Density Gradient Centrifugation Technique Research Articles

Chronic non-bacterial osteomyelitis and immune checkpoint molecules.

We aimed to investigate the plasma levels and cell surface expression of two checkpoint molecules, TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3) and PD-1 (programmed cell death protein 1), in pediatric patients with chronic non-bacterial osteomyelitis (CNO). Plasma samples of CNO patients were collected at diagnosis or during biologic agent treatment. Plasma levels of TIM-3 and PD-1 were measured using the sandwich enzyme-linked immunosorbent assay method, and the expression of the two immune checkpoint molecules on the cell surface was analyzed by isolating peripheral blood mononuclear cells by density gradient centrifugation technique. Twenty-seven patients with CNO (14 boys, 51.9%) and six healthy controls (3 boys, 50%) were enrolled in the study. There were no age differences between CNO patients and healthy controls (median age 14.5 vs. 13.5 years, respectively, p=0.762). Of the CNO patients, 18 were included at the time of diagnosis while 9 were receiving biologic treatment at enrollment. The median plasma PD-1 levels were significantly lower in the CNO group than in the healthy controls (p=0.011). However, no significant difference was found in the cellular expression of PD-1 and TIM-3 on CD3+CD4+ T cells in patients and healthy controls (p=0.083 and p=0.245, respectively). There was also no statistically significant difference in plasma TIM-3 levels of the patient and control groups (p=0.981). CNO is an autoinflammatory disease, and overall, our results suggest that T cell exhaustion may not be significant in CNO. Further research is needed to find out whether the immune checkpoints are mainly associated with autoimmunity but not autoinflammation. Key Points • The median plasma PD-1 levels were significantly lower in the CNO group than in the healthy controls. • No significant difference was found in the cellular expression of PD-1 and TIM-3 on CD3+CD4+ T cells in patients and healthy controls. •Our results suggest that T cell exhaustion may not be significant inCNO pathogenesis.

IMPACT OF MICROFLUID SPERM SORTING ON EMBRYO PLOIDY RATES IN COUPLES WITH HIGH SPERM DNA FRAGMENTATION?

Increased sperm DNA fragmentation (SDF) is one of the leading causes of male infertility. Conventional methods for sperm preparation have induced questioning of sperm recovery rates. The microfluidic sperm sorting (MSS) technique selects highly motile sperm with lower levels of SDF compared to density-gradient centrifugation (DGC) technique. This study aimed to compare MSS to DGC technique in couples with repeated implantation failure (RIF) and high SDF referred to a PGT-A (preimplantation genetic testing for aneuploidies) cycle.

Efficacy of density gradient centrifugation technique (DGC) in enhancing sperm cell DNA quality for assisted reproductive technique

ObjectiveDensity gradient centrifugation (DGC) is commonly used for sperm preparation before assisted reproductive technology (ART) procedures. This technique separates superior motile spermatozoa with normal morphology from the total sperm population. However, there is still controversy as to the effects of this sperm separation technique on sperm cell DNA integrity which is a determining element in the process of fertilization and embryonic development.The objective of this study was to determine the effects of DGC on sperm cell DNA integrity as assessed by a novel association between two cytogenetic tests.Study designsSemen samples were collected from 30 fertile donors and 40 patients being candidates for ART treatment. Each sample was divided into two parts: the first portion was subjected to selection by two layers of DGC (45% and 90%) and the second fraction was rinsed with phosphate-buffered saline solution and centrifuged without density gradient.Abnormal sperm chromatin structure as evaluated by a sperm chromatin dispersion (SCD) test and DNA denaturation as assessed by an acridine orange (AO) test were monitored in the initially washed sample and in the different layers of the density gradient centrifugation.ResultsDGC significantly improved the proportion of sperm progressive motility, total motility, and sperm morphology. Moreover, following density gradient centrifugation, the proportion of spermatozoa with denaturated DNA significantly decreased when compared with whole semen (p < 0.001). In addition, we found that spermatozoa isolated in the 90% layer possessed a significantly lower percentage of sperm chromatin decondensation when compared with those remaining in the 45% layer and unprocessed semen (p < 0.001).ConclusionsUsing double cytogenetic tests, our study shows that semen processing by density gradient centrifugation is useful in selecting sperm with higher double-strand DNA integrity and recommended to be used in sperm preparation for assisted reproduction.

