Density Gradient Centrifugation Method Research Articles

ANALYSIS OF SPERMATOZOA QUALITY USING PERCOLL DENSITY GRADIENT CENTRIFUGATION THROUGH THE ADMINISTRATION OF PHOSPHOLIPID + EGTA

The Percoll density gradient centrifugation (PDGC) method is frequently used in the sexing of spermatozoa. However, this method causes damage to the spermatozoa membranes, resulting in a decreased quality of spermatozoa. We analysed the impacts of phospholipid PC (phosphatidylcholine) and EGTA (ethylene glycol bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid) Ca2+ free buffer on the quality of bovine spermatozoa after the PDGC process, using semen from Friesian Holstein (FH) bulls aged 5 - 8 years. The following variables were observed: spermatozoa motility, spermatozoa viability, spermatozoa membrane integrity, spermatozoa that have not experienced capacitation, spermatozoa that have experienced capacitation and spermatozoa that have undergone acrosomal reaction. The results showed that the administration of phospholipid PC + EGTA Ca2+ free buffer to spermatozoa, followed by the PDGC process, could maintain or further improve the values of all variables. In the PDGC process, phospholipid PC 10% + EGTA Ca2+ free buffer at 1 mM was most suitable.

The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring.

Background and Aim:The production of male calf beef cattle is an agricultural innovation needed to increase the farm’s productivity as a provider of meat sources. This study aimed to determine the sex ratio of the offspring of cows inseminated with Y-bearing sperm enriched by Percoll density gradient centrifugation and swim-up, combined with delayed fixed-time artificial insemination (FTAI).Materials and Methods:Ejaculates of Simmental bulls were divided into four equal portions and grouped as T0 (control, non-sexed semen), T1 and T2 were sexed semen using Percoll density gradient centrifugation three and five levels, respectively, and T3 was sexed semen using swim-up. After the sex was sorted, the semen was diluted in a tris-egg yolk extender, packaged in French mini-straws containing 50 million live sperm cells, and frozen. Pre-sexed, post-sexed, and post-thawed spermatozoa were evaluated based on progressive motility, viability, intact plasma membrane, and abnormality. The post-thawed semen of T0 was artificially inseminated to recipient cows at 12 h after onset of estrus (not delayed FTAI). Meanwhile, the delayed FTAI was conducted 18-20 h after onset of estrus using the T0, the best of T1 and T2, and the T3 post-thawed semen.Results:The Percoll density gradient centrifugation reduced motility, viability, and intact plasma membrane but increased sperm abnormalities. Meanwhile, the swim-up process increased motility, viability, and intact plasma membrane of sperm cells but decreased sperm abnormalities. Post-thawed semen decreased motility, viability, and intact plasma membrane of sperm cells but increased sperm abnormalities. The sex ratio of the Simmental crossbred offspring was 96.08% and 100% in T1 and T3, respectively, compared to 48.25% and 67.39% in T0 not delayed and delayed FTAI, respectively.Conclusion:The Percoll density gradient centrifugation and swim-up methods are prospective for obtaining male offspring.

Giardia purification from fecal samples using rigid spiral inertial microfluidics

Giardia is one of the most common waterborne pathogens causing around 200 × 106 diarrheal infections annually. It is of great interest to microbiological research as it is among the oldest known eukaryotic cells. Purifying Giardia from fecal samples for both research and diagnostic purposes presents one of the most difficult challenges. Traditional purification methods rely on density gradient centrifugation, membrane-based filtration, and sedimentation methods, which suffer from low recovery rates, high costs, and poor efficiency. Here, we report on the use of microfluidics to purify Giardia cysts from mouse feces. We propose a rigid spiral microfluidic device with a trapezoidal cross section to effectively separate Giardia from surrounding debris. Our characterizations reveal that the recovery rate is concentration-dependent, and our proposed device can achieve recovery rates as high as 75% with 0.75 ml/min throughput. Moreover, this device can purify Giardia from extremely turbid samples to a level where cysts are visually distinguishable with just one round of purification. This highly scalable and versatile 3D printed microfluidic device is then capable of further purifying or enhancing the recovery rate of the samples by recirculation. This device also has the potential to purify other gastrointestinal pathogens of similar size, and throughput can be significantly increased by parallelization.

