Variation in recovery of motile sperm after preparation by a simple Percoll gradient technique

PURPOSE: Numerous techniques have been used to prepare sperm for assisted reproduction technology. Density-gradient centrifugation with Percoll is becoming a method of choice. This study reviewed the results of a simple two-layer discontinuous Percoll gradient. METHODS: We reviewed retrospectively results obtained from 208 semen specimens prepared for in vitro fertilization by a discontinuous Percoll gradient. RESULTS: Overall results (mean +/- SD) were a recovery of 21 +/- 16% of total sperm, recovery of 38 +/- 30% of motile sperm, and a motility in the final sperm preparation of 88 +/- 10%. Specimens with higher initial concentrations had higher motility in the final preparation. Higher total recovery and higher motility in the final preparation were found for specimens with a higher initial motility. CONCLUSIONS: This simple two-layer Percoll technique is rapid and inexpensive and reliably produces a final sperm preparation with desirable characteristics. Even though specimens with poor initial parameters yielded final preparations with excellent characteristics, fertilization rates were still related to the initial semen parameters.     

Dynamic aspects concerned with the mechanism of separating motile sperm from nonmotile sperm, leukocytes, and debris with the use of high-density Percoll gradients

OBJECTIVE: To investigate the dynamics and the efficacy of separating motile from nonmotile sperm with the use of Percoll gradients as a function of centrifugation time. DESIGN: In vitro laboratory experiments on human sperm. SETTING: Male Infertility Institute. PATIENT(S): Fresh sperm specimens collected from donors and patients. INTERVENTION(S): Aliquots of sperm specimens, spread on top of 40% and 80% Percoll layers in test tubes, were centrifuged for 5-60 minutes. MAIN OUTCOME MEASURE(S): After centrifugation, samples were aspirated from the bottom, interface, and top layers for microscopic analysis. RESULT(S): Within 5 minutes, only fast-moving sperm reached the bottom as a result of both centrifugation force and their own tail movements. From 10 minutes onward, nonmotile sperm, slowed down by Percoll, joined them and gradually reversed the separation process. Leukocytes, debris, and abnormal sperm remained in the upper levels and never reached the bottom. CONCLUSION(S): Separation of motile sperm with the use of Percoll gradients is strongly dependent on centrifugation time. The separation is the gap formed between down-swimming sperm, accelerated by centrifugation, and nonmotile sperm, decelerated by Percoll. This gap gradually disappears with extended spinning.     

Comparison of single- and two-layer Percoll separation for selection of motile spermatozoa

OBJECTIVE: Sperm recovery using a single-layer Percoll procedure is significantly better than using the swim-up technique for infertile men and patients with normal sperm characteristics; however, in normal men results have been contradictory. Some studies have shown further improvement in semen quality with multiple layers. Therefore, this study compared the effect of single-layer and two-layer Percoll procedures on sperm characteristics of normozoospermic men. METHODS: Semen specimens from 10 normal donors were processed by layering 1 mL of the liquefied ejaculate on a single layer of 80% Percoll or on a two-layer (47% and 90%) Percoll gradient. Computer-assisted semen analysis was done to examine total motile sperm, percentage of recovery of motile cells, percent motility, curvilinear velocity, linearity, and amplitude of lateral head displacement. Each specimen was evaluated by the hypo-osmotic swelling (HOS) test, bovine cervical mucus penetration test, viability (eosin-nigrosin stain), and sperm morphology (World Health Organization and Kruger's strict criteria). RESULTS: Specimens processed with the two-layer Percoll procedure had significantly better recovery of spermatozoa, and significantly better percentage motility, linearity, amplitude of lateral head displacement, percentage tail swelling, and percentage viability than those separated on single-layer Percoll. Results for sperm morphology using WHO and Kruger's criteria were similar between the two methods (P = 0.92 for both sets of criteria). CONCLUSIONS: In normozoospermic men, the two-layer Percoll separation procedure significantly improves semen characteristics compared with separation on a single layer.     

Diagnosis of canine babesiosis by Percoll gradient separation of parasitized erythrocytes

PURPOSE: To report the development of a bilateral ocular ischemic syndrome despite corticosteroid treatment in a patient with giant cell arteritis. METHOD: Case report. RESULTS: Despite receiving high-dose intravenous methylprednisolone and oral prednisone for biopsy-proven giant cell arteritis that presented as a severe anterior ischemic optic neuropathy in the right eye, a patient developed progressive ocular ischemia in that eye as well as an ocular ischemic syndrome in the fellow eye. CONCLUSIONS: Some patients with giant cell arteritis, possibly patients with other underlying systemic vasculopathies, are refractory to what should be adequate treatment with systemic corticosteroids and may develop a bilateral ocular ischemic syndrome.     

Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep II filtration, swim-up and Percoll density gradient methods

The purpose of this study was to compare the morphology/morphometry and fertilizing capacity of human spermatozoa recovered via swim-up method, Percoll density gradient method, and SpermPrep II filtration method. Thirty-three ejaculates were equally divided into 2 aliquots. Aliquot 1 was processed via the direct swim-up method, whereas aliquot 2 was filtered via a SpermPrep II column. The Percoll density gradient method was compared with the SpermPrep II method in a similar protocol using 43 ejaculates. Sperm populations recovered via the SpermPrep II filtration method showed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyperactivated spermatozoa than sperm fractions recovered by the swim-up method. Furthermore, significant differences were found in most of sperm morphometric parameters between the above sperm populations. However, sperm fractions recovered via the SpermPrep II method did not show significantly different values for these same tests and for most of sperm morphometric parameters compared to the Percoll density gradient method. These results suggest that the SpermPrep II filtration and Percoll density gradient method are equally efficient in isolating sperm subpopulations with better functional parameters than the swimup method.     

Intrauterine insemination in male subfertility: a comparative study of sperm preparation using a commercial Percoll kit and conventional sperm wash

Recent advances in restoring neural function to biological systems and incorporating neural function into bioartificial devices have been made possible by developments in biology, materials science, engineering, and physics. Further progress is being achieved in relating cellular function to overall system behavior through the application of quantitative experimental and theoretical techniques.     

Discontinuous Percoll gradient preparation for donor insemination: determinants for success

The use of cryopreserved donor spermatozoa for insemination has become necessary to decrease the risk from sexually transmitted infectious diseases. Lower fecundity rates have been reported with this practice. Efforts have been applied to increase success, including identification of those sperm characteristics which correlate with increased fecundity. Data from in-vitro fertilization have revealed sperm morphology, motility and zona pellucida binding as important sperm parameters. Discontinuous Percoll gradient preparation yields a high concentration of motile spermatozoa. Using this preparation for thawed donor spermatozoa, we have identified post-preparation motility and progression as factors associated with increased fecundity. Consideration should be given to screening sperm donors with a freeze-thaw Percoll gradient preparation prior to acceptance into a donor bank.     

Microsurgical epididymal sperm aspiration with motile trophozoite cells but no spermatozoa

This paper reports on a patient in whom the clinical diagnosis of obstructive azoospermia was made according to clinical observations, i.e. azoospermia, normal andrological examination, normal follicle stimulating hormone and a misleading histopathological report of a testicular biopsy. Microsurgical vasoepididymostomy failed to restore fertility, and as a last resort, microsurgical sperm aspiration was performed. Although flagellated cells were observed in the epididymal aspiration, no spermatozoa were observed and wet preparation of multiple testicular biopsies failed to demonstrate any spermatozoon. This patient was diagnosed to have a non-obstructive azoospermia, resulting from maturation arrest associated with trichomonas infection at the level of the epididymis.     

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity

Adenovirus DNA polymerase is one of three viral proteins and two cellular proteins required for replication of the adenovirus genome. During initiation of viral DNA synthesis the viral DNA polymerase transfers dCMP onto the adenovirus preterminal protein, to which it is tightly bound. The domain structure of the 140 kDa DNA polymerase has been probed by partial proteolysis and the sites of proteolytic cleavage determined by N-terminal sequencing. At least four domains can be recognised within the DNA polymerase. Adenovirus preterminal protein interacts with three of the four proteolytically derived domains. This was confirmed by cloning and expression of each of the individual domains. These data indicate that, like other members of the pol alpha family of DNA polymerases, the adenovirus DNA polymerase has a multidomain structure and that interaction with preterminal protein takes place with non-contiguous regions of the polypeptide chain over a large surface area of the viral DNA polymerase.     

The motile major sperm protein (MSP) from Ascaris suum is a symmetric dimer in solution

The major sperm protein (MSP) of Ascaris suum mediates amoeboid motility by forming an extensive intermeshed system of cytoskeletal filaments analogous to that formed by actin in many amoeboid cells. We have used a combination of biochemical and NMR methods to show that, in contrast to actin, MSP exist in solution as a symmetrical dimer. This result has important implications for the mechanism of both MSP filament assembly and the recognition of different MSP isoforms in vivo.     