Microfluidic chips as a method for sperm selection improve fertilization rate in couples with fertilization failure.

Sperm quality plays a vital role in successful fertilization and pregnancy. Patients with fertilization failure (total failure or low-fertilization rate) despite having normal semen parameters are a challenging group whose sperm cannot fertilize the oocyte via the intracytoplasmic sperm injection (ICSI) technique. Microfluidics is offered as a new method for proper sperm sorting. This study aimed to evaluate sperm parameters, DNA fragmentation index (DFI), expression of phospholipase C zeta 1 (PLCZ1), and transition nuclear proteins 1 (TNP1) mRNAs in sperm selected by microfluidic sperm sorting (MSS) chip compared with conventional density gradient centrifugation technique in patients with fertilization failure following ICSI. Subsequence fertilization rate and embryo quality were assayed. Normal morphology and total motility were significantly higher, and DFI was significantly lower in sperm selected by the MSS chip in fertilization failure and control groups. The RT-PCR results demonstrated a significant increase in the expression of PLCZ1 and TNP1 genes in sperm of both groups selected by MSS chips compared to the DGC method. In addition, with the selected sperm by MSS chip, an increase in fertilization rate and improvement of embryo quality was obtained. The present study findings show that sperm sorting by the microfluidic method improves fertilization rate in patients with poor fertilization outcomes following ICSI.

CD24/Siglec-10 “Don't Eat Me” Signal Blockade Is a Potential Immunotherapeutic Target in Mantle-Cell Lymphoma