The relationship of estrus character before double dose artificial insemination in Limousin Crossbred cow

Ongole cattle in smallholder farms on Java, Indonesia, have been crossed with Limousin, which is not controlled repeatedly. The character of estrus affects the success of Artificial Insemination. This study aimed to determine the relationship between estrus characters before double dose artificial insemination using frozen semen sexing. The research was conducted in Senggreng Village, Sumber Pucung District, Malang Regency. The material was 45 head of Limousin crossbred cow inseminated using sexing semen produced by the Singosari National Artificial Insemination Center (SNAIC). The sexed semen processed with the Percoll Density Gradient Centrifugation (PDGC) method. The average body value of cows between 3-5 (scale 1-9) with an average age of 1.8-7 years, normal reproduction, clear signs of estrus. The reproductive organs were examined by rectal palpation to confirm that the cows are not pregnant. Parameters of estrus character were vulvar color, cervical mucus, vaginal temperature, pH, vulvar swelling, and heat detector value. The result showed that the estrus characters, including vulva color, vulva swelling, cervical mucus, vaginal temperature, cervical mucus pH, and heat detector score in the first and second artificial insemination have a low relationship. The regression models produced can be used but are less precise to estimate the relationship between estrus characters because the coefficient of determination shows a low influence.

A new method of microglia sorting and functional characteristics of spinal microglia in aged rats

Objective: To establish an improved method of separating microglia from aged rats and to observe the biological characteristics of spinal microglia of aged rats. Methods: Young SD rats (2 months) were used as control group. Single cell suspension of rat microglia were prepared by trypsin, trypsin substitutes or mechanical net rubbing method. Then, by assessing the purity and survival rate of cells, and observing the morphological characteristics and analyzing the inflammatory functional characteristics, we optimized the isolation and purification method of microglia from aged rats (20 months old) , and observed the functional characteristics of spinal microglia in aged rats. Results: The survival rate of cells digested by pancreatic enzyme was low(young rats 83%, aged rats 60%). Although the survival rate of mechanical net rubbing method was higher than that of pancreatic enzyme digest methods (95%), the cell acquisition rate was lower(young rats(0.207±0.020)×106, aged rats(0.243±0.023)×106). Trypsin substitute dissociation combining density gradient centrifugation method was the best way to get abundant, active and higher survival microglia, and the purity reached more than 85%. We used this method to separate microglia from spinal cord of rats. Compared with the young rats, the spinal cord tissue of old rats was larger, the digestive fluid volume was higher, but the digestion time was shorter. Compared with the young rats, the aged rat spinal microglia had larger and rounder cell body, fewer and shorter protrusions, it tended to be activated morphologically, the level of proinflammatory cytokine IL-1β of microglia in aged rats was lower, and the level of antiinflammatory factor IL-10 was higher. Conclusion: The method of trypsin substitute dissociation combined with density gradient centrifugation was successfully established to isolate and purify microglia from spinal cord of rats, the spinal microglia of old rats showed anti-inflammatory phenotype.

Relationship of motility and acrosome reaction on sexing sperm in Ongole Crossbred bull

Artificial insemination using sexing semen is expected to produce calves with the desired sex. One sexing sperm method is the percoll density gradient centrifugation method. This study aimed to determine the changes and the relationship between motility and acrosome reaction after sexing process using percoll density gradient centrifugation. The material used was semen of ±5 years old Ongole crossbred bull with a bodyweight of ±700 kg as many as three bulls with mass motility 2+ and individual motility 70%. The method used was to compare fresh semen with sexed semen after the cooling process. Parameters measured were motility characters using CASA analysis, which included motility parameters, progressive motility, capacitation, and no acrosome reaction. Statistical analysis used paired T-test to distinguish among fresh semen, after sexing and cooling process. In comparison, regression and correlation were used to analyze the relationship of capacitation and hyperactivation sperm with no acrosomal reaction with motility and progressive motility. The results showed that motility and progressive motility decreased after the sexing and cooling process. Meanwhile, the acrosomal reaction, capacitation, and hyperactivity increased.

