Use Of Sperm Research Articles

Analysis of Fibronectin on Human Sperm

Purpose The purpose of this study was to localize fibronectin on human sperm and correlate its distribution with the morphological and functional integrity of sperm. Materials and Methods Semen samples were collected and sperm fractionated by swim-up. Subsets of the swim-up sperm were capacitated and acrosome reacted. Damage to swim-up sperm was induced by freezing and thawing. The presence of fibronectin on the surface of sperm was determined by immunocytochemistry. Results FN immunoreactivity was variable but staining on the sperm tail was consistently highest, whereas FN immunoreactivity over the acrosome and equatorial band was consistently lowest. Capacitation and acrosome reaction did not substantially change the distribution of FN staining. However, swim-up sperm had significantly less FN immunoreactivity (4%) than sperm that were unable to swim-up (12%; p < 0.01). Sperm that were deliberately damaged by freeze/thaw showed significantly increased FN binding (p < 0.01). FN immunoreactivity was inversely correlated with sperm viability (r = −0.68), motility (r = −0.70), and morphology (r = −0.63). Conclusions This study demonstrates that only a minority of the sperm in an ejaculate stain positive for FN and the localization of FN in positive sperm in primarily to the tail. Inferior sperm stain more frequently for FN leading to an inverse correlation between FN staining and sperm quality. Taken together, these results do not support a role for FN in sperm-egg binding. However, FN staining may provide a method for selecting the highest quality sperm for use in assisted reproduction techniques.

Vasal Aspiration of Sperm for Use in In-Vitro Fertilization

Vasal Aspiration of Sperm for Use in In-Vitro Fertilization

Detection of antispermal antibodies in female sera

The levels of antispermal antibodies in the sera of women varied within a wide range when spermatozoa from different donors were used. This was probably due to expression of different antigenic structures on the surface of spermatozoa of fertile and sterile men and their different specificity for the tested female serum. Spermatozoa from different donors did not influence the detection of antispermal antibodies in the sera of sterile men. The use of spermatozoa from the husband and a fertile donor is recommended for identification of antispermal antibodies in female serum when examining an infertile couple.

Fertilization and pregnancy using intentionally cryopreserved testicular tissue as the sperm source for intracytoplasmic sperm injection in 10 men with non-obstructive azoospermia.

Testicular tissue extraction (TESE) to obtain spermatozoa for use with intracytoplasmic sperm injection (ICSI) has recently been employed in patients with non-obstructive azoospermia. Standard protocol is to retrieve a new sample of testis tissue on the day of oocyte recovery. Unfortunately, approximately 30% of men will possess no spermatozoa in their tissue, making ICSI an impossibility. We investigated whether testicular tissue that was intentionally obtained well before any planned ICSI cycle and cryopreserved could then serve as an efficacious sperm source in a subsequent ICSI cycle. This study reports on 10 men with non-obstructive azoospermia who did have spermatozoa found within their testis tissue at the time of TESE and who chose to use their frozen samples as the source of spermatozoa for a later cycle of ICSI. In 19 cycles the overall fertilization rate was 48%. Embryo transfer occurred in 89% of cycles. Two couples have achieved pregnancy (one ongoing, one delivered). All patients except one had multiple vials of frozen tissue remaining following their first cycle. This approach is offered as an alternative to repeated testicular tissue sampling, as the availability of spermatozoa is assured prior to the initiation of ovulation induction. This tissue can be harvested at the same time as diagnostic biopsy, thereby minimizing the number of surgical procedures.

Development of a microchamber which spontaneously selects high-quality sperm for use in in vitro fertilization or micromanipulation.

A microchamber has been developed which allows motile sperm to swim from a central loading site to peripheral sidewells. The sidewells are designed such that oocytes may be placed within them for in vitro fertilization (IVF) or sperm may be harvested from them for use in standard IVF or micromanipulation. Because only motile sperm can reach the sidewells, the microchamber can select relatively high-quality sperm from a crude preparation. Moreover the steep walls of the sidewells create the potential to trap sperm. The present study was under taken to compare sperm in the sidewells to those found in randomly sampled regions of microchamber after various periods of incubation. We find that the sidewells concentrate motile sperm and that a higher percentage of sperm removed from sidewells is acrosome reacted. Motile sperm from oligospermic patients can be harvested from microchamber sidewells for use in micromanipulation after loading the microchamber with unprocessed specimens. Results suggest that this microchamber could be used to enhance sperm:egg interaction in IVF or to harvest sperm for micromanipulation.

