Double Density Gradient Centrifugation Research Articles

Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

New Approaches to Define The Functional Competency of Human Sperm Subpopulations and Its Relationship to Semen Quality.

BackgroundThis study aimed at comparing a comprehensive set of functional and structural sperm characteristics be- tween sperm motility fractions and correlating results to the standard semen parameters. By grouping related variables, our objective was to establish the predictive power of semen parameters and whether they accurately reflect the function- ality of sperm motility fractions or merely a small set of parameters within individual fractions.Materials and MethodsIn this non-invasive experimental study, donor semen samples (n=55) were separated via double density gradient centrifugation, isolating a high (HM) and low motile (LM) sperm fraction. Fractions were evaluated for percentage vitality, chromatin integrity, mature spermatozoa, motility and kinematic parameters, hyper- activation, positive reactive oxygen species, intact mitochondrial membrane potential (MMP) and acrosome reaction.ResultsHM fractions had significantly (P<0.001) enhanced percentages of induced acrosome reaction (HM, 55.6 ± 14.3%, LM, 25.0 ± 16.5%), motility and kinematic parameters, hyperactivation, vitality (HM, 70.4 ± 9.7%, LM, 47.9 ± 10.3%), mitochondrial membrane intactness (HM, 67.2 ± 10.4%, LM, 44.7 ± 15.0%) and mature spermatozoa (HM, 83.4 ± 10.0%, LM, 64.6 ± 8.2%) with intact chromatin (HM, 80.5 ± 8.1%, LM, 71.3 ± 8.0%). Various sperm mor- phology abnormalities correlated with LM fractions’ grouped motility parameters (range, 0.46 to 0.51; range -0.4 to -0.75), whereas combined semen traits of total motility, progressive motility, viscosity and mucus penetration (MPT) correlated with HM fractions’ grouped motility parameters (range, 0.44 to 0.84).ConclusionCollectively, total and progressive motility, viscosity and MPT may represent a reliable grouping of semen parameters for predicting the quality of HM sperm fractions. Separating the same donor semen samples into two signifi- cantly diverse motility sperm fractions could be a potential model in mimicking the qualities of fertile and sub-fertile males’ sperm populations and used for future research on the improvement of sperm subpopulations from males with different fertility statuses.

Progesterone, Myo-Inositol, Dopamine and Prolactin Present in Follicular Fluid Have Differential Effects on Sperm Motility Subpopulations.

Considering the challenges surrounding causative factors in male infertility, rather than relying on standard semen analysis, the assessment of sperm subpopulations and functional characteristics essential for fertilization is paramount. Furthermore, the diagnostic value of sperm interactions with biological components in the female reproductive tract may improve our understanding of subfertility and provide applications in assisted reproductive techniques. We investigated the response of two sperm motility subpopulations (mimicking the functionality of potentially fertile and sub-fertile semen samples) to biological substances present in the female reproductive tract. Donor semen was separated via double density gradient centrifugation, isolated into high (HM) and low motile (LM) sperm subpopulations and incubated in human tubal fluid (HTF), capacitating HTF, HD-C medium, progesterone, myo-inositol, dopamine and prolactin. Treated subpopulations were evaluated for vitality, motility percentages and kinematic parameters, hyperactivation, positive reactive oxygen species (ROS), intact mitochondrial membrane potential (MMP) and acrosome reaction (AR). While all media had a significantly positive effect on the LM subpopulation, dopamine appeared to significantly improve both subpopulations’ functional characteristics. HD-C, progesterone and myo-inositol resulted in increased motility, kinematic and hyperactivation parameters, whereas prolactin and myo-inositol improved the LM subpopulations’ MMP intactness and reduced ROS. Furthermore, progesterone, myo-inositol and dopamine improved the HM subpopulations’ motility parameters and AR. Our results suggest that treatment of sub-fertile semen samples with biological substances present in follicular fluid might assist the development of new strategies for IVF treatment.