IntroductionMantle-cell lymphoma (MCL) is a B-cell non-Hodgkin Lymphoma (NHL) characterized by heterogenous behavior, ranging from indolent phenotype to highly aggressive and drug resistant one with dismal prognosis. Drug resistance may be generated by Tumor Microenvironment (TME), owing that Tumor-Associated Macrophages (TAM) are pathologically functional in providing survival signals to MCL cells (Pham, Front Oncol. 2018).Recently, “Don't Eat Me” signal (DEMs) blockade with anti-CD47 monoclonal Antibody (moAb) showed promising activity in pretreated NHL, through increase of phagocytosis by TAM (Advani, NEJM. 2019).CD24 was also demonstrated to be involved in DEMs and, in a preclinical model of solid cancer, blocking the CD24/Siglec-10 interaction provided an improvement of M2-like TAM-mediated phagocytosis in vitro and an increase of survival in vivo (Barkal, Nature. 2019).CD24 can be expressed in some phases of B-cell differentiation and MCL derives from a B-cell precursor with upregulated CD24. To date, there are no functional studies showing an improvement of phagocytosis through CD24/Siglec-10 pathway inhibition in hematologic malignancies and MCL.Here, we present our in vitro results of CD24/Siglec-10 DEMs blockade in MCL subset.MethodsA panel of MCL cell lines (Jeko-1, Granta-519, Mino) has been analyzed for CD24 surface expression by flow cytometry (FC) (clone SN3). Consequently, we performed co-culture experiments with MCL cell lines and macrophages from healthy donors. Briefly, Peripheral Blood Mononucleated Cells (PBMC) were collected from healthy volunteers through density gradient centrifugation technique. CD14+ monocytes were isolated through CD14 Microbeads isolation kit and cultured in plates with 50 ng/ml human GM-CSF for 7-9 days. In order to create M2-like Siglec-10+ TAM, 50 ng/ml human IL-10 and 50 ng/ml human TGF-β 1 were added on days 3-4 of differentiation until use on days 7-9. Siglec-10 expression on TAM was checked by FC (clone 5G6).M2-like macrophages were then collected and co-cultured with CFSE-labelled MCL target cells for 1-2 hours in a serum-free medium. Anti-CD24 moAb (clone SN3) or the appropriate IgG 1 isotype control were added at a concentration of 10 μg/ml. Phagocytosis was then stopped on ice and CD11b-PE staining (anti-CD11b moAb, clone REA713) was performed to identify human macrophages by FC.Phagocytosis was measured as the number of CD11b+/CFSE+ macrophages, quantified as a percentage of the total CD11b+ macrophages. Each phagocytosis reaction was performed in technical triplicate and phagocytosis was normalized to the highest technical replicate per donor in order to consider raw phagocytic level among donor-derived macrophages.ResultsMCL cell lines express surface CD24 by FC, with higher levels in Mino cell line (Figure 1A). Differentiated M2-like macrophages showed an upregulation of Siglec-10 expression after immunosuppressive stimuli, which is fundamental owing that Siglec-10 is the ligand of CD24 (Figure 1B).As pertains to the phagocytic assay, we documented an improvement of phagocytosis when M2-like macrophages and MCL cell lines were co-cultured together with anti-CD24 moAb (Figure 2 and Figure 3A). Furthermore, it is worth mentioning that phagocytosis seemed to be much higher in MCL cell lines with higher surface levels of CD24 (e.g., Mino), presenting increased number of CD11b+/CFSE+ M2-like TAM by FC (Figure 3B).ConclusionsMCL was found to be sensitive to CD24/Siglec-10 DEMs blockade when co-cultured with M2-like macrophages in vitro. We can argue that most of the observed increase of phagocytosis after the addition of anti-CD24 moAb may be secondary to loss of CD24 signalling rather than Fc-mediated opsonization, as already documented in previous analysis about solid cancer (Barkal, Nature. 2019). We can therefore hypothesize that the blockade of this DEMs pathway can improve phagocytosis in a non-opsonization manner in NHL as well.Furthermore, CD24 surface density seemed to be positively correlated to the intensity of phagocytic activity, suggesting that MCL subtypes expressing higher CD24 levels are much more dependent on this DEMs pathway than others with low CD24 density.Overall, CD24 turned out to be a potential immunotherapeutic target in MCL, aiming at improving innate immune system through DEMs blockade. In vivo studies are needed to confirm the activity we documented in vitro in this NHL subset. [Display omitted] DisclosuresGambacorti-Passerini: Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

P–012 A new sperm preparation technique for ICSI

Abstract Study question Is there any difference in this technique(swim-in) according to clinical results in ICSI cycles? Summary answer Fertilization rates are significantly better with this new technique(swim-in). There is no difference in pregnancy and ongoing pregnancy rates between swim-in and DGS techniques. What is known already Sperm separation is crucial in assisted reproductive technologies, based on different principles like migration, filtration, or density gradient centrifugation techniques. However, there are some studies that centrifugation steps and using gradient solutions may increase sperm DNA fragmentation. On the other hand, multiple contacts to different plastic surfaces such as pipettes, plastic tubes, etc., and also pipetting the ejaculate in sperm preparation may have detrimental effects. The ideal sperm separation technique should be non-invasive, not time-consuming, easy, and cost-effective, not cause sperm damage or non-physiological alterations of the separated sperm cells, eliminate dead spermatozoa and other cells, including leukocytes. Study design, size, duration This is a prospective randomized study between 01/02/2019...01/01/2021. Sperms were prepared either with this new technique that we call “swim-in”(n = 359) or with density gradient centrifugation(n = 404) before microinjection (ICSI). Fertilization rates, clinical pregnancy rates, and on-going pregnancy rates were compared between groups. Sperm motility is less than%20 patients are not included in the study. t-test and Chi-square test used. p &amp;lt; 0.05 significant. Participants/materials, setting, methods For this new centrifugation free technique(swim-in), a Braun injector, 21G green needle, and sperm washing medium are used. First, we pulled a 0.5 ml sperm wash medium in a Braun injector. After sperm liquefaction, we put Braun injector with the needle in the sperm cap perpendicularly and waited 30 minutes at 37-degree Celcius. Sperms swam through the needle to the injector used for ICSI. For DGC sperms were prepared according to the 2015 WHO manual. Main results and the role of chance: We try to make a centrifugation-free, gentle, cost-effective, and easy sperm preparation technique and called it a swim-in technique. Groups were comparable according to mean age, collected oocyte numbers, and oocyte maturation ratios. Fertilization rates are%73,2(1786/2438) vs%66,9(2167/3237) respectively for swim-in and DGC groups. Clinical pregnancy rates are%45,1 vs%42,3 respectively for swim-in and DGC groups. On-going pregnancy rates are%41,5 vs%39,6 for swim-in and DGC respectively. There is a significant difference between the two techniques according to fertilization rates but no difference according to clinical pregnancy and ongoing pregnancy rates. Limitations, reasons for caution Sperms can move by their own motility from ejaculate to the sperm washing medium for that reason with severe sperm problem patients are not suitable for this technique. Wider implications of the findings: This technique is centrifugation free so there are no detrimental g-forces. The needle used may mimic the physiological environment like Uterine Tube. Alternative sperm washing mediums like follicular fluid or chemical attractor progesterone rich medium can be used.DNA fragmentation rates and live birth rates should be established for future aspect. Trial registration number Not applicable

