Autologous Seminal Plasma Research Articles

Effects of dead spermatozoa on motion characteristics and membrane integrity of live spermatozoa in fresh and cooled-stored equine semen

The aim of this study was to determine if dead spermatozoa reduced motility or membrane integrity of live spermatozoa in fresh and cooled-stored equine semen. Three ejaculates from each of three stallions were centrifuged and virtually all seminal plasma was removed. Spermatozoa were resuspended to 25×10 6 spermatozoa/ml with EZ-Mixin ® CST extender and 10% autologous seminal plasma, then divided into aliquots to which 0 (control), 10, 25, 50, or 75% (v/v) dead spermatozoa were added. Dead spermatozoa preparations contained 25×10 6 spermatozoa/ml and 10% seminal plasma from pooled ejaculates of the three stallions, in EZ-Mixin ® CST extender. Spermatozoa were killed in the pooled ejaculates by repeated freezing and thawing, then stored at −20 °C until warmed to 37 °C and mixed with aliquots of fresh spermatozoa to be cooled and stored in an Equitainer for 24 h. Motion characteristics (% total motility (MOT), % progressive motility (PMOT), and mean curvilinear velocity (VCL)) for fresh and 24 h cooled samples were determined using a computerized spermatozoal motion analyzer. The presence of up to 75% dead spermatozoa did not adversely affect MOT or PMOT of live spermatozoa in either fresh or cooled-stored semen. However, VCL and the percentage of membrane-intact spermatozoa were reduced compared to control samples when 75% (v/v) dead spermatozoa were added. Membrane integrity, as assessed by staining with carboxyfluoresein diacetate-propidium iodide, was highly correlated ( r>0.8; P<0.001) with MOT and PMOT in both fresh and cooled-stored semen samples. Results of this study have application to the processing of both cooled and frozen equine semen.

Autoimmune T cell responses to seminal plasma in chronic pelvic pain syndrome (CPPS).

The aetiology of chronic prostatitis is not understood. The aim of this study is to investigate an autoimmune hypothesis by looking for T cell proliferation in response to proteins of the seminal plasma. We studied peripheral blood mononuclear cell proliferation from 20 patients with chronic prostatitis and 20 aged-matched controls in response to serial dilutions of seminal plasma (SP) from themselves (autologous SP) and from a healthy individual without the disease (allo-SP). We found that the patients have a statistically greater lymphocyte proliferation to autologous SP at the 1/50 dilution on day 6 compared to controls (P = 0 x 01). They also have a greater proliferation to allo-SP on both day 5 (P = 0 x 001) and day 6 (P = 0 x 01) at the same dilution. Using a stimulation index (SI) of 9 to either autologous SP or allo-SP on day 6 at the 1/50 dilution as a definition of a proliferative response to SP, then 13/20 patients as compared to 3/20 controls showed a proliferative response to SP (P = 0 x 003, Fishers exact test). These data support an autoimmune hypothesis for chronic prostatitis.

Effect of frequency and interval of semen collection on seminal parameters and fertility of stallions