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

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

Sperm enrichment from poor semen samples by double density gradient centrifugation in combination with swim-up for IVF cycles

Sperm preparation in IVF cycles using density gradient centrifugation (DGC) in combination with swim-up (SU) has been widely adopted in reproductive centres worldwide. It is a fact that the sperm recovery rate following one DGC from poor semen samples (showing liquefaction defects/containing too many unresolvable clots or rare sperm) is relatively low. Our results showed that double DGC (DDGC) is effective at increasing the sperm recovery rate from poor semen samples. However, DDGC may increase the mechanical stress of sperm, thereby potentially impairing embryo development. Therefore, it is necessary to evaluate the safety of using sperm prepared by DDGC/SU for IVF cycles. In this study, we retrospectively analysed the data generated from a total of 529 IVF cycles (from June 2017 to June 2018), and these IVF cycles contributed 622 transfer cycles (from June 2017 to December 2018) in Changzhou Maternal and Child Health Care Hospital. Of them, 306 IVF cycles and the related 355 transfer cycles (normal semen samples prepared by DGC/SU) were set as the normal group, while 223 IVF cycles and the related 267 transfer cycles (poor semen prepared by DDGC/SU) were set as the observation group. The main outcome measures, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, clinical pregnancy rate and live birth rate, birth weight and duration of pregnancy, were compared between the two groups. Compared to semen in the DGC/SU group, semen in the DDGC/SU group showed increased levels of the DNA fragmentation index (DFI) and reduced sperm concentration, percentage of progressive motility (PR) sperm, and percentage of normal morphology sperm. The indicators reflecting in vitro embryo development and clinical outcomes were similar in the DGC/SU group and DDGC/SU group, including the normal fertilization rate, top D3 embryo formation rate, blastocyte formation rate, pregnancy rate, implantation rate, spontaneous abortion rate, live birth rate, birth weight and duration of pregnancy. Furthermore, we found that the 1PN zygote formation rate was significantly lower in the DDGC/SU group than that in the DGC/SU group. We concluded that oocytes fertilized by sperm from poor semen samples separated by DDGC/SU achieved the same outcomes as oocytes fertilized by sperm from normal semen separated by DGC/SU, suggesting that DDGC/SU is an effective and safe method of sperm enrichment for poor semen samples in IVF. The main contribution of the present study is the verification of the effectiveness of DDGC/SU in improving sperm recovery from poor semen samples and the safety of using sperm prepared by DDGC/SU for IVF.

Isolation of human monocytes by double gradient centrifugation and their differentiation to macrophages in teflon-coated cell culture bags.

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

Segregation of sperm subpopulations in normozoospermic infertile men

Sperm function is essential for fertilization and embryogenesis yet semen contain a heterogeneous population of sperm. This study was designed to evaluate two different sperm populations separated by the density gradient method. Semen from 25 idiopathic normozoospermic infertile men was processed by double density gradient centrifugation and evaluated for sperm present in the 50% (upper) layer and the 90% (lower) layer for reactive oxygen species (ROS), sperm chromatin integrity, and morphology. The population of sperm in the 90% layer showed significantly lower ROS levels (22.90 (0.92, 85.32) vs. 382.03 (158.30, 1409.51) and lower DNA fragmentation index (DFI) (24.26 (22.54, 25.50) vs. 29.93 (28.48, 31.25) and higher number of sperm with normal morphology (55 (45.0, 60.0) vs. 32.5 (20, 40) compared to sperm in the 50% layer. However, in the original raw semen, sperm DFI (27.02 (26.19, 27.76)) and percentage high DNA stainability (% HDS) (3.1 (2.40, 3.78)) cells were significantly higher compared to the 90% layer population. Density gradient separation of the sperm subpopulation from the original semen favors the selection of sperm with genome integrity, low levels of ROS, and normal morphology. Therefore presence of pathological sperm in the semen may disrupt the function of normal spermatozoa, and hence the selection of the normal sperm subpopulation may be a better candidate for assisted conception. Further studies are required to evaluate the gradient separated sperm population in assisted reproductive techniques (ART).