An effective, simplified, and practical approach to intracytoplasmic sperm injection at multiple IVF centers.

Our purpose was to use effectively a simplified approach for intracytoplasmic sperm injection (ICSI) that achieves high levels of fertilization, embryo cleavage, and subsequent establishment of clinical pregnancies. In a multicenter clinical analysis, 1814 mature oocytes were injected with a single sperm using a finely beveled (30 degrees) pipette without a spiked tip that was backfilled with an inert, silicone polymer. The injection system was composed of a sterile water-filled tubing interconnecting the pipette and a 20-gauge needle attached to a three-way stopcock and 5-ml disposable syringe. The efficiency of the ICSI procedure was assessed by analyzing fertilization, lysis, and cleavage rates, as well as pregnancy outcomes. High levels of normal fertilization (2PN; 56.4-73.5% of all eggs injected) and reasonably low lysis rates (9-16%) were produced. The efficiency of embryo formation was high (87-93%), with only 6 of 177 (3.4%) patients failing to produce an embryo. Clinical pregnancies were established by each IVF center (18-34% ongoing pregnancy/birth rate). Effective ICSI was achieved using a novel, simplified technique involving sharply beveled pipettes (without additional tip modification), simple sperm washing/handling procedures, and a disposable syringe injection system that offered extremely fine control for sperm manipulation.

Manipulation of sperm before intracytoplasmic sperm injection improves fertilization rates

To determine the effect of sperm manipulation before intracytoplasmic sperm injection (ICSI) on fertilization rates. Three methods of sperm manipulation before direct ICSI were compared in two sibling oocyte trials. In vitro fertilization unit within a teaching hospital. Patients undergoing infertility treatment using ICSI. Oocytes were inseminated by ICSI. Fertilization rate. In the first trial, a standard manipulation technique gave a 67% fertilization rate compared with 64% with a minimal manipulation technique. In the second trial the standard technique gave a significantly greater fertilization rate compared with no prior manipulation (67% versus 45%). Manipulation of sperm before direct ICSI is not mandatory for fertilization but significantly improves the fertilization rate.

Sex preselection by flow cytometric separation of X and Y chromosome-bearing sperm based on DNA difference: a review

Recent research on the flow cytometry of sperm for the purpose of predetermining gender of offspring has led to a validated method to separate X from Y chromosome-bearing sperm for use with in vitro fertilization and embryo transfer, intratubal insemination or intracytoplasmic sperm injection. The basis for the method is the sex chromosome-specific marker, DNA, which is present in greater amounts in X-bearing sperm than in Y-bearing sperm of mammals. Sperm are exposed to the vital dye Hoechst 33342 which binds to the minor groove of the DNA helix. Flow cytometric sorting of the sperm using a laser as the excitation source results in populations of Y- or X-bearing sperm that are 85-90% pure. Several hundred offspring have been produced from swine, rabbits, sheep and cattle that confirm the predicted sex. The method is currently being applied to the commercial embryo market. The method is not likely to be used in conjunction with standard cattle or swine artificial insemination practice in its current form since only about 4 x 10(5) sorted sperm can be produced per hour of sorting. The technology has also been applied to human sperm for use by couples that are at risk to sex-linked disease expression in their offspring. Populations of human sperm have been sorted with X and Y purities of about 80% as confirmed by DNA probe technology and fluorescence in situ hybridization.

A method of short-term cryostorage and selection of viable sperm for use in the various assisted reproductive techniques.