A Modified Flotation Density Gradient Centrifugation Technique Improves the Semen Quality of Stallions with a High DNA Fragmentation Index.

Simple SummarySperm DNA fragmentation has a negative impact on reproductive success, because it compromises fertilization and embryo development. Since spermatozoa lack the machinery to repair DNA damage, the likelihood of establishing a healthy pregnancy can be improved by processing ejaculates with a high sperm DNA fragmentation index. The current study examined a modified flotation density gradient centrifugation technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density, later layered between colloid layers of lower and higher density. This modified flotation technique was consistent in selecting viable, progressively motile, morphologically normal spermatozoa with intact acrosome and DNA. In reproductively normal stallions, sperm recovery was 57% and spermatozoa with damaged DNA decreased significantly (from 12% to 4%), with increase in the percentage of viable, progressively motile, morphologically normal spermatozoa with intact acrosome. In stallions with a high sperm DNA fragmentation index, this modified flotation technique markedly decreased the DNA fragmentation index (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, the stallion sperm DNA fragmentation index and other sperm quality parameters can be markedly improved using a modified flotation density gradient centrifugation technique employing a 40% Opti-prepTM cushion and a 20% top layer.Sperm DNA fragmentation compromises fertilization and early embryo development. Since spermatozoa lack the machinery to repair DNA damage, to improve the likelihood of establishing a healthy pregnancy, it is preferable to process ejaculates of stallions with a high sperm DNA fragmentation index (DFI) before artificial insemination or intracytoplasmic sperm injection. The aim of this study was to examine a modified flotation density gradient centrifugation (DGC) technique in which semen was diluted with a colloid solution (Opti-prepTM) to increase its density prior to layering between colloid layers of lower and higher density. The optimal Opti-prepTM solution (20–60%) for use as the bottom/cushion layer was first determined, followed by a comparison between a modified sedimentation DGC and the modified flotation DGC technique, using different Opti-prepTM solutions (20%, 25% and 30%) as the top layer. Finally, the most efficient DGC technique was selected to process ejaculates from Friesian stallions (n = 3) with high sperm DFI (>20%). The optimal Opti-prepTM solution for the cushion layer was 40%. The modified sedimentation technique resulted in two different sperm populations, whereas the modified flotation technique yielded three populations. Among the variants tested, the modified flotation DGC using 20% Opti-prepTM as the top layer yielded the best results; the average sperm recovery was 57%; the DFI decreased significantly (from 12% to 4%) and the other sperm quality parameters, including progressive and total motility, percentages of spermatozoa with normal morphology and viable spermatozoa with an intact acrosome, all increased (p < 0.05). In Friesian stallions with high sperm DFI, the modified flotation DGC markedly decreased the DFI (from 31% to 5%) and significantly improved the other semen quality parameters, although sperm recovery was low (approximately 20%). In conclusion, stallion sperm DFI and other sperm quality parameters can be markedly improved using a modified flotation DGC technique employing a 40% Opti-prepTM cushion and a 20% top layer.