Effect of recombinant canine interleukin‐6 and interleukin‐8 on tissue factor procoagulant activity in canine peripheral blood mononuclear cells and purified canine monocytes

Inflammation is a major cause of disseminated intravascular coagulation (DIC) in dogs, but underlying mechanisms for its initiation are unknown. We hypothesized that pro-inflammatory cytokines, interleukin (IL)-6 and IL-8, induce tissue factor (TF) expression on canine monocyte surfaces, which may contribute to DIC initiation. The objectives of this study were to determine if (1) IL-6 and IL-8 would induce TF activity on canine monocytes, (2) fetal bovine serum or autologous plasma was required for IL-6- or IL-8-induced TF responses in canine monocytes, and (3) these pro-inflammatory cytokines would enhance TF activity on canine monocytes in response to low concentrations of lipopolysaccharide (LPS). Canine monocytes were isolated from EDTA-anticoagulated blood as peripheral blood mononuclear cells (PBMC) by double-density gradient centrifugation and adhesion to plastic. Adherent cells were stimulated for 4 hours with recombinant canine (rc)-IL-6 or rc-IL-8 (10-5000 pg/mL) with or without 10% heat-inactivated (HI) fetal bovine serum, untreated autologous canine plasma (ACP), or HI-ACP. Lipopolysaccharide (100 ng/mL) served as a positive control. Cells were also costimulated with either cytokine (100 pg/mL) or low concentrations of LPS (0.1 and 1 ng/mL). Monocytes immunopurified from PBMC with anti-CD14 antibodies were also stimulated with both cytokines (100 and 5000 pg/mL). TF activity on cell surfaces was measured by a 2-stage amidolytic assay, based on activated factor X generation. Neither rc-IL-6 nor rc-IL-8 consistently stimulated TF procoagulant activity in canine PBMC or purified monocytes after 4 hours. Serum, plasma, or low concentrations of LPS did not enhance the TF response to these cytokines. IL-6 or IL-8 at evaluated concentrations may not play major roles in coagulation activation by induction of TF expression on monocytes in dogs with inflammation.

Interleukin-10 inhibits lipopolysaccharide-induced upregulation of tissue factor in canine peripheral blood monocytes

The potentially fatal hemostatic disorder of disseminated intravascular coagulation (DIC) is initiated in bacterial sepsis by lipopolysaccharide (LPS)-induced tissue factor (TF) expression on monocytes. Interleukin-10 (IL-10) is a potent inhibitory cytokine that downregulates monocyte inflammatory and procoagulant responses. We hypothesized that canine recombinant IL-10 (rIL-10) would inhibit LPS-induced TF upregulation on canine monocytes in a dose-dependent manner. Canine peripheral blood mononuclear cells (PBMC), obtained by double-density gradient centrifugation, and monocytes, purified from PBMC by immunomagnetic bead separation with an anti-canine CD14 antibody (Ab), were stimulated in suspension with LPS (0.1–1000ng/mL) for various times. Recombinant IL-10 (10–5000pg/mL) was added with LPS or up to 2h later. Tissue factor procoagulant activity was measured by cleavage of a chromogenic substrate by activated Factor X generated by the TF-factor VII complex. We found that rIL-10, when given concurrently or 1h after LPS, strongly inhibited LPS-induced TF procoagulant activity in canine PBMC and monocytes. This inhibition was dose-dependent and blocked by an anti-canine IL-10 Ab. Our results indicate that rIL-10 effectively inhibits LPS-induced TF upregulation in canine monocytes and could potentially be useful in limiting the development of DIC in dogs with endotoxemia.