OR18. Combination of ramiprilat and human umbilical cord blood-mesenchymal stem cells derived secretome increase endothelial progenitor cells migration

Abstract Background and Aims Stem cells therapy has been studied in the cardiovascular field, but the limitations in terms of survival and risk of rejection make cell-free therapy with secretome worth considering. This study aims to identify the effect of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) derived secretome, ramiprilat, and combination of both on endothelial progenitor cells (EPCs) migration. Methods and Results EPCs were collected from mononuclear cells (MNCs) of the peripheral blood of chronic coronary syndrome patient using density gradient centrifugation method, then cultured in Stemline II medium (Sigma Aldrich, USA). Secretom was made by taking the supernatant from the hUCB-MSCs cell line culture media that had been centrifuged. Cultured EPCs was divided into control group, treatment with 10 µmol Ramiprilat, various concentrations of hUCB-MSCs-derived secretome (2%, 10%, and 20%) and combination of 10 µmol Ramiprilat and each concentration of secretome. EPCs migration was assessed using Boyden chamber assay. Results showed that ramiprilat and all doses of secretome increased EPCs migration compared to the control group (p < 0.001). Secretome 20% had higher EPCs migration than ramiprilat (51.00±5.15 vs 33.80±2.49, p < 0.001), and the combination of ramiprilat and secretome 20% showed the highest number of migrated EPCs among all groups. Conclusion hUCB-MSCs-derived secretome and ramiprilat increase EPCs migration. The combination of those two substances furtherly increased the migrated cells. hUCB-MSCs-derived secretome has the potential as a cell-free cardiovascular treatment for coronary artery disease patients.

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

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.

P–220 Delayed-ICSI on day1-matured oocytes in low responders of different age groups

Abstract Study question Is the cycle outcome of day0-ICSI (day0-matured oocytes) and delayed-ICSI (day1-matured oocytes) in low responders affected by maternal age? Summary answer Delayed-ICSI improves cycle outcome of low responders in all age groups by providing additional blastocysts for embryo transfer and/or pre-implantation genetic testing for aneuploidies (PGT-A). What is known already We had previously compared the fertilization, blastulation, blastocyst utilisation, and euploidy rates between day0-ICSI and delayed-ICSI (Lee, C.S.S., 2018). We believed that patients who have <7 mature oocytes (low responders) can benefit by having their concomitant immature oocytes cultured to day 1. This study evaluates the benefit of delayed-ICSI in improving the cycle outcome of such low responders from different age groups. Study design, size, duration From January 2018 to December 2020, 434 IVF cycles in Alpha IVF & Women’s Specialists were classified as low responder (being <7 oocytes injected on day 0). The immature oocytes were further cultured and delayed-ICSI was done on day1-matured oocytes. Patients were divided into 3 groups: (A) ≤35 years old (n = 137; mean maternal age=32.5; range=23.0–35.0); (B) 36–40 years old (n = 208; mean maternal age=38.2; range=36.0–40.0); and (C) >40 years old (n = 89; mean maternal age=42.7; range=41.0–49.0). Participants/materials, setting, methods Semen samples were processed by density gradient centrifugation and/or swim-up method. Day0-matured oocytes were injected at 2.5–4.5 hours post-retrieval (PIEZO, Japan). Immature oocytes were further cultured for 18–24hours and delayed-ICSI was done immediately after maturity assessment. Injected oocytes were cultured up to 7 days and trophectoderm biopsy for PGT-A screening (IonTorrent, USA) was performed on selected blastocysts prior to vitrification (Cryotec, Japan). The cycle outcomes of day0-ICSI and delayed-ICSI were analysed and compared. Main results and the role of chance In Group A, the fertilisation, blastulation, blastocyst utilisation and euploidy rates of day0-ICSI and day1-ICSI were 80.0% vs. 70.9%, 74.7% vs. 33.6%, 57.1% vs. 17.9%, and 52.3% vs. 30.8% respectively. The fertilisation (p = 0.0024), blastulation (p = 0.0001), utilization (p = 0.001) and euploidy (p = 0.0205) rates of delayed-ICSI were significantly lower compared to day0-ICSI. In Group B, the fertilisation, blastulation, blastocyst utilisation and euploidy rates of day0-ICSI and day1-ICSI were 77.3% vs. 64.1%, 75.0% vs. 37.5%, 52.7% vs. 20.6% and 26.0% vs. 32.1% respectively. Delayed-ICSI showed significantly lower fertilisation (p = 0.0001), blastulation (p = 0.0001) and utilization (p = 0.0001) rates than day0-ICSI, but the euploidy rate was comparable (p = 03997). In Group C, the fertilisation, blastulation and blastocyst utilisation rates of day0-ICSI and delayed-ICSI were 76.6% vs. 56.4%, 67.1% vs. 22.8%, 34.3% vs. 7.9% respectively. Similar to Group B, the fertilization (p = 0.0001), blastulation (p = 0.0001) and utilisation (p = 0.0003) rates in day0-ICSI was significantly higher than delayed-ICSI. No significant difference was observed between the euploidy rates of day0-ICSI and delayed-ICSI (18.9% vs. 0.0%, p = 0.3452). Despite all blastocysts derived from delayed-ICSI in this group being aneuploid, it could still increase the chances of these patients obtaining an euploid blastocyst. Limitations, reasons for caution Analysis on Group B was done on a larger sample size compared to Group A and C. A larger sample size in Group A and C is needed to further support our results. Wider implications of the findings: Delayed-ICSI generates additional blastocysts for low responders from all age groups. Therefore, delayed-ICSI could be a routine procedure in low responder IVF patients in order to optimise the cycle and clinical outcomes. Trial registration number Not applicable