Structural and molecular characterization of dynein in a gall-midge insect having motile sperm with only the outer arm

The dipteran Monarthropalpus flavus possesses a peculiar sperm axoneme, characterized by multiple rows of microtubular doublets linked by the outer dynein arms only, lacking any equivalent of the central pair/radial spoke complex. The structure of these dynein molecules was studied by electron microscopy (EM). Using the quick-freeze, deep-etch method of EM, they were found to be similar to outer dynein arms described previously. Two globular "heads," each subdivided by a cleft, are clearly discernible. "Stalks" extend from proximal head to contact the B-tubule of the adjacent doublet. Unlike the situation in vertebrate sperm, the stalks sometimes branch into two thinner strands that contact the B-tubule at different sites. Treatment of demembranated sperm cells with ATP and vanadate induces conformational changes in the dynein outer arms. These are interpreted as the result of rotation of the dynein head with respect to what is observed in axonemes in rigor condition (after ATP depletion). SDS-PAGE indicates that the high-molecular-weight complement of this molecule comprises a single heavy chain. Specific dynein heavy chain-related DNA sequences corresponding to the catalytic-phosphate binding region were amplified by RT-PCR. Only one axonemal dynein sequence was identified among all amplified fragments. Southern blot analysis performed on genomic DNA using this sequence as a probe identified two hybridizing genes, only one of which is able to encode a functional product. Thus, genetic analysis indicates that this axonemal outer arm dynein is a homodymer of a single heavy chain subunit. In vivo, spermatozoa of this species are stored in a rolled configuration in female spermatheca, where they move rapidly with a wave-like motion. This movement could not be reproduced in vitro, except when spermatozoa were constrained in a bent configuration by some mechanical impediment. We propose that, in the absence of both the central pair/radial spoke complex and the inner arms, a curvature-dependent activation acts to trigger motility in these spermatozoa.     

A simple method for recovering the motile spermatozoa from extremely low quality sperm samples

Recovery of motile spermatozoa from extremely low quality samples for use in the intracytoplasmic sperm injection (ICSI) procedure is difficult. To solve this problem we developed a simple method to recover the motile spermatozoa using a 3% polyvinylpyrrolidone (PVP) droplet. After depositing a sperm pellet into this slightly viscous droplet, motile spermatozoa readily swam out to the clear area while immotile spermatozoa dispersed to a lesser extent, so that motile and immotile cells became clearly separated from each other. A total of 36 ICSI cycles using spermatozoa with extremely low quality characteristics were performed. We recovered the motile spermatozoa from all sperm samples from two sources of poor quality spermatozoa. Thirty-one cycles of ICSI with ejaculate resulted in fertilization and pregnancy rates of 54 and 29% respectively. Five cycles of ICSI with frozen-thawed epididymal spermatozoa resulted in fertilization and pregnancy rates of 70 and 60% respectively. The 3% PVP droplet method is very simple and easy to perform, so it may be useful for recovering the motile spermatozoa from extremely low quality sperm samples used for ICSI.     

Pregnancy rates after intracytoplasmic sperm injection in relation to sperm recovery techniques

Xanthogranulomatous pyelonephritis (XGP) is a rare and aggressive form of chronic pyelonephritis for which partial or complete nephrectomy is mandatory [1.2]. The diagnosis and, in particular, the differentiation from hypernephroma is mostly established by the histopathological examination only. We have reviewed the literature and present an unusual case of XGP with septic spreading into the lungs mimicking pulmonary metastasis and with an inflammatory infiltration of the descending colon.     

2.5 A resolution crystal structure of the motile major sperm protein (MSP) of Ascaris suum

We have determined the structure of the Ascaris major sperm protein (MSP) to 2.5 A resolution using X-ray crystallography. The MSP polypeptide chain has an immunoglobulin-like fold based on a seven-stranded beta sandwich. In two strands, cis-proline residues impart distinctive kinks, and overall the structure most closely resembles that of the N-terminal domain of the bacterial chaperonin, PapD. In the C2 crystal form which we have solved here, two MSP chains are tightly associated in the asymmetric unit and are related by a non-crystallographic 2-fold rotation axis. This arrangement almost certainly represents the MSP dimer that is present in solution. Additionally, the arrangement of two MSP dimers at one of the crystallographic 2-fold axes in the 215 A unit cell suggests a possible mode for the assembly of MSP into the filaments which promote cell movement. This dimer-dimer association is based on a beta sheet extension mechanism between adjoining MSP monomers which resembles the interaction between PapD and its protein substrate.     