Comparision in the yield of fetal nucleated red blood cell between the first-and second-trimester using double density gradient centrifugation

Objective: The aim of our study was to make a practical comparative evaluation of the first and second trimesters in order to determine the period during which a higher yield of fetal nucleated red blood cells (FNRBCs) can be obtained. Methods: NRBCs were isolated from maternal blood during the first and second trimesters of pregnancy using double Percoll gradients with different osmolarities. Magnetic activated cell sorting was performed with Kleihauer-Betke stain. We isolated fetal NRBCs from 10 mL of samples of maternal blood and determined fetal sex and fetal aneuploidy by fluorescence in situ hybridization (FISH). Results: The average number of NRBCs was 9.85 in samples obtained during the first trimester and 14.88 in samples obtained during the second trimester (P=0.07). The average number of NRBCs with Y chromosome signals was 5.73 in the first trimester and 8.22 in second trimester (P=0.56). However, the percentage of NRBCs with Y chromosome signals in the first trimester (70.6%) was significantly higher than in the second trimester (59.8%) (P=0.049). We diagnosed the blood samples from 7 pregnant women having fetal aneuploidy using this method and the number of NRBCs was 18.4. Conclusion: The method using Percoll osmolarity and a double density gradient system may be a very useful method for separation of NRBCs in the first trimester of pregnancy and also in the second trimester.

Sperm preparation by magnetic-activated cell sorting (MACS) reduced apoptotic sperm and improved acrosome reaction for unexplained infertility

OBJECTIVE: This study was designed to evaluate the effect of magnetic-activated cell sorting (MACS) using Annexin V-conjugated microbeads in sperm preparation for patients with unexpalined infertility and repeated intrauterine inseminaiton (IUI) failure. DESIGN: This study collected semen samples from male partners of couples with unexplained infertility and repeated IUI failure. The rearch design was a self-matched case control study. MATERIALS AND METHODS: This study was approved by institutional review board of Chung Shan Medical University Hospital. Semen samples (n=41) with normal semen analysis (sperm count≥20X10, motility>50% and morphology>14%) were collected. After double density gradient centrifugation (DGC), the sperm cell suspension was divided into two separate fractions with or without subsequent MACS. And then, we checked the induced acrosome reaction and the apoptosis of sperm by AnnexinV/PI, TUNEL assay (terminal dUTP nick-end labeling) and mitochondrial membrane potential using JC-1. Wilcoxon signed rank test was utilized to calculate the difference between samples. RESULTS: Following MACS, the induced acrosome reaction was significantly improved (45.9±5.3 vs. 32.8±4.3, respectively, p<0.001). The proportion of sperm DNA fragmentation(9.2±0.7% v.s. 12.5±1.0%), mitochondrial membrane potential disruption (18.7±1.9% v.s. 27.2±3.0%) and the externalization of phosphatidylserine (5.9±1.3% v.s 8.2±2.0%) subsequent to MACS was significantly reduced compared to the DGC. CONCLUSIONS: Magnetic-activated cell sorting using annexin-V microbeads effectively separated apoptotic and non-apoptotic spermatozoa based on the externalization of phosphatidylserine (PS) to the outer membrane leaflet. The acrosome reaction percentage was also significantly elevated. MACS in combination with DGC could be used as a sperm preparation technique to enhance the sperm quality and function for unexplained infertility.

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

To develop a method whereby sperm with phosphatidylserine externalization can be separated from those without this feature. Because annexin V binds phosphatidylserine, this study is using this feature to select functional spermatozoa. In addition, the relationship between annexin V binding in human spermatozoa and normal sperm morphology according to strict criteria was to be assessed. Prospective study. Department of Obstetrics and Gynaecology of Stellenbosch University at Tygerberg Academic Hospital, Tygerberg, South Africa. Semen from 14 healthy sperm donors. Exclusion criterion was the presence of less than 20 x 10(6)/mL total motile spermatozoa in the original sample. Annexin V-negative sperm, annexin V-positive sperm, normal sperm morphology. An aliquot of a semen sample after double density gradient centrifugation was incubated with annexin V fluorescein isothiocyanate conjugate (FITC). Cell fluorescence signals were determined using a FACScalibur flow cytometer equipped with a FACSSort fluidic sorting module. The sorting procedure delivered two sperm subpopulations: annexin V-negative and annexin V-positive. Morphology slides were made and the sperm morphology was assessed according to strict criteria. There was a significant enrichment of annexin V-negative sperm as well as morphologically normal sperm in the annexin V-negative subgroup after separation with flow cytometry. The percentage of morphologically normal sperm increased from 8.3% in the control to 11.9% in the annexin V-negative fraction, whereas the percentage of annexin V-positive sperm decreased to 5.7%. The annexin V-negative sperm subpopulation had morphologically superior quality sperm compared to annexin V-positive sperm. It is important to select morphologically normal sperm during intracytoplasmic sperm injection (ICSI) as it may contribute to increased implantation and pregnancy rates (PR).