IT TAKES TWO TO TANGO: COMPARING SPERM PREPARATION TECHNIQUES AND THE EFFECT ON EMBRYO GRADING

Male factor infertility accounts for 40-50% of infertility among couples seeking assisted reproductive technology (ART). Research on a new parameter of sperm quality, known as DNA fragmentation, suggests that fragmentation rates >27% may hinder embryo development and increase rates of miscarraige, while lower fragmentation rates are associated with improved outcomes. Recently, microfluidic sperm sorting as a sperm selection method has shown to decrease DNA fragmentation rates by positively selecting healthy sperm, compared to the standard method of density gradient centrifugation.

Towards a Closed System for Extracting Lymphocytes from Whole Blood

Introduction: Cellular therapies promise new treatment options and potentially curative efficacy for a wide array of acute and chronic diseases. The ability to obtain viable, uncontaminated lymphocyte concentrates from peripheral blood is in high demand. A major barrier for translating lymphocyte-based therapies into clinical practice is the poor scalability of manual, labor- and time-consuming ‘open system’ methods (e.g. Ficoll density gradient centrifugation) currently being used for isolating lymphocytes. To address the well-known limitations of existing separation methods, we developed a two-stage cell isolation approach that enables highly-efficient extraction of lymphocytes from whole blood (WB) without the use of centrifugation or any open-air manipulations by (1) depleting ~99% of red blood cells (RBCs) from the sample via rapid sedimentation induced by HES (6% hydroxyethyl starch in 0.9% sodium chloride), and then (2) passing the leukocyte-rich supernatant through a high-throughput microfluidic device (based on our newly-developed approach of ‘controlled incremental filtration,‘ CIF) to separate lymphocytes from platelets and residual RBCs.Materials and Methods: WB was collected from healthy consenting volunteers under an IRB approved protocol. An optimization study was performed to identify parameters of the sedimentation protocol with the highest lymphocyte yield and minimal contamination. This optimal protocol was then used to compare performance of (i) the standard Ficoll density gradient separation method, and (ii) our new two-stage separation approach. Each WB sample was divided into two equal parts - one half was processed via the conventional density gradient method, and the other half was first mixed with HES, allowed to sediment, and then the supernatant was passed through a CIF-based microfluidic device (designed for high-precision separation of lymphocytes from smaller cells, and ~15-fold reduction of the sample volume) at a flow rate of 0.5 mL/min. For both separation methods, product yield and purity were quantified by performing CBC with a 5-part differential on all samples. Flow cytometry was used to measure viability of isolated lymphocytes, and activation level of residual platelets via P-selectin and phosphatidylserine (PS) exposure. Separated lymphocytes were cultured for 4 days using standard proliferation techniques. Total number of viable cells was determined using Trypan Blue exclusion dye and manual counting with a hemocytometer.Results and Discussion: The optimized sedimentation protocol, which involved mixing WB with 10% HES (v/v) and waiting 30 min to complete, yielded the best combination of lymphocyte recovery (72.1±2.2%) and RBC depletion (99.48±0.07%) for the first stage of our separation approach. In the split-sample study, our two-stage method overall recovered 70.4±0.7% of lymphocytes from the initial WB sample, with 98.2±0.6% viability, while depleting 92.4±0.4% of platelets and 99.62±0.1% of RBCs. In comparison, the Ficoll density gradient centrifugation method removed 99.95±0.2% of RBCs and 94.2±0.1% of platelets, while recovering 74.5±2.40% of lymphocytes, with 98.3±0.7% viability. Lymphocytes produced by our method showed a significnatly higher prolifiration capacity than cells separated via density gradient centrifugation (Fig. 1). Markers of activation for residual platelets in the lymphocyte concentrate produced by our two-stage method were several fold lower than for Ficoll density gradient separation: 1.9±0.1% (our method) vs. 8.4±7.0% (Ficoll) for P-selectin, and 2.0±1.5% (our method) vs. 4.4±1.5% (Ficoll) for PS exposure. Importantly, lymphocyte isolation using our two-stage method was 3-fold faster than the Ficoll density graduate separation for the same amount of WB sample: <60 min (our method) vs. >180 min (Ficoll).Conclusion: We developed an easy-to-use approach for extracting lymphocytes from peripheral blood with purity, viability and yield meeting the current standard for cellular therapies. Since our new approach is scalable, compatible with ‘closed system’ operation, produces lymphocytes with higher proliferation capacity, and is much faster than conventional density gradient separation, it could have a potentially transformative impact by accelerating research into new cellular therapies, and simplifying translation of these treatments into clinical practice. [Display omitted] DisclosuresGifford:Halcyon Biomedical Incorporated: Employment, Equity Ownership. Shevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Selection of high-quality sperms by nanotechnological method of magnetic activation in Brazilian cervids

