P–069 microfluidic sperm sorting vs density gradient to yield sperm with reduced DFI for patients undergoing IVF-ICSI

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
D P Makwana ◽  
S Makwana ◽  
T Sen
Keyword(s):  
Density Gradient ◽  
Gradient Method ◽  
Fluid Dynamic ◽  
Sperm Concentration ◽  
Semen Sample ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Preparation ◽  
Sperm Sorting

Abstract Study question To compare the effect of sperm preparation methods on the DFI of semen sample for patients undergoing ICSI. Summary answer On comparing the results, microfluidic sperm sorting yielded sperms with significantly less DFI as compared to density gradient method of sperm preparation. What is known already The DNA integrity of the sperm plays an important role to ensure formation of good quality embryos with increased potential of fertilization, growth and ultimately implantation.. Centrifugation has shown to add stress to the sperm and leading to DNA damage, therefore there is a need to develop techniques of sperm preparation which help in retrieving as many sperms with intact DNA from the unprocessed sample as possible. Microfludic is fluid dynamic based technique of sperm preparation. in this study, we evaluated if microfluidic sperm sorter can recover motile sperm with better DNA integrity compared to density gradient preparation method. Study design, size, duration Prospective randomized study conducted in 80 patients undergoing IVF-ICSI with normal semen parameters (based WHO criteria 2010). DFI was done using Sperm Chromatin Dispersion (SCD) test in split semen samples prepared by microfluidic sperm sorter and density gradient method. Sperm morphology and motility were also recorded and evaluated based on the WHO 2010 criteria. Participants/materials, setting, methods Semen parameters of the sample were assessed by microscopic examination. DFI of each unprocessed sample was carried out using SCD test, following that the sample was split and sperm preparation was done using microfluidic sperm sorter and density gradient. the recovered sperm were tested for DFI and the results were compared. Main results and the role of chance Mean DFI in unprocessed semen samples was 23%. the analysis of split semen samples post preparation showed that the DFI was significantly reduced with the use of microfluidic sperm sorter (mean DFI 0.6%) as compared to density gradient (mean DFI 9%). Limitations, reasons for caution A major limitation of the microfluidic sperm sorter is the use sperm concentration and motility of the semen sample. In oligospermic and asthenospermic samples, density gradient is the preferred method of preparation. Lack of data showing improvement in clinical outcomes with reduced DFI is also a major limitation. Wider implications of the findings: Microfluidics has shown to significantly reduce the DFI of the semen sample, it requires no extra equipment and cost and is relatively easy to pick up. Density gradient method of sperm preparation continues to be the preferred method due to its versatility and recovery of good quality sperm. Trial registration number Not applicable

scholarly journals Sperm Selection and Embryo Development: A Comparison of the Density Gradient Centrifugation and Microfluidic Chip Sperm Preparation Methods in Patients with Astheno-Teratozoospermia

Life ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 933
Author(s):  
Cagla Guler ◽  
Sureyya Melil ◽  
Umit Ozekici ◽  
Yaprak Donmez Cakil ◽  
Belgin Selam ◽  
...  
Keyword(s):  
Density Gradient ◽  
Embryo Development ◽  
Microfluidic Chip ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sperm Concentration ◽  
Gradient Methods ◽  
Semen Parameters ◽  
Preparation Methods ◽  
Sperm Preparation

In recent years, microfluidic chip-based sperm sorting has emerged as an alternative tool to centrifugation-based conventional techniques for in vitro fertilization. This prospective study aims to compare the effects of density gradient centrifugation and microfluidic chip sperm preparation methods on embryo development in patient populations with astheno-teratozoospermia. In the study, the semen samples of the patients were divided into two groups for preparation with either the microfluidic or density gradient methods. Selected spermatozoa were then used to fertilize mature sibling oocytes and the semen parameters and embryo development on days 3 and 5 were assessed. While the density gradient group was associated with a higher sperm concentration, motility (progressive and total) was significantly higher in the microfluidic chip group. No significant differences were observed in the fertilization rates or grade 1 (G1) and grade 2 (G2) proportions of the third-day embryos. Furthermore, while the proportions of the poor, fair and good blastocysts on day 5 did not differ significantly, excellent blastocysts (indicating high-quality embryos) were observed in a significantly higher proportion of the microfluidic chip group. When compared to the classical density gradient method, the microfluidic chip sperm preparation yielded sperm with higher motility and higher quality blastocysts at day 5; in patients with astheno-teratozoospermia.

