A novel approach for the selection of human sperm using annexin V-binding and flow cytometry

2009 ◽  
Vol 91 (4) ◽  
pp. 1285-1292 ◽  
Author(s):  
Christiaan F. Hoogendijk ◽  
Theunis F. Kruger ◽  
Patric J.D. Bouic ◽  
Ralf R. Henkel
Keyword(s):  
Flow Cytometry ◽  
Annexin V ◽  
Human Sperm ◽  
Novel Approach ◽  
Selection Of

8 Effects of magnetic-activated cell sorting on human sperm motility and DNA fragmentation index

2021 ◽  
Vol 33 (2) ◽  
pp. 111
Author(s):  
Y. M. Toishibekov ◽  
S. B. Baikoshkarova ◽  
Y. A. Assanova ◽  
M. K. Otarbayev ◽  
A. N. Komogortsev ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Motility ◽  
Cell Sorting ◽  
Annexin V ◽  
Human Sperm ◽  
Fragmentation Index ◽  
Fresh Semen ◽  
Selection Of ◽  
Magnetic Activated Cell Sorting ◽  
Dna Fragmentation Index

Selection of spermatozoa before their use for assisted reproductive techniques is an important step in therapy of human infertility. The DNA fragmentation index of sperm plays a major role in pregnancy rates following IVF and intracytoplasmic sperm injection (ICSI). Sperm analyses and standard sperm selection methods in many cases do not eliminate a sufficient proportion of sperm with apoptosis and DNA fragmentation. Magnetic-activated cell sorting (MACS) is a selection method that eliminates apoptotic spermatozoa based on the presence of externalized phosphatidylserine residues. The aim of our study was to evaluate the effect of MACS on human sperm motility and DNA fragmentation index (DFI) in a patient population. The participants were 63 male patients of an IVF clinic, 34 to 45 years old, with 3 years of primary infertility due to male factor. Semen analysis was performed according to the World Health Organization guidelines (2010) and revealed oligoasthenoteratozoospermia in 63 patients. The DFI of fresh semen samples was evaluated using the sperm chromatin structure assay (SCSA) test and revealed DFI 32.4±5.9%. The SCSA test was done on a flow cytometer CyFlow Space (Sysmex-Partec; Evenson 2016 Anim. Reprod. Sci. 169, 56-75; https://doi.org/10.1016/j.anireprosci.2016.01.017). Sperm motility was studied on Hamilton Thorne IVOS. For MACS, we used the MACS® ART Annexin V system (Miltenyi Biotec). The semen sample was diluted to a concentration 10×106 spermatozoa mL−1. After double-density gradient centrifugation, the pellet was resuspended in 100µL of MACS Art Annexin V reagent and added MACS Art Binding Buffer (BB) to 500µL. The sample was gently mixed and incubated for 15min at room temperature. After rinsing the column with BB, the sperm-bead suspension was added on top with BB and, immediately after that, the annexin V-negative and annexin V-positive fractions were obtained (MiniMACS; Miltenyi Biotec). Data were evaluated by ANOVA Student’s t-test. Fresh semen samples collected from the patients had an average sperm concentration of 29.7±5.7×106 mL−1, motility of 32.7±5.9%, and DFI of 32.4±5.9%. Motility of spermatozoa after MACS for the annexin-negative fraction was 47.2±6.3% and for the annexin-positive fraction was 3.5±2.3% (P<0.003). Similarly, the annexin-negative spermatozoa had a lower DFI (10.5±3.8%) rate than did the annexin-positive fraction (67.8±5.9%; P<0.003). The MACS technique allowed a significant reduction of DNA fragmentation levels (from 32.4% for the original sample to 10.5% for the annexin-negative; P<0.01). The separation of a distinct population of nonapoptotic spermatozoa with intact membranes may optimize outcomes from IVF and ICSI procedures. Magnetic-activated cell sorting of human sperm using annexin-V microbeads results in selection of a population with enhanced motility and reduced DFI rates.

scholarly journals Time-Lapse Flow Cytometry: A Robust Tool to Assess Physiological Parameters Related to the Fertilizing Capability of Human Sperm

2020 ◽  
Vol 22 (1) ◽  
pp. 93
Author(s):  
Arturo Matamoros-Volante ◽  
Valeria Castillo-Viveros ◽  
Paulina Torres-Rodríguez ◽  
Marcela B. Treviño ◽  
Claudia L. Treviño
Keyword(s):  
Flow Cytometry ◽  
Calcium Concentration ◽  
Human Sperm ◽  
Time Lapse ◽  
Physiological Parameters ◽  
Clinical Validation ◽  
Artificial Reproduction ◽  
Membrane Hyperpolarization ◽  
Novel Strategy ◽  
Fertilizing Capability