The role of phagocytic leukocytes following flexible ureterenoscopy, for the treatment of kidney stones: an observational, clinical pilots-study

BackgroundThe number of patients undergoing flexible ureterenoscopy (FURS) for the treatment of kidney stones (renal calculi) is increasing annually, and as such the development of post-operative complications, such as acute kidney injury (AKI), haematuria and infection is likely to increase. Phagocytic leukocytes are white blood cells that help fight foreign material such as bacteria and viruses, and they are intrinsically involved in the inflammatory reaction. Investigating the role of phagocytic leukocytes following FURS has not been widely researched. The main aim of the study was to evaluate the role phagocytic leukocytes (neutrophils and monocytes) function, in patients undergoing FURS for the treatment of kidney stones (renal calculi).MethodsFourteen consecutive patients aged between 27 and 70 years (median 49.5 years) undergoing FURS for the treatment of kidney stones were recruited (seven males, seven females). Blood samples were collected from each patient at four time points: baseline (pre-operatively) followed by 30, 120 and 240 min post-operatively. Mononuclear (MN) and polymorphonuclear (PMN) leukocyte sub-populations were isolated by density gradient centrifugation techniques. Neutrophil and monocyte cell function was investigated by measuring the cell surface expression of CD62L (L-selectin), CD11b (Mac-1), CD99 and the intracellular production of hydrogen peroxide (H2O2), via flow cytometry.ResultsSignificant increases was observed in monocyte CD62L expression post FURS for the treatment of kidney stones (p ≤ 0.05); while significant decreases were observed in neutrophil CD62L. The levels of the other activation markers CD11b, CD99 and H2O2 corresponded to the increases and decreases seen in CD62L for monocytes and neutrophils respectively, though the changes were not statistically significant (p > 0.05). Limiting factors for this study were the relatively small sample size, and restriction on the recruitment time points.ConclusionsThis study demonstrates that following FURS for the treatment of kidney stones, monocytes are rapidly activated and produce potent reactive oxygen intermediates. Interestingly, the pattern of expression in neutrophils suggests that these cells are deactivated in response to the treatment. The leukocyte biomarkers assessed during this investigation may have a role in monitoring the ‘normal’ post-operative response, as no complications occurred in any of the patients; or may help predict potential infectious complications (e.g. urosepsis) that can occur during the post-operative period. This data, however, will need to be validated and reproduced in larger multi-centre studies.

Five Layered Optiprep based Density Gradient Model is a Promising Model for Enrichment of Viable X Chromosome Bearing Spermatozoa in Bubalus bubalis

Background: A reliable method for controlling the sex of farm animals has the potential to revolutionize dairy farming. This can only be achieved by use of a method capable of separating X and Y chromosome bearing spermatozoa at lower cost and causing no significant damages to sperm viability. Realizing significance of buffaloes in Indian dairy farming, present study was aimed to develop appropriate density gradient model for enrichment of X chromosome bearing spermatozoa population in semen of Murrah buffalo bull. Methods: Density gradient centrifugation (DGC) technique was employed for enriching X chromosome spermatozoa in buffalo bull semen using four gradient media viz., Percoll, Optiprep, Ficoll and Sucrose. Percentage enrichment of X chromosome bearing spermatozoa in all the DGC models was determined by SYBR green based Real Time PCR. Result: Our investigations revealed that number of layers and centrifugation speed (g) factors in a density gradient centrifugation model, have significant effect on the percentage enrichment of X chromosome content in semen samples.Three layers, four layers and five layers density gradient centrifugation models showed significant differences (P less than 0.05) in the sex ratio towards X spermatozoa population in semen (61.72%±0.81, 64.55%±0.26 and 67.31±0.33%) respectively. Density gradient centrifugation models with centrifugation speed (g) 200x g and 300x g also showed significant differences (P less than 0.05) in the sex ratio towards X spermatozoa population in semen (66.36%±0.25 and 62.69±0.22%) respectively. Out of all DGC models, Optiprep density gradient model with 5 layers and centrifugation at 200xg (O3) yielded maximum enrichment (72.4%±1.38). In conclusion, O3 poses to be a promising model for enrichment of X spermatozoa in buffalo bull semen.