313 ASSESSMENT OF SPERM DNA FRAGMENTATION IN CANINE EJACULATES USING THE Sperm-Halomax® KIT: PRELIMINARY RESULTS

2010 ◽  
Vol 22 (1) ◽  
pp. 312 ◽  
Author(s):  
M. Hidalgo ◽  
M. R. Murabito ◽  
M. J. Gálvez ◽  
S. Demyda ◽  
L. J. De Luca ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Sperm Concentration ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Sperm Dna Fragmentation ◽  
Fragmentation Index ◽  
Sperm Dna ◽  
Commercial Kit ◽  
Sperm Dna Fragmentation Index ◽  
Dna Fragmentation Index

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for humans and different mammalian species, using a commercial kit based on the sperm chromatin dispersion (SCD) test; however, a descriptive study in canine semen has not been performed. The aim of this work was to assess the sperm DNA fragmentation in canine ejaculates using the SCD test and 2 different staining techniques. For this purpose, ejaculates were collectedby digital manipulation from4 healthy dogs of different breeds (1 German Pointer, 2 Spanish Greyhounds, and 1 Crossbreed). After collection, the sperm-rich fraction of the ejaculates from 3 dogs were pooled each time (n = 4) and then extended in Dulbecco’s phosphate buffered saline. All the pooled semen samples presented physiological values concerning routine semen parameters (motility, morphology, and sperm concentration). The sperm DNA fragmentation was assessed using the Sperm-Halomax® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain). Two semen aliquots of the diluted pooled semen samples were processed on each pre-treated slide provided in the kit following the manufacturer’s instructions. The last step was the staining technique. We stained each slide with 2 different staining procedures. The first half of the slide was stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) mixed in a proportion 1 : 1 with an antifading solution. The second half of the slide was stained for 15 min in Wright solution (1.01383.0500, Merck, Whitehouse Station, NJ, USA) 1 :1 in Phosphate Buffer pH 6.88 (1.07294.1000, Merck). The stained slides were observed using fluorescence and light microscopy, respectively. Five hundred sperm per slide were counted. Spermatozoa with fragmented DNA showed a large and spotty halo of chromatin dispersion. Unfragmented sperm only showed a small and compact halo. Statistical analyses were performed using the Statistical Package for Social Science version 12.0 (SPSS Inc., Chicago, IL, USA). The sperm DNA fragmentation index was compared between Wright and fluorescence staining methods by ANOVA. Results were expressed as mean ± standard error of the mean. The first report of the sperm DNA fragmentation index in canine ejaculates was 2.26 ± 0.53% for Wright staining and 1.99 ± 0.10% for fluorescence technique. No differences were found between staining procedures. In conclusion, it was possible to assess the sperm DNA fragmentation of canine ejaculates using 2 different staining procedures, expecting that continuous research could be useful in defining the role of DNA fragmentation SCD test in canine semen evaluation and cryopreservation.

Impacts administration of Rifampicin on sperm DNA integrity and Male Reproductive System parameters in rats

2021 ◽  
pp. 4897-4902
Author(s):  
Furqan Mohammed Al-Asady ◽  
Dalia Abdulzahra Al-Saray
Keyword(s):  
Sperm Motility ◽  
Sperm Concentration ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Function ◽  
High Dose ◽  
Adult Rats ◽  
Male Adult ◽  
Sperm Dna Integrity ◽  
Sperm Dna

Objective: Evaluate the impacts of rifampicin on certain sperm function parameters and to determine whether rifampicin has an impact on chromatin quality or sperm DNA integrity. Materials and Methods: Forty two male adult rats were subjected to this study. The entire rats were subjected to random division into six groups; four rifampicin- treated groups and two control groups. Rifampicin- treated groups were treated with a dose of either (27mg/kg/day) or (54mg/kg/day) and for each treatment dose, the treatment persists for either 14 days or 28 days. Certain parameters of sperm function including sperm concentration and sperm motility were assessed. Furthermore, analysis of sperm DNA integrity and chromatin quality were also studied. Results: No significant changes related to sperm concentration were observed when rifampicin was given in different doses and different durations. A significant change in sperm motility were recorded only when rifampicin was given in high dose for 28 days and there was a significant reduction in sperm progressive and total motility. Rifampicin showed a significant increase in sperm DNA staining capability when the dose and duration was increased. Administration of rifampicin in high dosage for 28 days represented in larger adverse impact on structure of sperm chromatin. Conclusion: Rifampicin could negatively affect male fertility potential in rats mainly through affecting the quality of sperm chromatin structure.