Phenotypic and Genetic Characterization of Circulating Tumor Cells by Combining Immunomagnetic Selection and FICTION Techniques

The presence of circulating tumor cells (CTCs) in breast cancer patients has been proven to have clinical relevance. Cytogenetic characterization of these cells could have crucial relevance for targeted cancer therapies. We developed a method that combines an immunomagnetic selection of CTCs from peripheral blood with the fluorescence immunophenotyping and interphase cytogenetics as a tool for investigation of neoplasm (FICTION) technique. Briefly, peripheral blood (10 ml) from healthy donors was spiked with a predetermined number of human breast cancer cells. Nucleated cells were separated by double density gradient centrifugation of blood samples. Tumor cells (TCs) were immunomagnetically isolated with an anti-cytokeratin antibody and placed onto slides for FICTION analysis. For immunophenotyping and genetic characterization of TCs, a mixture of primary monoclonal anti-pancytokeratin antibodies was used, followed by fluorescent secondary antibodies, and finally hybridized with a TOP2A/HER-2/CEP17 multicolor probe. Our results show that TCs can be efficiently isolated from peripheral blood and characterized by FICTION. Because genetic amplification of TOP2A and ErbB2 (HER-2) in breast cancer correlates with response to anthracyclines and herceptin therapies, respectively, this novel methodology could be useful for a better classification of patients according to the genetic alterations of CTCs and for the application of targeted therapies.

Evaluation of poly (ADP-ribose) polymerase cleavage (cleaved-PARP) in sperm fractions after sperm apoptosis induction

Sperm cell apoptosis is an energy and protein dependent process that results in sperm DNA damage. PARP cleavage (cPARP) is stimulated by DNA breaks for help in repair process, so it is considered as an early marker for apoptosis. Staurosporine (STS) is a PARP inducer while 3-aminobenzamide (3ABA), is a PARP inhibitor. Sperm DNA damage due to oxidative stress is considered as one of the causes of male infertility. Conflicting reports exist regarding the presence of whole or cPARP in human ejaculated spermatozoa. We evaluated: 1) the presence of cPARP in mature ejaculated spermatozoa and 2) if we can control its level in sperm fraction using apoptosis inducers with or without PARP inhibitor. Prospective-controlled study. Semen samples were collected from 10 healthy donors. The neat semen samples were divided into mature and immature sperm fractions using double density gradient centrifugation. Mature and immature sperm fractions were resuspended into HTF media and divided in 5 aliquots (control, H2O2 treated, H2O2 + 3ABA, STS treated, and STS + 3ABA) incubated at 37°C with 5 % CO2 for 1 hour. The final concentrations were 2.5 μM for STS, 30 μM for H2O2, and 0.3 mM for 3ABA. After incubation, the mature and immature aliquots were evaluated for the early and late apoptosis by 1) annexin V, activated caspase-3, 2) sperm DNA damage by TUNEL and 3) cPARP by FACS. cPARP was detected in both neat and mature fractions. There were no significant differences between mature versus immature fractions regarding most of molecular apoptosis markers for treated and untreated sperm fractions for cPARP modulation.Table 1Correlation between the measured parameters in different treatment groupsUntreated sperm fractionsTreatmentActivated Caspase-3 (a C-3)TUNEL + sperm%Cleaved PARP + %Early apoptotic sperm %Late apoptotic sperm %rPrPrPrPrPSTS treated sperm0.960.120.30.50.720.90.420.130.60.12STS + 3ABA treated sperm0.940.0040.60.570.80.170.240.040.16<0.001H2O2 treated sperm0.940.110.80.20.80.26-0.190.002-0.13<0.001H2O2 + 3ABA treated sperm0.970.0020.510.560.81-0.7-0.20.003-0.06<0.001Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Open table in a new tab Matched pairs' correlation for the between each measured parameter in various treated versus untreated groups. Cleaved PARP is present in neat as well as in washed ejaculated human spermatozoa. Its level is related to the extent of sperm DNA damage. PARP inhibitors can not prevent/repair oxidative or chemical-induced sperm DNA damage.