Plasma membrane (PM) hyperpolarization, increased intracellular pH (pHi), and changes in intracellular calcium concentration ([Ca2+]i) are physiological events that occur during human sperm capacitation. These parameters are potential predictors of successful outcomes for men undergoing artificial reproduction techniques (ARTs), but methods currently available for their determination pose various technical challenges and limitations. Here, we developed a novel strategy employing time-lapse flow cytometry (TLFC) to determine capacitation-related membrane potential (Em) and pHi changes, and progesterone-induced [Ca2+]i increases. Our results show that TLFC is a robust method to measure absolute Em and pHi values and to qualitatively evaluate [Ca2+]i changes. To support the usefulness of our methodology, we used sperm from two types of normozoospermic donors: known paternity (subjects with self-reported paternity) and no-known paternity (subjects without self-reported paternity and no known fertility problems). We found relevant differences between them. The incidences of membrane hyperpolarization, pHi alkalinization, and increased [Ca2+]i were consistently high among known paternity samples (100%, 100%, and 86%, respectively), while they varied widely among no-known paternity samples (44%, 17%, and 45%, respectively). Our results indicate that TLFC is a powerful tool to analyze key physiological parameters of human sperm, which pending clinical validation, could potentially be employed as fertility predictors.

Development of a Performance-Related Framework for Asphalt Mixture Design for the Illinois Tollway

2021 ◽  
pp. 036119812110148
Author(s):  
Behnam Jahangiri ◽  
Punyaslok Rath ◽  
Hamed Majidifard ◽  
William G. Buttlar
Keyword(s):  
Asphalt Mixture ◽  
Field Performance ◽  
Compact Tension ◽  
T Test ◽  
Novel Approach ◽  
Final Adjustment ◽  
Development And Validation ◽  
High Degree ◽  
Selection Of

Various agencies have begun to research and introduce performance-related specifications (PRS) for the design of modern asphalt paving mixtures. The focus of most recent studies has been directed toward simplified cracking test development and evaluation. In some cases, development and validation of PRS has been performed, building on these new tests, often by comparison of test values to accelerated pavement test studies and/or to limited field data. This study describes the findings of a comprehensive research project conducted at Illinois Tollway, leading to a PRS for the design of mainline and shoulder asphalt mixtures. A novel approach was developed, involving the systematic establishment of specification requirements based on: 1) selection of baseline values based on minimally acceptable field performance thresholds; 2) elevation of thresholds to account for differences between short-term lab aging and expected long-term field aging; 3) further elevation of thresholds to account for variability in lab testing, plus variability in the testing of field cores; and 4) final adjustment and rounding of thresholds based on a consensus process. After a thorough evaluation of different candidate cracking tests in the course of the project, the Disk-shaped Compact Tension—DC(T)—test was chosen to be retained in the Illinois Tollway PRS and to be presented in this study for the design of crack-resistant mixtures. The DC(T) test was selected because of its high degree of correlation with field results and its excellent repeatability. Tailored Hamburg rut depth and stripping inflection point thresholds were also established for mainline and shoulder mixes.

scholarly journals Gradient-based selection of cross sections: a novel approach for optimal frame structure design

2017 ◽  
Vol 56 (5) ◽  
pp. 959-972 ◽  
Author(s):  
Christian Krogh ◽  
Mathias H. Jungersen ◽  
Erik Lund ◽  
Esben Lindgaard
Keyword(s):  
Cross Sections ◽  
Structure Design ◽  
Frame Structure ◽  
Novel Approach ◽  
Gradient Based ◽  
Selection Of

scholarly journals Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Christo J. Botha ◽  
Sarah J. Clift ◽  
Gezina C.H. Ferreira ◽  
Mxolisi G. Masango
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Ethical Issues ◽  
Sesquiterpene Lactones ◽  
Annexin V ◽  
Metabolic Activation ◽  
Suitable Model ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern Africa. The toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. Because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. The objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (C2C12) using methyl-thiazol-tetrazolium (MTT) and lactate dehydrogenase (LDH) assays, annexin V and propidium iodide (PI) flow cytometry and transmission electron microscopy (TEM). Mouse myoblasts were exposed to 2.0 mM, 2.5 mM and 5.0 mM geigerin for 24, 48 and 72 h. A concentration-dependent cytotoxic response was observed. Apoptosis was detected by means of annexin V flow cytometry during the first 24 h and apoptotic bodies were also visible on TEM. According to the LDH and PI flow cytometry results, myoblast cell membranes were not injured. We concluded that the murine myoblast cell line (C2C12) is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts.

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