scholarly journals Are semen quality parameters sufficient for biomonitoring spermatozoa DNA integrity and oxidatively damaged DNA

Biomonitoring ◽  
2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Hueiwang Anna Jeng ◽  
Ruei-Nian Li ◽  
Wen-Yi Lin
Keyword(s):  
Dna Damage ◽  
Dna Fragmentation ◽  
Semen Quality ◽  
Quality Parameters ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Phase System ◽  
Sperm Dna Fragmentation ◽  
Sperm Dna

Abstract:The present study aimed to investigate the relationship between semen quality parameters and DNA integrity, and determine whether semen quality parameters could serve as a reliable biomarker for monitoring sperm DNA damage. Conventional semen parameters from a total of 202 male human subjects were analyzed. DNA fragmentation and 8-oxo-7,8-dihydro-2′- deoxyguanosine (8-oxoGuo) were used to assess sperm DNA integrity. DNA fragmentation was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and sperm chromatin structure assay (SCSA), while 8-oxodGuo was quantified by the liquid chromatography/tandem mass spectrometry (LC-MS/MS) coupled with an on-line solid phase system. The levels of 8-oxodGuo levels in sperm were related to the percentages of DNA fragmentation measured by both the TUNEL and SCSA (r = 0.22, p = 0.048; r = 0.12, p = 0.039). Sperm vitality, motility and morphology from all of the participants exhibited a weak correlation with the levels of 8-oxodGuo and the percentages of DNA fragmentation. Semen quality parameters may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Semen quality parameters may be insufficient to monitor sperm DNA fragmentation and oxidative damage. DNA damage in sperm is recommended to be included in routine measurements.

scholarly journals Effect of Alcohol Consumption on the Sperm DNA Integrity: A Systematic Review

2017 ◽  
Vol 9 (13) ◽  
pp. 136
Author(s):  
Farah Hanan Fathihah Jaafar ◽  
Khairul Osman ◽  
Jaya Kumar ◽  
Siti Fatimah Ibrahim
Keyword(s):  
Dna Damage ◽  
Alcohol Consumption ◽  
Alcohol Withdrawal ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Damage ◽  
Sperm Dna Integrity ◽  
Sperm Dna ◽  
Keywords Alcohol

There is no solid conclusion on the conventional sperm parameters in association with alcohol consumption, evaluation of sperm DNA integrity thus become a more reliable parameter. Hereby, this literature search was performed to summarize alcohol consumption on the sperm DNA integrity. A computerized database search was done through MEDLINE via Ovid (since 1946 until August 2017) and Cochrane was used. The following set of keywords: ‘alcohol consumption OR alcohol intake OR alcohol diet OR drinking alcohol OR ethanol diet’ AND ‘sperm DNA OR sperm chromatin OR sperm genome OR sperm histone OR sperm protamine’ were utilised. 24 articles were retrieved where only five studies conform to the inclusion criteria All studies demonstrated a negative effect of alcohol consumption on sperm DNA integrity, regardless of various range of alcohol doses and duration of alcohol consumption. Out of five studies reviewed, four studies were using a different approach to measure the sperm DNA damage. Hereby, this review identified a need to use a single approach of DNA damage test by having various method of alcohol administration and/or vice versa so that the extension of sperm DNA damage to alcohol consumption will have a better conclusion. On the same note, a few studies have reported the reversibility on conventional semen parameters, none has been done on the sperm DNA damage upon alcohol withdrawal. Therefore, the role of alcohol withdrawal on the reversibility of sperm DNA damage needs to be as well investigated further.

scholarly journals Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls

2015 ◽  
Vol 67 (2) ◽  
pp. 417-423 ◽  
Author(s):  
J.T. Carreira ◽  
J.T. Trevizan ◽  
B.H. Kipper ◽  
S.H.V. Perri ◽  
I.R. Carvalho ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Cell Damage ◽  
Sperm Concentration ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Dna Breaks ◽  
Mitochondrial Potential ◽  
Sperm Dna Damage ◽  
Sperm Dna

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.