Assessment of sperm motility, viability and apoptosis in human spermatozoa after hydrogen peroxide exposure

ObjectiveMale infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2).DesignProspective study.Materials and methodsSemen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence.ResultsTableComparing different sperm parameters after exposure to H2O2Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. Open table in a new tab ConclusionsExposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage. ObjectiveMale infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2). Male infertility has been linked to excessive production of ROS in semen. ROS is involved in sperm cell apoptosis signaling pathway. Apoptosis is distinguished from necrosis, or accidental cell death, by characteristic morphological and biochemical changes. Our objective was to investigate sperm motility, viability, and incidence of apoptosis after exposure to hydrogen peroxide (H2O2). DesignProspective study. Prospective study. Materials and methodsSemen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence. Semen samples were collected from 12 semen donors. The neat samples were subjected to double density gradient centrifugation to separate mature and immature sperm subpopulation. Each was resuspended in buffer and divided into 2 aliquots. One aliquot was incubated with 50 uL of freshly prepared H2O2 added to 1 mL of sperm suspension at a final concentration of 20 uM for 15 min at 37°C, and the other aliquot served as control. The incidence of apoptosis and necrosis was assessed by flowcytometry using the YoPro-1 dye which enters apoptotic cells giving green fluorescence, and propidium iodide (PI), which stains necrotic sperm cells giving red fluorescence. ResultsTableComparing different sperm parameters after exposure to H2O2Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. Open table in a new tab Values are mean (±SD). Paired Student t-test was utilized to compare different variables in different spem fractions. P<0.05 was considered significant. ConclusionsExposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage. Exposure of human spermatozoa to H2O2 is associated with a decrease in sperm viability. The increase in the proportion of apoptotic sperm reached significance only in the immature fractions. There was no apparent effect on sperm motility. Thus sperm motility may not serve as a marker for early oxidative sperm damage.

Relationship between sperm apoptosis signalling and oocyte penetration capacity

Human sperm have been documented to display apoptosis-like features such as externalization of phosphatidylserine (EPS), disruption of the transmembrane mitochondrial potential (MMP) and activation of caspases. Our aim was to evaluate possible association between activation of the apoptosis cascade in human sperm and its oocyte penetration capacity using the zona free hamster oocyte penetration assay (SPA). Semen specimens from 76 unselected donors were subjected to double density gradient centrifugation followed by incubation under capacitating conditions for 3 h and SPA. Apoptosis signalling was monitored by assessment of EPS, disruption of MMP and activation of caspase-3 by flow cytometry. Semen samples with subnormal SPA values (<20% penetrated oocytes) contained significantly higher amounts of spermatozoa with EPS, disrupted MMP and activated caspase-3 compared with those samples with normal SPA values (>20% penetrated oocytes, p < 0.01). All three apoptosis markers showed a significantly negative correlation with the percentage of penetrated oocytes (p < 0.01). Apoptosis-related signalling appears to have a negative association with sperm-oocyte penetration. The exclusion of sperm presenting with those apoptosis-related features during assisted reproduction may improve success rates of procedures such as intrauterine insemination and in vitro fertilization.