Establishing a density-based method to separate proliferating and senescent cells from bone marrow stromal cells.

To assist in the study of cellular aging, we established a new method of enriching physiologically aged bone marrow stromal cells (BMSCs) in animals of any age using a Percoll density gradient centrifugation technique. BMSCs from mice 2 months of age were isolated, and their cellular age determined (over 80% Scal-1+ CD29+ CD11b- CD45- CD105- and able to differentiate into osteoblasts, adipocytes, and chondrocytes). As proof –of principle, cells were aged in vitro and confirmed by low bromodeoxyuridine (BrdU) incorporation and senescence-associated β-galactosidase (SA-β-gal) staining. Proliferating cells were enriched in high-density gradient layers, and senescent cells were enriched in low-density gradient layers. We confirmed that over 80% of cells from the low-density gradient layer were senescent with SA-β-gal staining and telomere dysfunction-induced foci (TIF) assay. This density-based method, which can separate proliferating and senescent BMSCs, could be used to study mechanisms of physiologic cell aging and may have implications for the use of BMSCs in clinical transplant applications.

Comparative Efficacy of PercollTM Discontinuous Density Gradient Centrifugation and Glass Wool Filtration Techniques for Spermatozoa Selection in Buffalo (Bubalus bubalis)

Comparative Efficacy of PercollTM Discontinuous Density Gradient Centrifugation and Glass Wool Filtration Techniques for Spermatozoa Selection in Buffalo (Bubalus bubalis)

The Motility and Ratio of X and Y Sperm Filial Ongole Cattle Using Different Sexed Semen Methods

Artificial Insemination (AI) using sexed semen produce sex of calf as expected. There are various methods of sexed semen has been found. The aim of this research is to determine the effect of sexed semen methods using egg white sedimentation and percoll density gradient centrifugation toward motility and ratio of X and Y sperm Filial Ongole Cattle. Semen was collected using Artificial Vagina (AV) from Filial Ongole Cattle, evaluated and then separated using egg white sedimentation and percoll density gradient centrifugation technique. Only fresh semen with a minimum of 70% individual motile sperm and 2+ mass motility used in this study. Andromed as a based extender was diluted using aquabidest with 1:4 ratio. The obtained data were analyzed with analysis of variance (ANOVA) and continued by Duncan test if there was significant or very significant different. The result showed that the sexing methods (non sexing sperm, upper and under fraction using percoll density gradient centrifugation, upper and under fraction using egg white sedimentation technique) had very significant effect (P<0.01) on motility (64.25±3.94%; 48.55±8.28%; 53±7.93%; 56.9±8.22%; 49.75±8.19% respectively) and significant effect (P<0.05) on the ratio of X and Y sperm. The ratio of X and Y using percoll density gradient centrifugation and egg white sedimentation methods for the upper fraction were 28.0±2.26%:72.0±2.26% and 77.5±1.26%:22.5±1.26%. The ratio of X and Y using percoll density gradient centrifugation and egg white sedimentation technique for the under fraction were 69.0±15.35%: 26.0±4.37% and 22.9±1.44%: 77.1±1.44%. The study concludes that the sexed with egg white sedimentation technique is better than percoll density gradient centrifugation on sperm motility and ratio of X and Y sperm.