61 Swim-up and microfluidic techniques improve the kinetic parameters of selected bovine spermatozoa for invitro fertilization: Preliminary results

2021 ◽  
Vol 33 (2) ◽  
pp. 137
Author(s):  
J. Vega ◽  
M. Rodriguez ◽  
D. Dipaz-Berrocal ◽  
J. Rivas ◽  
C. Huayhua ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Dna Fragmentation ◽  
Density Gradient ◽  
Gradient Method ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Sperm Selection ◽  
Embryo Production ◽  
Microfluidic Sorting ◽  
Microfluidic Technique

For invitro embryo production, spermatozoa with fertilizing capacity must possess optimal kinetic, morphometric, vitality, and DNA integrity characteristics. The objective of this study was to evaluate the effect of 3 sperm selection methods on sperm quality and invitro embryonic development in bovine. Frozen commercial semen (0.5 mL/straws) from one bull with known fertility was thawed at 37°C for 20s and was divided for 3 sperm selection techniques: density gradient, swim-up, and microfluidic sperm sorting. The sperm kinetic parameters (VCL=curvilinear velocity, VSL=straight line velocity, VAP=average path velocity, ALH=lateral displacement of sperm head, BCF=beat frequency cross, STR=path straightness) were assessed using computer-assisted sperm analysis (CASA). Sperm morphometric parameters were evaluated using Diff-Quick staining followed by automated analysis. To assess vitality, the sperm were stained with propidium iodide and acridine orange, then analysed under a fluorescence microscope. In addition, DNA fragmentation was assessed using sperm chromatin dispersion method. Last, the fertilizing capacity of the selected sperm was tested by fertilizing cumulus–oocyte complexes (2×106 sperm mL−1) obtained from slaughterhouse ovaries and matured invitro for 24h. A standardized invitro embryo production protocol was used with commercial medium from Vitrogen. The cleavage rate and blastocyst yield were measured on Day 2 and 7, respectively (fertilization=Day 0). The results were calculated with analysis of variance and Tukey’s test (P&lt;0.05). The values of sperm kinetic parameters obtained with swim-up (VCL 132.5µm/s; VSL 73.5µm/s) and microfluidic technique (VCL 129.5µm/s; VSL 64.4µm/s) were significantly higher (P&lt;0.05) than those obtained by density gradient (VCL 98.3µm/s; VSL 45.01µm/s). However, the total and progressive motility by density gradient method was slightly higher (89% and 57%) compared with that assessed by swim-up (64% and 43%) or microfluidic technique (74% and 54%) respectively. Microfluidic sorting (11.3%) showed lower (P&lt;0.05) DNA fragmentation levels compared with density gradient method (16.6%), whereas the swim-up technique (12.5%) was similar between both groups. No significant difference was detected between the 3 groups for sperm morphometric and vitality parameters. Moreover, cleavage rates were similar (P&gt;0.05) between the 3 sperm selection techniques: density gradient (84.0%), swim-up (75.2%), and microfluidic sorting (67.3%). However, blastocyst yield was significantly higher (P&lt;0.05) using sperm selected by density gradient (28.1%) and swim-up (21.9%) compared with microfluidic sorting (15.3%). In conclusion, sperm selection using microfluidic and swim-up techniques improved kinetic parameters with lower levels of DNA fragmentation, without affecting sperm morphometry. However, both the density gradient and swim-up techniques are efficient systems for producing invitro bovine embryo.

scholarly journals Sperm DNA Integrity Assessment: A New Tool in Diagnosis and Treatment of Fertility

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Mona Bungum
Keyword(s):  
Semen Quality ◽  
Significant Proportion ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Dna Integrity ◽  
Sperm Dna Fragmentation ◽  
Integrity Assessment ◽  
Sperm Dna Integrity ◽  
Sperm Dna

Infertility affects 15% of all couples. Although male infertility factors with reduced semen quality are contributing to about half of all involuntary childlessness, the value of standard semen parameters in prediction of fertilityin vivoand choice of proper method for assisted reproduction is limited. In the search for better markers of male fertility, during the last 10 years, assessment of sperm DNA integrity has emerged as a strong new biomarker of semen quality that may have the potential to discriminate between infertile and fertile men. Sperm DNA Fragmentation Index (DFI) as assessed by the flow cytometric Sperm Chromatin Structure Assay (SCSA) can be used for evaluation of sperm chromatin integrity. The biological background for abnormal DFI is not completely known, but clinical data show that DFI above 30% is associated with very low chance for achieving pregnancy in natural way or by insemination, but notin vitro. Already when the DFI is above 20%, the chance of natural pregnancy may be reduced, despite other sperm parameters being normal. Thus this method may explain a significant proportion of cases of unexplained infertility and can be beneficial in counselling involuntary childless couples need ofin vitrofertilisation.

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