Cervids show a high degree of abnormalities in their sperm cells. Thus, this study aimed to select high-quality spermatozoa using magnetic-activated sperm sorting (MASS) compared to density gradient centrifugation (DGC) by assessing the post-selection cell quality. Semen from six Mazama deer was collected by electroejaculation after chemical restraint. The semen was analyzed in four samples: Fresh, DGC, SEMgood - non-apoptotic fraction, and SEMpoor - apoptotic fraction. The material was analyzed for motility and vigor (light microscopy), concentration (Neubauer chamber), semen morphology (phase contrast), and supravital staining test (eosin/ nigrosine). The DGC method used 20 x 106 cells in 90% and 45% percoll® gradient. The MASS used 10 x 106 cells with 20 μl of iron nanoparticles attached to Annexin V and filtration in a magnetic separation column. Both processing methods (DGC and MASS) were effective in producing high-quality sperm samples, with a marked reduction in abnormalities from 41.83 ± 10.25 (fresh) to 14.83 ± 3.17(DGC) and 12 ± 3.01(SEMgood), with 80.3% ± 2.06 livings cells. These findings suggest that this nanotechnological method, using nanoparticles, effectively produces high-quality semen samples in cervids for use in assisted reproduction.

Functional Sites of Ribosome Modulation Factor (RMF) Involved in the Formation of 100S Ribosome.

One of the important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosomal activity is dependent on the environmental situation of the cell. Bacteria form 100S ribosomes, lacking translational activity, to survive under stress conditions such as nutrient starvation. The 100S ribosome is a dimer of two 70S ribosomes bridged through the 30S subunits. In some pathogens of gammaproteobacteria, such as Escherichia coli, Yersinia pestis, and Vibrio cholerae, the key factor for ribosomal dimerization is the small protein, ribosome modulation factor (RMF). When ribosomal dimerization by RMF is impaired, long-term bacterial survival is abolished. This shows that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important survival strategy for bacteria. According to the results of several structural analyses, RMF does not directly connect two ribosomes, but binds to them and changes the conformation of their 30S subunits, thus promoting ribosomal dimerization. In this study, conserved RMF amino acids among 50 bacteria were selectively altered by mutagenesis to identify the residues involved in ribosome binding and dimerization. The activities of mutant RMF for ribosome binding and ribosome dimerization were measured using the sucrose density gradient centrifugation (SDGC) and western blotting methods. As a result, some essential amino acids of RMF for the ribosomal binding and dimerization were elucidated. Since the induction of RMF expression inhibits bacterial growth, the data on this protein could serve as information for the development of antibiotic or bacteriostatic agents.

