by GISA and MRSA.

scholarly journals Accuracy of linear measurements using low dose cone beam computed tomography protocol versus direct skull linear measurements: An in vitro study

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 25
Author(s):  
Nora Aly Al Abbady ◽  
Reham Mohamed Hamdy ◽  
Sahar Hosny El Dessouky
Keyword(s):  
Computed Tomography ◽  
Cone Beam Computed Tomography ◽  
Low Dose ◽  
In Vitro Study ◽  
Cone Beam ◽  
High Dose ◽  
Anatomical Landmarks ◽  
Linear Measurements ◽  
Significant Difference

Background: Cone beam computed tomography (CBCT) imaging has been widely used for different dental applications over the last few years. It delivers a high dose of radiation compared to conventional imaging modalities. This study aimed to compare the accuracy of linear measurements conducted using a low dose CBCT protocol in comparison with direct skull linear measurements. Methods: Ten dry human skulls were included in the study. 12 linear measurements were measured directly on each skull between 23 chosen anatomical landmarks using a digital calliper. Radio-opaque markers were then glued on these anatomical landmarks. Each skull was then scanned using low dose CBCT protocol operated at 90 kVp, 7.1 mA, for 9 sec. Results: There was no statistically significant difference in the accuracy of linear measurements conducted using the low dose CBCT protocol when compared with direct linear measurements. Relative Dahlberg Error value ranged from 0.8% to 1.9%. Conclusion: Reducing mAs using a low dose CBCT protocol does not affect the accuracy of linear measurements used in craniofacial imaging tasks as compared with those taken directly on the skull by a digital calliper.

scholarly journals Moxifloxacin efficacy against Mycobacterium abscessus in vivo using zebrafish model

2019 ◽  
Author(s):  
Wenjuan Nie ◽  
Shan Gao ◽  
Tianlu Teng ◽  
Wenqiang Zhou ◽  
Yuanyuan Shang ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Mycobacterium Abscessus ◽  
High Dose ◽  
Good Activity ◽  
Zebrafish Model ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
Significant Difference

AbstractMoxifloxacin (MFX) showed good activity in vitro against Mycobacterium abscessus (M. abscessus) and was suggested as one of the antibiotic regimens for adults with M. abscessus disease. However, some other studies showed that MFX showed less or none activity against M. abscessus. In our study we aim to evaluate MFX activity against M. abscessus using zebrafish (ZF) model in vivo. MIC of each drugs were determined by broth microdilution method. M. abscessus labeled by CM-DiI, were micro-injected into ZF. Survival curves were determined by recording dead ZF every day. After 4 days of incubation ZF were lysed. Colony-forming unit (CFU) were enumerated and results are expressed as mean log10 CFU per ZF. Bacteria dissemination and fluorescence intensity in ZF were observed and analyzed. Inhibition rate was also calculated. In our study MFX showed good activity in vitro. But in vivo MFX showed limited restriction to M. abscessus. The association between increased survival and high dose of MFX is not significant. Same results were observed in bacterial fluorescence intensity and inhibition rates, with no significant difference when compared with no drug group (P > 0.05). However, significant difference was observed in azithromycin (AZM) group. MFX showed limited efficacy on Mycobacterium abscessus in vivo using ZF model. MFX’s activity in vivo need to be confirmed.

319 REDUCED SUPEROVULATION EFFICIENCY BY HIGH-DOSE TREATMENT OF DEHYDROEPIANDROSTERONE IN MICE

2013 ◽  
Vol 25 (1) ◽  
pp. 307 ◽  
Author(s):  
O. Suzuki ◽  
M. Koura ◽  
Y. Noguchi ◽  
K. Uchio-Yamada ◽  
J. Matsuda
Keyword(s):  
Testosterone Level ◽  
Plasma Testosterone ◽  
High Dose ◽  
Dose Treatment ◽  
Mouse Oocytes ◽  
High Dose Treatment ◽  
Significant Difference ◽  
Dhea Treatment ◽  
High Testosterone Level