Human herpesvirus 8 genoprevalence in populations at disparate risks of Kaposi's sarcoma

The prevalence of human herpesvirus 8 (HHV-8) in populations at different risks of developing Kaposi's sarcoma (KS) was assessed using a protocol involving immunomagnetic fractionation of CD45+ blood cells prior to detection of the HHV-8 genome by nested PCR. Preliminary studies using blood of eight gay men infected by human immunodeficiency virus-1 (HIV-1) revealed that, for the detection of HHV-8 DNA derived from open reading frame (ORF) 26 of the HHV-8 genome, this protocol provided substantially higher rates (100%) compared to one involving red blood cell (RBC) lysis (0%) and to another requiring double density gradient centrifugation (DDGC) of leukocytes (13%). When the CD45+ fractionation protocol was applied to samples from 103 other HIV-1-infected patients (the vast majority of whom were gay men) and 100 blood donors, the ORF 26 DNA detection rates obtained were 37% and 8%, respectively. When DNA from the variable region 1 of ORF K1 was additionally amplified from samples of the blood donors, a detection rate of 9% was achieved. This rate was highly concordant with the ORF 26 DNA detection rate. Furthermore, the ORF K1 sequences were predominantly unique, assignable to genotypes A1, A4, and C3. When assays for anti-HHV-8 and anti-herpes simplex viruses (HSV) 1 and 2 were applied, significant concordances between HHV-8 DNA detection rates and those relating to anti-HHV-8 and to anti-HSV 1 and 2 were more frequently observed for HIV-1-infected patients than for blood donors. The higher-than-expected HHV-8 genoprevalence rate in blood donors requires further confirmation in view of its implications for post-transfusion HHV-8 transmission.

P-988: Association of intermediate staining patterns using the Toluidine blue test with late apoptosis and necrosis in human spermatozoa

P-988: Association of intermediate staining patterns using the Toluidine blue test with late apoptosis and necrosis in human spermatozoa

P-989: Comparison of DNA contents of apoptotic sperm cells in neat and processed semen

Semen processing is used during assisted reproductive techniques to eliminate unwanted, damaging cells and debris from semen samples. To date little is known about the sperm DNA content and its relation to apoptosis or DNA fragmentation in neat semen and following density gradient centrifugation. Sperm DNA content can be evaluated by flow cytometry using propidium iodide (PI) stain. Flow cytometric analysis could quantitatively differentiate between intensities of haploid spermatid, spermatozoa with condensed chromatin and sub-haploid with low DNA content. Our objective was to investigate the DNA content of the late apoptotic and necrotic sperm cells in neat and processed semen following density gradient centrifugation. Prospective-controlled study. Semen samples were collected from 22 donors. The neat samples were divided into mature and sperm immature fractions using double density gradient centrifugation (90% and 47% layers). Both fractions as well as an aliquot of the neat sample were evaluated for the incidence of early apoptosis and necrosis using the annexin V/ PI assay using flow cytometry. To evaluate the DNA content, PI-positive cells were gated and subjected to analysis. In addition, the DNA fragmentation index (%DFI) and high DNA stainability index (%HDS) were measured using the sperm chromatin structure assay. Mature sperm fractions showed significantly lower abnormal DNA content parameters compared to neat samples: sub-haploid (p=0.003) and spermatid (p=0.03), while the number of sperm with condensed chromatin was comparable (Table). Neat semen and mature fractions showed significantly higher proportion of spermatozoa with condensed chromatin compared with immature fraction (p=0.02 and p=0.01, respectively). There was no significant difference between mature and immature fractions regarding the number of sub-haploid cells. In neat semen, there was a significant negative correlation between haploid apoptotic cells and %HDS (r=-0.48, p=0.04), while positive correlation was found between immature and sub-haploid apoptotic cells with %DFI (r=0.6, p<0.01). In mature and immature fractions there was strong positive correlation between each apoptotic haploid and spermatid with DFI (r=0.5, p<0.01 and r= 0.6, p=0.005, respectively).Tabled 1 Mature sperm fractions in processed semen contain more normal DNA content than neat and mature semen. The %DFI in neat semen could be related to immature and low DNA content cells, while %HDS could be related to diploid immature cells.