Optimization of liver glycogen extraction when considering the fine molecular structure

Liver glycogen is a branched glucose polymer that functions as a blood-sugar buffer in animals. Previous studies have shown that glycogen’s molecular structure affects its properties. This makes it important to develop a technique that extracts and purifies a representative sample of glycogen. Here we aim to optimize the sucrose density gradient centrifugation method for preserving glycogen’s molecular structure by varying the density of the sucrose solution. The preservation of glycogen’s structure involves: 1) minimizing molecular damage and 2) obtaining a structurally representative sample of glycogen. The addition of a 10-minute boiling step was also tested as a means for denaturing any glycogen degrading enzymes. Lower sucrose concentrations and the introduction of the boiling step were shown to be beneficial in obtaining a more structurally representative sample, with the preservation of smaller glycogen particles and decreased glycogen chain degradation.

Evaluating differentiation potential of the human menstrual blood-derived stem cells from infertile women into oocyte-like cells

One of the most intricate infertility problems among women is the number and quality of the oocytes. Menstrual blood-derived stem cells (MenSCs) are a recently discovered source of mesenchymal stem cells which is known as a suitable source of cells for regenerative medicine. We aimed to investigate whether MenSCs as autologous cell source from endometriosis, PCOS, and healthy women have different characteristics regarding their morphology, CD marker expression pattern, differentiation potential into oocyte-like cells, and oocyte-related genes expression.Menstrual blood samples (1−2 ml) from healthy and infertile women (PCOS and endometriosis) in the age range of 22–35 years were collected. Isolated MenSCs by the Ficoll-Paque density-gradient centrifugation method was characterized by flow cytometry. MenSCs were induced under 20 % follicular fluid (FF), and then they were evaluated for differentiation by Real time-PCR and immunocytochemistry assay.MenSCs derived from endometriosis women had different morphology from PCOS and healthy women, but similar regarding their CD marker pattern. All induced MenSCs showed morphological changes and expressed oocyte related genes (STELLA, GDF9, STRA8, PRDM, LHR, FSHR, SCP3, DDX4, and ZP2) in the 2nd week of culture, but there was a significant difference between the groups. Endometriosis-derived MenSCs showed higher levels of both gene and protein expressions.These findings propose that MenSCs derived from endometriosis and PCOS patients under 20 % FF, not only could differentiate into oocyte-like cells, but also showed more differential potential in comparison with healthy women. This indicates the possibility of using the patients’ own MenSCs to differentiate into the oocyte-like cells.

Sperm selection of cryopreserved milt of catfish (Rhamdia quelen) by density gradient centrifugation with AllGrad® 90%

Density gradient centrifugation is a technique used to wash or separate samples of cryopreserved milt, mainly in humans and bovines allowing, for example, reducing the concentration of cryoprotectants or choosing the best portion of sperm. The proposed method seeks to reduce the presence of cryoprotectant in the cryopreserved milt of the Rhamdia qhelen and to obtain a fraction of better quality sperm. Gradient centrifugation was formed from 90% AllGrad® and different centrifugation times and forces were compared. The separated sperm presented a low increase in motility and decreased head damage and presence of gout, however, it was better compared to the non-separated samples. The speed of 1000 × g for 10 min, 4 °C, allowed 22.25 ± 4.64% of normal spermatozoa, that is, 9.25% more than the non-centrifuged milt (p = 0.0013).•The centrifugation method allows a fraction of spermatozoa morphologically less affected by cryopreservation.•Density gradient centrifugation with AllGrad® 90% is proposed as a tool of easy adaptation and application for the separation of cryopreserved sperm of R. quelen.•Density gradient centrifugation method at 1000 × g for 10 min allows obtaining a better fraction of normal sperm.