The objective of this study was to determine if spermatozoa, following short-term cryostorage at 5 degrees C in Test-Yolk buffer (TYB; ZBL, Inc., Lexington, KY, USA), could be recovered and improved via the SpermPrep filtration method and to assess the possibly enhanced fertilizing capacity of the selected spermatozoa. Semen specimens from 20 men were collected, evaluated, diluted 1:1 (v/v) with TYB, divided into aliquots and cooled to 5 degrees C for 24 and 48 hr. Semen samples were assessed for volume, sperm count, percentage and grade of motility, percentage of morphologically normal spermatozoa and outcome of the sperm penetration assay (SPA). After storage, aliquots were rewarmed at 37 degrees C, centrifuged, and the pellet was resuspended in 1.0 ml of SpermPrep media (ZBL, Inc.). Following 15 min of incubation, the rewarmed spermatozoa were filtered via the SpermPrep I filtration column (ZBL, Inc.) and assessed accordingly. The results obtained in this study indicate that the short-term cryostorage procedure yielded spermatozoa of adequate qualitative characteristics when compared to the fresh spermatozoa. Furthermore, filtration of rewarmed specimens yielded spermatozoa of significantly higher qualitative characteristics and superior fertilizing capacity following a short-term cryostorage period in TYB when compared to fresh and rewarmed spermatozoa (p < 0.05). This method of short-term cryostorage in TYB and selection of superior spermatozoa via the SpermPrep filtration method could further enhance the fertilizing ability of patients who produce spermatozoa characterized by deficient capacitation, acrosome reaction and subsequent fertilization.

Sex selection: Human sex pre-selection by sperm manipulation

Sex selection: Human sex pre-selection by sperm manipulation

MICROMANIPULATION OF GAMETES FOR MALE FACTOR INFERTILITY

Techniques such as PZD, SZI, and ICSI are advantageous in cases of extreme male factor infertility. They are adjunctive techniques, applicable in the laboratory only during an IVF cycle. They can be used either simultaneously on sibling oocytes or, if indicated, individually. The present results demonstrate a lack of correlation between quantitative sperm parameters and outcome of assisted fertilization procedures. Preliminary results strongly suggest that ICSI will replace SZI and PZD for treatment of severe male factor infertility. Hundreds of viable pregnancies have been established well below the normal cut-off values for regular assisted conception procedures. Fertilization and pregnancy occurred following the use of spermatozoa without progressive motility and without normal morphology. In some patients sperm counts were correspondingly reduced and spermatozoa could be visualized only after centrifugation of the semen specimen. The current results provide evidence that spermatozoa from extremely oligoasthenoteratozoospermic men can produce normal offspring after the application of micromanipulation techniques, even when fertilization previously failed following standard IVF. Currently, investigators have not been able to identify, on the basis of conventional semen evaluations, subgroups of patients who would not benefit from microsurgical techniques. Findings published to date suggest approximately 24% clinical pregnancy rates per cycle when SZI and PZD are used together, but 35% clinical pregnancy rate per cycle for ICSI. Preliminary results suggest that the application of these techniques does not increase the risk of birth defects for subsequent offspring. However, continued observation of the potential effects of these procedures on progeny will be required.

IMMUNOLOGIC INFERTILITY

The existence of immunologic infertility is well grounded in the theories of humoral and cell-mediated immunology. Both of these immune system arms can theoretically and experimentally be shown to play roles in states of male factor infertility. Although much progress has been made in relating immunopathology to infertility, several problems plague the investigation and diagnosis: the use of multiple, different assay systems; the lack of properly controlled experimental work; a need to oversimplify a tremendously complex immunologic network for clinical purposes; and a dearth of communication between basic science and clinical investigators. Unlike the established role of humoral or antibody-mediated infertility, the way cell-mediated immunity induces reproductive failure is just beginning to be understood. Two active areas of investigation include a delineation of active immunosuppression in the normal testicular environment and the pathologic deficits induced by cytokines in cases of leukospermia. Other research seeks to define functional, sperm-specific antigens to which autoantibodies are directed and to elucidate the mechanisms of antibody-mediated infertility at various sites in the reproductive tract. The diagnosis of immunologic infertility still remains one of exclusion. When the diagnosis is made, it is necessary to define precisely which functional reproductive deficits exist, through an analysis of semen parameters, postcoital tests, sperm penetration assays, and tests of acrosomal reactivity. This is essential because the therapeutic options are broad and can involve significant side effects and expense. Proven modalities include sperm manipulation, direct sperm insemination, donor insemination, systemic immunosuppression, and assisted reproductive techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Localisation and capacitation-dependent loss of buffalo sperm-coating antigens shared with rat sperm