Insemination with chilled transported semen has become distinctly important in the horse-breeding industry. To ensure cell survival during cooled storage, semen is diluted with an appropriate extender and the concentration of seminal plasma (SP) is reduced. Nevertheless, SP plays an important immunomodulatory role in the female genital tract and supports sperm fertility. The aim of the present study was to evaluate the effect of the addition of autologous SP after cooled storage to highly concentrated stallion semen. Therefore, SP was removed by simple centrifugation of extended semen, aspiration of the supernatant, and resuspension of the sperm pellet with semen extender. Motion characteristics were evaluated after cooled storage for 48 hours at concentrations of 333 × 106 sperm/mL in comparison with stored samples at concentration of 25 × 106 sperm/mL (control). The highly concentrated semen samples were diluted with an extender containing 0%, 5%, 20%, and 80% SP directly before motility analysis. Dilution of the cooled semen with a fresh semen extender without SP (0%) increased kinematic parameters (curvilinear velocity [VCL] 137.3 vs. 151.8; straight-line velocity [VSL] 49.0 vs. 57.5; average path velocity [VAP] 69.5 vs. 79.4 μm/second; amplitude of lateral head [ALH] 3.1 vs. 3.3 μm; beat cross frequency [BCF] 31.6 vs. 33.5 Hz; P < .05) but not total motility (51% vs. 43%) and progressive motility (46% vs. 36%) compared with controls. The addition of SP after storage for 48 hours decreased sperm total motility and progressive motility regardless of SP concentration: 5 (38% and 34%), 20 (37% and 33%), and 80% SP (27% and 22%; P < .05). In contrast, kinematic parameters were enhanced by extenders containing 5% and 20% SP (VCL: 148.0 and 155.6; VSL: 59.2 and 60.9; VAP: 78.7 and 81.9; BCF: 33.4 and 35.7; ALH: 3.4 and 3.4; P < .05). However, using an extender containing 80% SP was detrimental to kinematic parameters (VCL: 151.2; VSL: 52.2; VAP: 76.9; BCF: 34.8; P < .05) except for ALH, which increased (3.5; P < .05). In conclusion, cooled storage at concentrations of 333 × 106 sperm/mL did not affect sperm motility. The addition of a fresh extender or an extender containing small concentrations of SP to highly concentrated ejaculated sperm increased kinematic values after storage; however, increasing concentrations of SP decreased sperm motility.

Post-thaw motility and viability of fractionated and frozen stallion ejaculates

During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 106 sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P < .05), but there was no difference in poor freezing stallions among groups (P > .05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P < .05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P < .05), there was no difference among groups in stallions with poor freezability (P > .05). Average lateral head displacement did not change among groups of fair freezer stallions (P > .05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P < .05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P < .05), but only in group SP1 than in group FT in poor freezer stallions (P < .05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions.

Influence of seminal plasma on cryopreservation of human spermatozoa in a biological material-free medium: study of normal and low-quality semen.

The objective was to evaluate the efficiency of a biological material-free medium and the role of seminal plasma (SP) in the cryopreservation of human spermatozoa. Normal semen samples and low-quality semen samples were used for this study. After centrifugation of 300 microL fractions of whole semen, pellets were resuspended either in autologous SP or in a chemically defined medium (BM) supplemented or not with 3% bovine serum albumin (BSA); after 15 min at 37 degrees C, the samples were diluted (V/V) with cryoprotective medium (30 mM NaCl; 22 mM sodium citrate, 19.4 mM fructose; 80 mM glutamine; 14%, V/V, glycerol) and maintained for 15 min at room temperature before freezing. Assessment of viability and motility was performed using fresh semen (T0), after centrifugation and resuspension prior to adding the cryoprotectant (T15), after adding the cryoprotectant (T30) and after freezing and thawing (Tpost). In all three resuspending media used, sperm viability and motility (forward and total) decreased (p < 0.05) during both the equilibration period especially before addition of the cryoprotective medium (between T0 and T15) and during the freeze-thaw process comparison between T30 and Tpost. The recovery of viable and motile spermatozoa (post-thaw values/values of fresh samples) was higher (p < 0.05) in normal semen than in low-quality semen. In both groups, the recovery was slightly, but significantly, higher with SP than with BM and the presence of BSA has no beneficial effect. To conclude, these data suggest that SP may reduce the deleterious effects of cryopreservation. Nevertheless cryopreservation of spermatozoa in a medium containing neither SP nor biological substances could offer an acceptable cryoprotection of spermatozoa to be used in assisted fertilization procedures, especially for intracytoplasmic sperm injection.