Strain differences in in vitro fertilizability still constitute a serious problem in mouse reproduction. To improve the in vitro fertilizability of mouse oocytes, we examined the effect of implanting time-release pellets of dehydroepiandrosterone (DHEA), a testosterone precursor, in female mice on oocyte fertilizability. The DHEA pellets (0.25, 1.5, or 5 mg pellet–1, 21-day release form, Innovative Research of America, Sarasota, FL, USA) or corresponding placebo pellets were implanted subcutaneously in 9-week-old female 129X1/SvJJmsSlc mice. On Day 18 of implantation, superovulation was induced in these females by injections of pregnant mare serum gonadotropin (PMSG) and hCG 48 h apart. On Day 21, IVF was conducted using oocytes collected from the oviducal ampullae of these females and epididymal sperm from Slc : ICR male mice using TYH medium. Then, the embryos were cultured in vitro in Whitten’s medium for 96 h. Plasma steroid levels and expression of 5 ovarian proteins (PTEN and receptors for FSH, androgen, oestrogen, and progesterone) at oocyte collection were measured by enzyme immunoassay and quantitative Western blots, respectively. Embryo development into 2-cell and blastocyst stages at each dose at 24 h and 96 h after insemination, respectively, were compared between DHEA and placebo groups using weighted ANOVA with angular transformations. Other observed values were compared using Student’s t-test. Treatment with DHEA suppressed the numbers of ovulated oocytes in the 1.5 and 5 mg groups (DHEA v. placebo: 21.0 ± 2.6 v. 32.5 ± 2.8 and 19.9 ± 1.4 v. 27.1 ± 1.6, respectively, n = 10; P < 0.05), but not in the 0.25 mg group (26.6 ± 3.2 v. 24.8 ± 2.4). Treatment with DHEA did not affect the percentages of 2-cell embryo formation at any dose, ranging 50 to 60%. In the 0.25 mg group, DHEA treatment tended to increase blastocyst formation rate (1.8 ± 0.8% v. 0.4 ± 0.4%; not significant). However, the treatment at 1.5 mg suppressed the rate (0.0 ± 0.0% v. 3.1 ± 0.7%; P < 0.05) and treatment at 5 mg did not affect the rate (1.3 ± 0.9% v. 0.8 ± 0.6%). Plasma testosterone levels were increased by DHEA at 1.5 and 5 mg (338.2 ± 39.8 v. 197.0 ± 8.9 pg mL–1 and 534.9 ± 111.4 v. 241.8 ± 34.4 pg mL–1, respectively, n = 6; P < 0.05), but not at 0.25 mg (247.4 ± 22.0 v. 252.5 ± 35.6 pg mL–1, n = 6). No significant difference was induced by DHEA in plasma DHEA, progesterone, or oestradiol at any doses. Ovarian PTEN protein was more abundant in DHEA group than in placebo group at 5 mg (P < 0.05), tended to be more abundant at 1.5 mg (P ≈ 0.14), and was not different at 0.25 mg (P ≈ 0.35). The amounts of the 4 receptor proteins were not significantly changed by DHEA at any dose. These results suggest that DHEA at a low dose (e.g. 0.25 mg pellets for 21 days) might have a potential to improve in vitro fertilizability of mouse oocytes. Higher doses of DHEA reduced superovulation efficiency, perhaps because of the high testosterone level induced by the high-dose treatment of DHEA. The high testosterone level might upregulate ovarian PTEN expression, which might suppress ovarian primordial follicle activation. A more detailed study is needed to determine the optimal dose, timing, and duration of DHEA treatment for the improvement of female fertilizability.

29 Development and survival of bovine vitrified sexed IVF-derived embryos in vitro matured with pituitary or human recombinant follicle-stimulating hormone

2019 ◽  
Vol 31 (1) ◽  
pp. 140
Author(s):  
L. B. Ferré ◽  
C. Fresno ◽  
M. E. Kjelland ◽  
P. J. Ross
Keyword(s):  
Embryo Development ◽  
Production Efficiency ◽  
Mixed Model ◽  
Linear Mixed Model ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Development Rate ◽  
High Dose ◽  
Significant Difference

Improving in vitro production efficiency involves the development of effective oocyte in vitro maturation conditions. Although &gt;80% of cumulus-oocyte complexes (COC) undergo nuclear maturation, only approximately 30 to 35% of immature bovine COC develop to the blastocyst stage. Also, animal-sourced FSH is typically used in IVF, so an effective alternative using recombinant DNA technology is desirable. The aim of this study was to compare the effect of porcine (p) and recombinant human (rh)FSH concentrations on in vitro performance and post-thaw survival. Ovaries were collected from an abattoir and oocytes were aspirated from 2- to 6-mm follicles. The COC with compact and complete cumulus cell layers were selected and matured in groups of 25 COC in 200µL of M199 medium supplemented with alanyl-glutamine (0.1mM), Na pyruvate (0.2mM), gentamicin (5µg mL−1), epidermal growth factor (50ng mL−1), pLH (5µg mL−1), cysteamine (0.1mM), and 10% FBS for 22 to 24h in humidified air and 5% CO2. Oocytes were divided into the following groups: 1× pFSH (2µg mL−1), 1× rhFSH (0.01 UI mL−1), 1× pFSH+1× rhFSH, 2× pFSH, and 2× rhFSH. After 22 to 24h, fertilization (Day 0) was carried out using female sexed-sorted semen selected with a mini single-continuous 80% layer (PureSperm, Nidacon International AB, Mölndal, Sweden) and diluted to 1×106 sperm mL−1. The SOF-FERT medium was supplemented with fructose (90µg mL−1), penicillamine (3µg mL−1), hypotaurine (11µg mL−1), and heparin (20µg mL−1). After 18h, presumptive zygotes were denuded and cultured under low oxygen tension in groups of 15 to 20 in 50-µL drops of SOF-BSA for 7 days. Also, 2% FBS was added post-fertilization on Day 3.5. Expanded blastocysts were selected based on IETS standards at Day 6.5 to 7 of culture. Only grade 1 expanded blastocysts were vitrified (Cryotop, Kitazato, Tokyo, Japan). Vitrification medium was 15% (vol/vol) ethylene glycol+propylene glycol. Vitrified embryos were thawed in a solution of H199+20% FBS and 0.25M sucrose at 39°C. Thawed embryos were cultured in SOF-BSA+10% FBS under cumulus/granulosa cell monolayer co-culture. Embryo assessment involved post-thaw survival (0h), re-expansion and development progress (24-48h), and hatching of the zona pellucida (72h). A minimum of 4 replicates were performed. Data were analysed using a generalized linear mixed model with logit-link binomial distribution. Media treatment differences were determined using Fisher’s least significant difference test with the Bonferroni correction (α-level=0.05). The FSH origin affected cleavage and embryo development rate but not cryotolerance (Table 1). The results support previous research on low dose versus high dose rhFSH effectiveness and interspecies interaction of FSH on follicular receptors. Table 1.Cleavage, embryo development rate, and cryotolerance of FSH of various origins

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