The heterodimeric sperm-coating protein CFS was previously localised on the middle-piece region of rat spermatozoa by anti-CFS rabbit antibodies. CFS-immunorelated antigens were detected in the secretion of the water buffalo seminal vesicle by protein electrophoresis and Western blotting. Spermatozoa from buffalo epididymal cauda were incubated with the rat antigen and, upon immunostaining with anti-CFS antibodies and goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated IgGs, CFS was found attached on both the post-acrosomal region and the tail. Indirect immunofluorescence analysis permitted the localisation of CFS-related antigens on the same domains of buffalo ejaculated spermatozoa. These results suggest that the buffalo antigens not only share some epitopes with the homologous rat antigen but may also have some of its functional properties. Ejaculated spermatozoa were capacitated in vitro and then assayed for their content of CFS-like antigens. An inverse relationship was found between the levels of capacitation and the amounts of antigens detected, thus suggesting that the in vitro treatment was effective at removing CFS-related proteins from the cell surface. Titration of these proteins to monitor plasma membrane changes during sperm manipulation or to evaluate sperm quality is proposed.

Effect of sperm washing on levels of reactive oxygen species in semen.

The possibility was evaluated that the production of reactive oxygen species (ROS) by human sperm is stimulated by the repeated cycles of centrifugation and resuspension involved in conventional sperm preparation. ROS generation by human sperm was monitored before and after the washing of sperm from 55 men (43 men with suspected subfertility and 12 normal volunteers). The ROS activity of all 55 specimens before washing was inversely correlated with original sperm motility (r = .278, p < .05). The mean level of ROS activity was significantly higher after washing than before processing (p < .05) for the 26 specimens with normal sperm motility, the 20 specimens with normal sperm morphology, and the 12 specimens with both normal motility and normal morphology. In contrast, the mean ROS level was not significantly changed after washing in the 27 specimens with poor sperm motility, the 16 specimens with abnormal sperm morphology, or the 13 specimens with both abnormal motility and abnormal morphology. It would appear that repeated centrifugation, resuspension, and vortexing cause excessive generation of ROS in the motile sperm population of the washed specimen. Washing procedures involving excessive manipulation of sperm may, in fact, cause the most harm to motile sperm, i.e., those that the method is trying to select. Procedures that minimize multiple centrifugation, resuspension, and vortexing steps should therefore be used for the preparation of semen specimens for assisted-reproduction techniques.

Microencapsulation of bovine spermatozoa for use in artificial insemination: a review.

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus of potential use in artificial insemination. The polymers poly-l-lysine, polyvinylamine and protamine sulfate have proven best for membranes. Encapsulation has been successful with capsules ranging in size from 0.75 to 1.5 mm, and with sperm concentrations from 45 to 180 x 10(6) cells mL-1. Successful extenders include CUE, CAPROGEN, and egg yolk-citrate-glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under ageing and physical stress) is negatively related to membrane thickness which ranges from 1.92 to 5.32 microns (depending on the concentration of polymer used) and positively related to concentration of sperm encapsulated. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when cows are inseminated at conventional times. Uterine retention of inseminates is favoured by capsules having a 'sticky' membrane. Using current procedures, preliminary homospermic fertility studies indicate that sperm encapsulated with poly-l-lysine or protamine sulfate may achieve normal fertility.

Diploid Gynogenesis in Lampam Jawa Puntius gonionotus Using UV Irradiated Sperm of Puntius schwanenfeldii Followed by Temperature Schok.

The effects of genetic manipulation of eggs and sperm on the yields of gynogenetic fry of Puntius gonionotus were investigated. Gynogenesis was achieved by cold-and heat-shocking eggs fertilized with ultraviolet irradiated sperm of P. schwanenfeldii at various times after fertilization and at different duration intervals. 66.6% viable, gynogenetic fry were obtained when eggs were inseminated with irradiated sperm and cold shocked at 2°C for 5 minutes duration 1 minute after fertilization. At warm water temperature shocks the fertilized eggs performed best at 42°C, with percent survival rates of 20.0% 1 minute after fertilization for 1.0 minute duration and 17.2% and 14.7% 13 and 23 minutes after fertilization respectively for a duration of 1.5 minutes.