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

Pregnancy Rate after Intrauterine Insemination with the Presence or Absence of Leukocytospermia in Sperms Prepared using Density Gradient Method

Objective: To examine the association between different concentrations of leukocyte and sperm recovery rate after sperms are prepared using density gradient method and pregnancy rate after intrauterine insemination (IUI). Increased leukocytes in semen have been associated with increased reactive oxygen species (ROS) that reduces sperm quality. Methods: Semen samples that were collected from 31 male partners of couples undergoing infertility investigation were analyzed for sperm concentration, motility, and leucocytes concentration. Semen samples were then divided in two groups based on their leucocytes concentrations (category A: >0 to 1 x 106/mL. Semen samples were processed using density-gradient centrifugation technique. Results: There was a significant difference in the number of sperms harvested and sperm motility after preparation. Interestingly, pregnancy rate after IUI was higher (p<0.05) in non-leukocytospermia semen (39%) when compared to leukocytospermia semen (30%). Conclusions: Seminal leukocytes (PMNL) concentration affects pregnancy rate after intrauterine insemination. Keywords: Density gradient method, sperm recovery rate, intrauterine insemination, pregnancy rate DOI: 10.15850/ijihs.v6n2.1318

Low-stress Microfluidic Density-gradient Centrifugation for Blood Cell Sorting.

Density gradient centrifugation exploits density differences between different blood cells to accomplish separation of peripheral blood mononuclear cells (PBMCs) from polymorphonuclear (PNM) cells, and erythrocytes or red blood cells (RBCs). While density gradient centrifugation offers a label-free alternative avoiding the use of harsh lysis buffers for blood cell isolation, it is a time-consuming and labor-intensive process during which blood cells are subject to high-levels of centrifugal force that can artifactually activate cells. To provide a low-stress alternative to this elegant method, we miniaturized and automated this process using microfluidics to ensure continuous PBMCs isolation from whole blood while avoiding the exposure to high-levels of centrifugal stress in a simple flow-through format. Within this device, a density gradient is established by exploiting laminar flow within microfluidic channels to layer a thin stream of blood over a larger stream of Ficoll. Using this approach we demonstrate successful isolation of PBMCs from whole blood with preservation of monocytes and different lymphocyte subpopulations similar to that seen with conventional density gradient centrifugation. Evaluation of activation status of PBMCs isolated using this technique shows that our approach achieves minimal isolation process induced activation of cells in comparison to conventional lysis or density gradient centrifugation. This simple, automated microfluidic density gradient centrifugation technique can potentially serve as tool for rapid and activation-free technique for isolation of PBMCs from whole blood for point-of-care applications.

The thermo-sensitive gene expression signatures of spermatogenesis

BackgroundSpermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen. The present study investigates the heat-sensitive transcriptome and miRNAs in the most vulnerable germ cells (spermatocytes and round spermatids) that are primarily targeted at elevated temperature in a bid to identify novel targets for contraception and/or infertility treatment.MethodsTestes of adult male rats subjected to surgical cryptorchidism were obtained at 0, 24, 72 and 120 h post-surgery, followed by isolation of primary spermatocytes and round spermatids and purification to > 90% purity using a combination of trypsin digestion, centrifugal elutriation and density gradient centrifugation techniques. RNA isolated from these cells was sequenced by massive parallel sequencing technique to identify the most-heat sensitive mRNAs and miRNAs.ResultsHeat stress altered the expression of a large number of genes by ≥2.0 fold, out of which 594 genes (286↑; 308↓) showed alterations in spermatocytes and 154 genes (105↑; 49↓) showed alterations in spermatids throughout the duration of experiment. 62 heat-sensitive genes were common to both cell types. Similarly, 66 and 60 heat-sensitive miRNAs in spermatocytes and spermatids, respectively, were affected by ≥1.5 fold, out of which 6 were common to both the cell types.ConclusionThe study has identified Acly, selV, SLC16A7(MCT-2), Txnrd1 and Prkar2B as potential heat sensitive targets in germ cells, which may be tightly regulated by heat sensitive miRNAs rno-miR-22-3P, rno-miR-22-5P, rno-miR-129-5P, rno-miR-3560, rno-miR-3560 and rno-miR-466c-5P.