Intrauterine insemination after ovarian stimulation with clomiphene citrate: predictive potential of inseminating motile count and sperm morphology

This retrospective study aimed to evaluate the prognostic value of the inseminating motile count (IMC) and sperm morphology (using strict criteria) on success rates after homologous intrauterine insemination (IUI) combined with clomiphene citrate (CC) stimulation. A total of 373 couples underwent 792 IUI cycles in a predominantly (87.4%) male subfertility group. The overall cycle fecundity (CF) and baby take-home rate (BTH) was 14.6 and 9.9% respectively. The cumulative CF and BTH (per couple) after three cycles were 30.6 and 21.1% respectively. Overall, sperm morphology and IMC were of no prognostic value using receiver operating characteristic (ROC) curve analysis, but after classifying the study population into different subgroups according to IMC, sperm morphology turned out to be a valuable prognostic parameter in subgroup 1, i.e. IMC <1 x 10(6). In this subgroup, no pregnancies were seen when the morphology score was <4% and the mean value of sperm morphology was significantly different in the pregnant (8.3%) versus non-pregnant group (5.0%; P <0.05). The cumulative CF and BTH after three IUI cycles were comparable for all couples with the exception of those cases in which the IMC was <1 x 10(6) with a morphology score of <4% normal forms. We recorded only two twin pregnancies (2.5%) and no moderate or severe ovarian hyperstimulation syndrome. We conclude that in a selected group of patients without CC resistance and normal ovarian response following CC stimulation [maximum of three follicles with a diameter of >16 mm at the time of administration of human chorionic gonadotrophin (HCG)], IUI combined with CC-HCG can be offered as a very safe and non-expensive first-line treatment, at least with an IMC of >1 x 10(6) spermatozoa. In cases with <1 x 10(6) spermatozoa, CC-IUI remains important as a first-choice therapy provided the morphology score is > or =4%.     

Testicular sperm recovery in nine 47,XXY Klinefelter patients

Klinefelter's syndrome is generally characterized by hypergonadotrophic hypogonadism and azoospermia. The clinical features, however, are variable, and occasionally severe oligozoospermia may be present. Usually in these cases a 46,XY/47,XXY mosaic karyotype is involved. However, focal spermatogenesis and severe oligozoospermia have been reported in 47,XXY individuals too. In the present study we investigated whether testicular spermatozoa can be recovered in 47,XXY patients with a view to intracytoplasmic sperm injection (ICSI). In four out of nine apparently non-mosaic 47,XXY patients, spermatozoa were recovered from the wet preparations of testicular tissue and ICSI was performed in three couples. In one patient in whom spermatozoa were successfully recovered and used for ICSI, no spermatozoa were retrieved at a second trial. Although these results show that in some 47,XXY individuals testicular spermatozoa can be successfully recovered and even used for ICSI, at present this approach should be considered experimental. There may indeed be some concern about the chromosomal normality of the embryos generated through this infertility treatment. Patients with Klinefelter's syndrome should therefore be counselled about the complexity of this treatment, which involves multiple testicular biopsies from hypogonadal testes, ICSI and preimplantation diagnosis by fluorescence-in-situ hybridization.     

Subcellular fractionation on Percoll gradient of mossy fiber synaptosomes: morphological and biochemical characterization in control and degranulated rat hippocampus

A method for preparation of hippocampal mossy fiber synaptosomes directly from the postnuclear pellet is presented. This method represents an adaptation of that previously described for the isolation of synaptosomes by centrifugation through Percoll gradients directly from the supernatant fraction. We have characterized by electron microscopy two fractions, PII and PIII, enriched in mossy fiber synaptosomes; fraction PIII had 75% mossy fiber synaptosomes with well-preserved morphology (large size 3 microns, complex morphology, high synaptic vesicle density, multisynapses), whereas fraction PII contained 12%. These fractions were enriched in lactate dehydrogenase activity indicating that the integrity of synaptosomes was preserved. Compared with the other synaptosomal fractions, these fractions showed greater levels of dynorphin A (1-8) immunoreactivity and endogenous zinc, which are particularly concentrated in hippocampal mossy fiber terminals. Furthermore, we prepared synaptosomes from adult hippocampus after neonatal irradiation, which destroys the majority of granule cells and associated mossy fibers. The levels of dynorphin and zinc decreased by 88 and 70% in fraction PII and by 95 and 90%, respectively, in PIII. These results suggest that the rapid Percoll procedure is convenient for the purification of mossy fiber synaptosomes.     