The Use of Laser Optical Traps for Sperm Manipulation

The Use of Laser Optical Traps for Sperm Manipulation

Protective role of superoxide dismutase in human sperm motifity: superoxide dismutase activity and lipid peroxide in human seminal plasma and spermatozoa

The levels of superoxide dismutase (SOD), a highly specific scavenging enzyme for superoxide anion radicals (O2-), and lipid peroxide produced by oxygen free radicals were measured in human seminal plasma and spermatozoa. Seminal plasma contained 366.8 +/- 20.9 U/ml (mean +/- SE) of SOD activity. SOD activity in human spermatozoa showed a significant correlation to the number of motile spermatozoa, while the activity in seminal plasma did not relate to the sperm concentration or motility. The lipid peroxide concentration in seminal plasma was 6.22 +/- 0.46 nmol/ml and had no significant relationship to sperm concentration or motility. The malondialdehyde (MDA) concentration in spermatozoa was significantly related to the number of immotile spermatozoa. A decrease in the motility of spermatozoa incubated in medium without seminal plasma was observed after 120 min, while the MDA concentration of the spermatozoa increased. Addition of exogenous SOD (400 U/ml) to the sperm suspension significantly decreased this loss of motility and the increase of the MDA concentration. These data suggest a significant role for SOD in sperm motility. It seems that lipid peroxidation of human spermatozoa may cause loss of motility and that SOD may inhibit this lipid peroxidation. These results suggest that SOD may have a possible clinical application in the use of spermatozoa for in-vitro fertilization (IVF) or artificial insemination.

Encapsulation of porcine spermatozoa in poly-lysine microspheres

Three experiments were conducted to examine the effects of incubating porcine spermatozoa in concentrated samples, to determine the viability of sperm encapsulated in microspheres and to evaluate the potential of microencapsulating porcine spermatozoa for use in artificial insemination. In Experiment 1, sperm incubated at 4, 15, 20 or 37°C and at concentrations of 7.5, 15, 30, 60 or 120 × 10 6 sperm/ml lost motility over a 16-h incubation period. Sperm motility was significantly lower at 4°C than at 15, 20 or 37°C and was significantly higher in more concentrated samples. In Experiment 2, sperm were encapsulated in poly-lysine microspheres at concentrations of 30, 60 or 120 × 10 6 sperm/ml and incubated in vitro at 4, 15 or 20°C. Unencapsulated samples were incubated at similar concentrations and temperatures and served as controls. Motility and percentage of sperm with intact acrosomes were estimated at 2, 4, 8 and 16 h of incubation. The procedure of encapsulation did not affect sperm motility or acrosomal morphology; however, there was an accelerated loss of motility in encapsulated samples. There were no differences in acrosomal morphology between the two groups across time. In Experiment 3, sperm were encapsulated at a concentration of 120 × 10 6 sperm/ml and 20 ml of capsules were inseminated into estrous sows. Uterine contents were flushed at 3, 6 and 24 h after insemination and examined for capsules. Capsules containing motile sperm were recovered from sows at 3 and 6 h, but not at 24 h. These results demonstrate that porcine spermatozoa can be encapsulated in microspheres and that these capsules can be inseminated into estrous females, but the sperm undergo an accelerated loss of motility in vitro and in vivo.

Efficacy of Fertilization in Artificially Inseminated Turkey Hens

Research was conducted to develop an artificial insemination protocol optimizing the use of spermatozoa by turkey breeder hens. Large White turkey hens were inseminated on Days 14 and 17 postphotostimulation with 200 million spermatozoa from one male phenotype to fill the oviductal storage sites. Artificial inseminations were then performed weekly for 20 wk with different spermatozoa numbers of another male phenotype. Fertility and phenotype of each poult were determined at hatch to ascertain which insemination, initial or subsequent, was responsible for fertility.Inseminating weekly with 200 million viable spermatozoa cells resulted in better fertility but did not optimize the hen’s utilization of spermatozoa from the initial inseminations. When fewer spermatozoa were inseminated weekly (50 million cells), more progeny were fertilized by spermatozoa already residing in the oviduct than would be expected. When the number of spermatozoa inseminated weekly was increased at intervals during a laying cycle, spermatozoa from the initial inseminations were utilized more efficiently, but fertility was depressed at times during the laying cycle. Gradually increasing weekly inseminated numbers of spermatozoa from 50 to 200 million viable cells/hen as the hens age results in nearly equivalent fertility to that resulting from insemination by 200 million cells each week. This represents a savings of 1.4 billion spermatozoa/hen over a 20-wk laying period.