Studies on Separation of Macerals from Coal by Froth Flotation

In the past coal maceral separation have been carried out based on density difference e. g., sink-float analysis, density gradient centrifugation (DGC) techniques etc. These approaches are of limited success. Froth flotation is a physico-chemical process separating particles on the basis of difference in surface properties in a vessel consisting of two distinct zones viz., pulp and froth zones. Rate of floatability of individual size range particles varies with their ash percent and the maceral content present. Therefore, the flotation rate constant eventually becomes a function of maceral percentage of a coal particle. In the present paper, an attempt has been made by performing size-wise flotation tests using a sub-bituminous rank coal and flotation kinetics have been established based on maceral percentage reporting to concentrate at different time intervals of flotation. These results have been related to the kinetic behaviour of the process.

Effect of semen preparation technique and its incubation on sperm quality in the Moroccan population.

In in vitro fertilisation (IVF), sperm preparation as critical part and influencing the sperm quality is especially dependent on the chosen technique itself and incubation parameters including temperature and CO2. In this study, we compared firstly density-gradient centrifugation technique (DGC) to the adapted DGC using the sperm pellet of 80% fraction (DGC/80P) in order to improve the sperm yield. Secondly, this study led to evaluate different sperm incubation conditions based on temperature effect (room temperature (RT=23°C) versus 35°C) and in the other hand, with or without 5% CO2 during 24hrs. Based on evaluating sperm conventional parameters and the DNA damage using TUNEL assay, our result showed that DGC/80P increased sperm quality compared to DGC with 25% of improvement. For temperature incubation effect after 24hrs, 35°C increased the DNA damage and decreased the sperm quality while RT could improve sperm motility by 38%. Moreover, the sperm incubation with 5% CO2 after 24hrs realised a negative impact on sperm parameters and its DNA damage. Indeed, for current IVF practice, a good sperm quality can be maintained for several hours at room temperature, while the sperm preparation is processed using the DGC/80P without CO2.

Purification of HIV-1 gag virus-like particles and separation of other extracellular particles

Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100⿿200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1ÿ109 particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity.

Combination of density gradient centrifugation and swim-up methods effectively decreases morphologically abnormal sperms.

Density gradient centrifugation (DGC) and swim-up techniques have been reported for semen preparation in assisted reproductive techniques in humans. We investigated whether semen preparation using a combination of DGC and swim-up techniques could effectively decrease morphologically abnormal human sperms at the ultrastructural level. Semen samples were obtained from 16 infertile males and fractionated by swim-up following DGC. Ultrastructural abnormalities of sperms obtained from original semen, lower layer of swim-up following DGC, and upper layer of swim-up following DGC were analyzed by transmission electron microscopy. The correlation among ultrastructural head abnormality in sperms from the upper layer of swim-up, fertilization in in vitro fertilization, and pregnancy after embryo transfer was also investigated. Furthermore, sperms with DNA fragmentation in the samples processed via a combination of DGC and swim-up was assessed in a sperm chromatin structure assay. Ultrastructural abnormalities in sperm heads and tails in the upper layer after swim-up following DGC was the lowest among the three groups. Sperms with nuclear vacuoles were the most difficult to eliminate using a combination of DGC and swim-up in all types of head abnormalities. A negative correlation was confirmed between the fertilization rates of intracytoplasmic sperm injection and head abnormality of sperms obtained from the upper layer of the swim-up following DGC. Sperms with DNA fragmentation were effectively decreased using the combination of two techniques. In conclusion, the combination of DGC and swim-up effectively decreased the number of sperms with ultrastructural abnormalities both in the head and in the tail. However, sperms with ultrastructural abnormalities that cannot be completely decreased using a combination of DGC and swim-up may impair fertilization in some cases of intracytoplasmic sperm injection.