Improved motility recovery of human spermatozoa after freeze preservation via a new approach

A new sperm isolation technique was studied in conjunction with the short-term freeze preservation of human spermatozoa. The isolation procedure yielded subpopulations of spermatozoa with very high percentage motility and progressive motility score, and which were virtually free of seminal debris. For 24 semen samples from 13 donors, the mean prefreeze values of percentage motility and motility score were 53% (2.8), 73% (3.1), and 88% (3.5) for the parent semen, 7.5% BSA (middle) fractions, and 17.5% BSA (bottom) fractions, respectively. These samples were used without a priori constraint on semen quality. Eleven samples were preselected on the basis of good motility in the semen. These yielded prefreeze motility values of 66% (3.2), 70% (3.4), and 87% (3.9) for the semen and two fractions, respectively. Sperm motility in the middle fractions was thus intermediate to that in the semen and in the bottom fractions, although closer to the former in these 11 cases. The sperm freeze preservation procedure involved dilution of the semen samples and separated sperm fractions with a cryoprotective semen extender, and freezing and thawing in a conventional manner. Post-thaw percentage motility, motility score, and percentage survival were substantially higher for the separated fractions than for the parent semen. For the 24 cases, the bottom fractions yielded mean values of 57% motility, 3.0 motility score, and 70% survival, in comparison with the respective values of 20%, 2.3, and 34% for the semen. In the 11 preselected cases, the bottom fractions yielded post-thaw mean values of 60% motility, 3.3 motility score, and 72% survival; the middle fractions yielded respective values of 45%, 3.1, and 64%; and the parent semen yielded respective values of 27%, 2.3, and 41%. It was concluded that the major factor in improving post-thaw motility recovery was the separation process as a whole, rather than the degree of separation.     

Microstructures induced by a stress gradient in a nickel-based superalloy

The evolution of the microstructure of single crystals of a nickel-based superalloy during high temperature (1323 K, 1050°C) creep in bending has been studied. Bending provides both tensile and compressive stress gradients; consequently the effects of varying stress conditions on the evolution of the morphology of the γ′ precipitates can be determined from a single specimen. The morphological changes were analysed by scanning electron microscopy using image analysis techniques and by transmission electron microscopy, then described by dimensionless parameters. We discuss the dependence of the morphological changes in the superalloy on the stresses acting in the sample (magnitude and sign). We also discuss the driving mechanisms for the observed morphological changes.     

Use of Percoll gradient centrifugation for the isolation of diatoms from Wadden Sea sediments; diatom yields, species recoveries and population diversity

Platelet-activating factor (PAF) production is carefully controlled in inflammatory cells. The specific removal of arachidonate (AA) from 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (GPC), thought to be mediated by CoA-independent transacylase (CoA-IT), is required to generate the PAF precursor 1-O-alkyl-2-lyso-GPC in human neutrophils. Exposure of A23187-stimulated human monocytes to the CoA-IT inhibitors SK&F 98625 and SK&F 45905 inhibited PAF formation (IC50s of 10 and 12 microM, respectively), indicating that these cells also need CoA-IT activity for PAF production. Because CoA-IT activity transfers arachidonate to a 2-lyso phospholipid substrate, its activity is obligated to an sn-2 acyl hydrolase to form the 2-lyso phospholipid substrate. SB 203347, an inhibitor of 14 kDa phospholipase A2 (PLA2), and AACOCF3, an inhibitor of 85 kDa PLA2, both inhibited AA release from A23187-stimulated human monocytes. However, AACOCF3 had no effect on A23187-induced PAF formation at concentrations as high as 3 microM. Further, depletion of 85 kDa PLA2 using antisense (SB 7111, 1 microM) had no effect on PAF production, indicating a lack of a role of 85 kDa PLA2 in PAF biosynthesis. Both SB 203347 and the 14 kDa PLA2 inhibitor scalaradial blocked PAF synthesis in monocytes (IC50s of 2 and 0.5 microM, respectively), suggesting a key role of 14 kDa PLA2 in this process. Further, A23187-stimulated monocytes produced two forms of PAF: 80% 1-O-alkyl-2-acetyl-GPC and 20% 1-acyl-2-acetyl-GPC, which were both equally inhibited by SB 203347. In contrast, inhibition of CoA-IT using SK&F 45905 (20 microM) had a greater effect on the production of 1-O-alkyl (-80%) than of 1-acyl (-14%) acetylated material. Finally, treatment of U937 cell membranes with exogenous human recombinant (rh) type II 14 kDa PLA2, but not rh 85 kDa PLA2, induced PAF production. Elimination of membrane CoA-IT activity by heat treatment impaired the ability of 14 kDa PLA2 to induce PAF formation. Taken together, these results suggest that a 14 kDa PLA2-like activity, and not 85 kDa PLA2, is coupled to monocyte CoA-IT-induced PAF production.