Chemical looping gasification of maceral from low-rank coal: Products distribution and kinetic analysis on vitrinite

The product distribution and kinetic analysis of low-rank coal vitrinite were investigated during the chemical looping gasification (CLG) process. The acid washing method was used to treat low-rank coal, and the density gradient centrifugation method was adopted to obtain the coal macerals. By combining thermogravimetric analysis and online mass spectrometry, the influence of the heating rate and oxygen carrier (Fe2O3) blending ratio on product distribution was discussed. The macroscopic kinetic parameters were solved by the Kissinger-Akahira-Sunose (KAS) method, and the main gaseous product formation kinetic parameters were solved by the iso-conversion method. The results of vitrinite during slow heating chemical looping gasification showed that the main weight loss interval was 400–600 °C, and the solid yield of sample vitrinite-Fe-10 at different heating rates was 64.30%–69.67%. When β = 20 °C·min−1, the maximum decomposition rate of vitrinite-Fe-10 was −0.312%·min−1. The addition of Fe2O3 reduced the maximum decomposition rate, but by comparing the chemical looping conversion characteristic index, it could be inferred that the chemical looping gasification of vitrinite might produce volatile substances higher than the pyrolysis process of vitrinite alone. The average activation energy of the reaction was significantly reduced during chemical looping gasification of vitrinite, which was lower than the average activation energy of 448.69 kJ·mol−1 during the pyrolysis process of vitrinite alone. The gaseous products were mainly CO and CO2. When the heating rate was 10 °C·min−1, the highest activation energy for CH4 formation was 21.353 kJ·mol−1, and the lowest activation energy for CO formation was 9.7333 kJ·mol−1. This study provides basic data for exploring coal chemical looping gasification mechanism and reactor design by studying the chemical looping gasification process of coal macerals.

Phenotypic Changes of PD-1 and GITR in T Cells Are Associated With Hepatitis B Surface Antigen Seroclearance.

Regulatory T cells (Tregs) possess hepatitis B virus (HBV)-specific immunoregulatory effects in chronic HBV infection. The role of Tregs in spontaneous seroclearance of hepatitis B surface antigen (HBsAg) remains to be determined. We recruited treatment-naive chronic HBV patients achieving spontaneous HBsAg seroclearance (experimental group) and matched HBsAg-positive controls. Peripheral blood mononuclear cells were isolated using the Ficoll-Paque density gradient centrifugation method. The frequency of Tregs and inhibitory phenotypes and immunoregulatory cytokines of Tregs were detected by flow cytometry. Twenty-seven patients with HBsAg seroclearance (mean age: 52.40±6.00 y, 55.6% male) and 27 matched controls were recruited. Median HBsAg and HBV DNA levels in the control group were 2.80 (1.24 to 3.43) and 3.16 (1.68 to 3.85) log IU/mL, respectively. Mean frequencies of Tregs and expressions of FoxP3+ Tregs were comparable in both groups (both P>0.05). The mean expression of programmed death 1 (PD-1) and glucocorticoid-induced TNFR family-related gene (GITR) in total CD4+ T cells were significantly downregulated in the experimental group when compared with the control group (10.62% vs. 13.85%, P=0.003; 16.20% vs. 27.02%, P=0.002, respectively). When compared with the control group, PD-1+CD4+ Tregs expression in the experimental group was significantly downregulated (13.85% vs. 10.62%, P=0.003). A similar phenomenon was noted for GITR+CD8+ Tregs (20.16% vs. 14.08%, P=0.049). Intracellular cytokine productions showed no significant differences (all P>0.05). The reduced expression of PD-1 and GITR might attenuate the immunosuppressive capability of Tregs. Decreased expression on CD4+ T cells might represent an enhanced antiviral function, playing a role in initiating the "functional cure" of chronic HBV infection.

Differential expression of apoptosis related genes in the peripheral blood mononuclear cells of acute lymphoblastic/lymphocytic leukemia (ALL) patients