Effects of seminal plasma and three extenders on canine semen stored at 4 °C

Semen preservation and artificial insemination (AI) in the canine has become a common practice in veterinary medicine. Chilled dog semen is easy to handle, and several extenders can be used. The aim of this study was to compare the effects on canine spermatozoa of seminal plasma and 3 extenders commonly used for chilled semen preservation in clinical practice. The characteristics evaluated were sperm motility; velocity; plasma membrane status (assessed with a fluorescence staining technique and hypo-osmotic swelling test); acrosome morphology; semen pH; and semen osmolarity. These criteria were monitored daily in the ejaculates of 11 dogs. The ejaculates were divided into 4 aliquots. Each aliquot was extended in autologous seminal plasma, egg-yolk Tris, egg-yolk milk or egg-yolk cream and preserved at 4 °C for 4 d. In 10 of 11 semen samples extended in autologous seminal plasma, motility had already decreased to 0% by Day 2, and the percentage of spermatozoa with intact membranes was lower than in the 3 extenders (P < 0.05). Motility up to Day 4 was higher in egg-yolk Tris-stored spermatozoa (53.6%) than in those preserved in egg-yolk milk (30.4%) and egg-yolk cream (14.1%). Spermatozoa stored in egg-yolk Tris also had the highest sperm velocity, whereas no difference was found in plasma membrane or acrosome status (P>0.05). Egg-yolk Tris extender seems to be superior to the other extenders tested, to preserve dog semen at 4 °C, although differences were not significant for all the parameters.

White Cell Subpopulations in Autoimmune Infertile Men and Their Wives with Cytotoxic Sperm Antibodies

Sixty six infertile men with cytotoxic sperm antibodies had fewer and lower percentages of OKT4-positive helper/inducer T lymphocytes p = 0.004 and 0.003, respectively), than the fertile (n = 14) and infertile (n = 12) antibody-negative controls. Ratio of OKT4/OKT8 was decreased (p less than 0.01), while numbers of OKDR and BD-Leu12 positive predominantly B lymphocytes were increased (p less than 0.0001) in infertile men with sperm antibodies. T lymphocytes positive for OKT3, OKT11 and OKT4 and the OKT4/OKT8 ratio were decreased in infertile women with sperm antibodies, while B lymphocytes positive for OKDR and BD-Leu12 were increased in infertile women with or without sperm antibodies (p less than 0.0001, versus fertile controls). Lymphocyte responses to phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM), however, were similar in 20 fertile and 136 infertile couples. Lymphocytes from infertile men with sperm antibodies had an enhanced stimulatory response to autologous sperm and seminal plasma. It is concluded that sperm antigens from autoimmune infertile men stimulate heightened immune responses to sperm antigens and altered distribution of white cell populations in both partners.

Cytotoxic sperm antibodies inhibit sperm penetration of zona-free hamster eggs

Zona-free hamster egg sperm penetration assay was used to study the effects of cytotoxic sperm antibodies on egg penetration by the sperm of fertile and infertile men. Twenty-nine fertile and 9 infertile men did not have significant cytotoxic sperm antibodies in their serum and seminal plasma; 7 infertile men were positive for these antibodies in serum and seminal plasma. Two others were positive in sera, and 14 were positive in seminal plasma. Sperm from 18 of 23 (78%) infertile men with sperm antibodies had poor egg penetration (less than or equal to 20%) compared with only 6 of 38 (16%) nonautoimmune men (P less than 0.0001). Sperm from nonautoimmune fertile men were coated with seminal plasma and serum of autoimmune men and serum of isoimmune women, resulting in a significant decrease in hamster egg penetration. Sixteen of 21 (76%) seminal plasma samples with cytotoxic sperm antibodies reduced the control sperm penetration of hamster eggs by greater than or equal to 50%. Coating of sperm from fertile men with serum and seminal plasma samples from non-sperm-immune fertile and infertile subjects did not alter their penetration of hamster eggs. Coating of sperm from autoimmune men with cytotoxic antibody-positive autologous seminal plasma samples resulted in a significant decrease of egg penetration. The inhibitory effect of antibody-positive seminal plasma samples on egg penetration by control sperm was abrogated when the samples were preabsorbed with sperm. It is concluded that cytotoxic sperm antibodies, especially those in seminal plasma, inhibit hamster egg penetration by autologous and control sperm. This may explain in part the incidence of infertility associated with sperm antibodies.