Apoptosis is the cell’s intrinsic death program which plays a crucial role in the regulation of many normal physiological processes in the body’s tissues. In leukemia patients, the extent of cancer cell susceptibility to apoptosis is correlated with clinical responses to chemotherapy and disease prognosis. The aim of this study was evaluation of the expression of apoptosis related genes in the peripheral blood mononuclear cells (PBMCs) of acute lymphoblastic/lymphocytic leukemia (ALL) patients. Peripheral blood samples were obtained from 20 patients with ALL and 20 healthy individuals. PBMCs were isolated using Ficoll-Paque density gradient centrifugation method. After RNA extraction and cDNA synthesis, gene expression levels of apoptosis related genes including caspase-3, 8, 9, BAX, and BAK genes were measured by real-time RT-PCR technique. Gene expression analysis showed that the expression levels of the initiator caspases-8 and -9 are increased in the PBMCs of adult ALL patients when compared with that in PBMCs of healthy individuals. Increased gene expression levels of the proapoptotic protein BAK was also detected in PBMCs of ALL patients. In contrast, decreased expression levels of the proapoptotic BAX and the executioner caspase-3 were observed in the PBMCs of ALL patients. These results suggest that the expression of genes involved in both extrinsic and intrinsic signaling pathways of apoptosis are induced in PBMCs of ALL patients while the gene expression of other proapoptotic molecule, BAK, and the executioner caspase-3 diminished in PBMCs cells of ALL patients. This findings indicate that resistance to apoptosis may be one of the hallmarks of ALL cells.

A NOVEL METHOD TO ATTENUATE EMBRYO ANEUPLOIDY DUE TO PATERNAL INHERITANCE

To test the efficacy of a sperm-selection method to generate genetically healthy embryos in couples with recurrent embryo aneuploidy. Consenting men with high sperm DNA fragmentation (SCF) in their spermatozoa who were treated at our center during the last three years were included. Spermatozoa were selected by density gradient centrifugation (DGC) for ICSI cycles, which frequently yielded aneuploid embryos, resulting in no embryo transfer or failed implantation. In subsequent ICSI cycles, their spermatozoa were processed by microfluidics sperm selection (MFSS). SCF, including the assessment of double-stranded DNA breaks (DSBs), was performed before and after each selection method. SCF, embryo aneuploidy, and clinical outcomes were assessed and compared. A total of 29 men underwent semen analysis according to WHO 2010 criteria. SCF was carried out by TUNEL on at least 500 spermatozoa with an established threshold of 15%. DSBs were measured by a neutral Comet test using a modified in-house protocol. A threshold of ≤3% was considered normal, with at least 200 spermatozoa assessed per patient. Clinical outcomes were compared between ICSI cycles performed with the DGC and MFSS methods. Preimplantation genetic testing for aneuploidy (PGT-A) was carried out on the resulting blastocysts. Euploid embryos were replaced into the uterine cavity, and implantation and clinical pregnancy rates (CPR) were recorded. A total of 29 men (40±7 years) had the following semen parameters: a concentration of 38.7 ± 39 x106/mL, 31 ± 17% motility, and 2.1 ± 1% morphology. After DGC and MFSS, the sperm concentration was 18±29 and 7.3±11 x106/mL, with 55.2±30% and 98.3±2% motility, respectively (P<0.0001). The average SCF decreased from 22±9% in the raw samples to 16±3% following DGC, and dropped to 1.8±1% after MFSS processing (P<0.0001). The DSB rate was 3.8±2% in the raw samples, 2.8±1% after DGC, and only 0.3±0.1% after MFSS. These couples (female partner, 37.2±5 years) underwent 42 ICSI cycles with DGC-selected spermatozoa and achieved a fertilization rate of 61.8% (254/411) with 23.8% (26/109) of the embryos identified as euploid. The replacement of these euploid embryos yielded an implantation rate of only 4.3% (1/23) and a CPR of 8.3% (1/12), with 8.3% (1/12) resulting in a pregnancy loss. When these couples underwent subsequent ICSI cycles with MFSS, a fertilization rate of 80.2% (334/416) (P<0.00001) was achieved, with 48.9% (90/184) of the embryos identified as euploid (P<0.0001). A total of 29 embryos were replaced, resulting in an implantation rate of 65.5% (19/29; P<0.0001) and a CPR of 73% (19/26) (P<0.001), resulting in 5.2% (1/19) pregnancy loss and 69.2% (18/26) ongoing pregnancy or deliveries (P<0.0001). Spermatozoa with DNA damage, particularly double-stranded nicks/breaks, may impair the ability of the oocyte to repair the male genome. Because the male genome contributes to embryo aneuploidy, selecting spermatozoa with the highest motility and chromatin integrity may enhance the chances of obtaining a euploid embryo for transfer and boost embryo implantation.