Recovery Of Motile Sperm Research Articles

LESS NO PRODUCTION AND BETTER MOTION PARAMETER IN HUMAN SPERM BY SWIM-UP PROCESSING

Fifteen semen specimens were obtained from men for semen analysis; each was divided into two aliquots for prepararation. The motile sperm recovery rate, percentage motility, and motion parameters were measured for each semen specimen (n = 15) before and after preparation with the use of the two methods, and cultured with different time courses (1 hr, 3 hr, and 6 hr). Nitric oxide (NO) was measured using the chemiluminscence method after centrifugation. Recovery rate of motile cell was significantly higher in direct swim-up method (69.5 +/- 42.4% versus 49.3 +/- 29.3%, p < 0.05). In motility, direct swim-up method in the different time courses was significantly better than IxaPrep method. (1 hr: 91.1 +/- 5.2% vs 65.6 +/- 16.4%, 3 hr: 87.2 +/- 7.9% vs 65.2 +/- 16.5%, 6 hr: 86.1 +/- 7.5% vs 60.8 +/- 17.6% and prewash: 61.6 +/- 16.2%, p < 0.05). In VAP and VSL, the sperm prepared by the above two methods all improved compared to pre-wash sperm (p < 0.05), but there was no statistical significance between the two methods. NO production in the direct swim-up group was significantly lower than IxaPrep group in the first hour of culture (0.09 +/- 0.09 uM vs 0.15 +/- 0.09 uM, p < 0.05). NO production increased as the culture time increased in swim-up group, but conversed in IxaPrep group. The lower level of NO produced in the swim-up group may suggest that better sperm quality achieved is due to the decreased NO production.

Intracellular sperm and seminal plasma Ca+2 concentrations affect cryodamage susceptibility after a cycle of freezing and thawing

Objective: Sperm cells can be affected in viability, fertilizing ability and motility by freezing and subsequent thawing in assisted reproduction techniques. Ca+2 is an important cellular metabolism messenger. In other mammals it has been noted that high calcium concentrations in the external medium may decrease the motility and metabolism of sperm and “calcium intoxication” may be a factor in cryodamage. Subsequently, the intracellular and seminal plasma Ca+2 contents are potential predictors of sperm quality after freezing-thawing process. Our aim with this study is to increase the information of the parameters of the cell physiology that are affecting sperm survival after freezing/thawing process, and to determine the predictive value of plasma and cellular Ca+2 determinations. Materials and Methods: This study was performed in 94 human sperm samples after freezing-thawing process, studied according with WHO statements for concentration of motile and alive sperm, both fresh and after thawing. We measured intracellular Ca+2 by incubation with Fluo Ca+2 indicator Fluo-4 AM (Molecular Probes) (30′ 37°C) by fluorometry (Excitation: 485nm Emmision: 538nm) (Fluoroskan, Labsystems). Seminal plasma Ca+2 were measured with an Alcyon analyzer (Abbot). One way analysis of variance was used to detect differences between Ca+2 and sperm rate survival after thawing. Linear regression analysis was used to determine the extent of correlation between parameters compared. The diagnostic performance of the tests performed was evaluated using Receiver Operating Characteristic (ROC) curve analysis. Results: Intracellular sperm Ca+2 was expressed as fluorescence arbitrary units/50 million cells and Ca+2 in seminal plasma was expressed as mg/dl. AUCROC: area under the curve in a receiver operating characteristic analysis. CI: confidence interval. PPV: positive predictive value. NPV: negative predictive value. Tabled 1 A significant negative relationship between intracellular Ca+2 (r=−0.36) and seminal plasma Ca+2 (r=−0.30) concentrations and post-thaw motile sperm recovery rate was observed. Predictive values demonstrated that Ca+2 has good diagnostic accuracy. ∗ denotes statistically significant relationship, p<0.05. Conclusions: There was a negative correlation between the concentrations of intracellular and plasma seminal Ca+2 with the percentage of sperm survival after thawing. This findings strongly suggests that high intracellular sperm Ca+2 and seminal plasma Ca+2 negatively affect sperm metabolism and increase the possibilities of cryodamage after a freezing/thawing process. Research should be focused now in introducing Ca+2 quelators and phospholipids in the freezing media to prevent increased calcium flux into the sperm.

Sperm glutathione antioxidant defense system protects from cryodamage after a cycle of freezing and thawing

Sperm glutathione antioxidant defense system protects from cryodamage after a cycle of freezing and thawing

Predictive factors for motile sperm recovery using testicular biopsy in nonobstructive azoospermic patients.

The objective of the present study was to assess the predictive value of hormone values, histological analysis for the finding of motile spermatozoa on testicular biopsy in nonobstructive azoospermia. This cross-sectional study assessed serum follicle-stimulating hormone (FSH), luteinizing hormone, prolactin and total testosterone values in 50 patients undergoing testicular biopsy prior to ICSI. P < 0.05 was considered significant. Recovery was successful in 28 cases, and motile spermatozoa were retrieved in 7. Significant differences were detected between presence of motile spermatozoa and absence of spermatozoa in terms of FSH values (P = 0.003, one-way ANOVA). The other variables did not present statistical differences. A receiver operating characteristic curve showed that FSH levels below 17.00 IU/L were predictive of motile spermatozoa recovery. FSH values can serve as a predictive factor for the recovery of motile spermatozoa using biopsy in azoospermic patients. On the other hand, histological analysis and other hormone values were not helpful in the prediction of motile sperm.

Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium

Experiments were carried out to investigate the cryoprotecting potential of dextrans (ranging from 10 to 2000 kDa) using a synthetic model system developed recently in this laboratory. Goat spermatozoa from the cauda epididymidis were extracted in a chemically defined medium (modified Ringer's solution) and assayed for motility using a phase-contrast microscope. The sperm cells were subjected to a freezing protocol in a computer-controlled biofreezer (cooling at 0.25 degrees C min(-1) to 5 degrees C; 5 degrees C min(-1) to -20 degrees C; 20 degrees C min(-1) to -100 degrees C) and plunged into liquid nitrogen. The frozen cells were thawed rapidly at 37 degrees C in a thermostatic waterbath. In the absence of dextran, all the spermatozoa lost their motility. The cryoprotecting efficacy of each dextran was found to be biphasic in nature. Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the least active. Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility to the extent of approximately 23%, 21%, 19%, 18%, 16%, 15% and 8%, respectively. Optimum concentrations of these dextrans for cryoprotection of sperm motility were 8.42, 2.50, 1.09, 0.37, 0.31, 0.10 and 0.04 mmol l(-1), respectively. It thus appears that each dextran has a characteristic cryoprotection profile. The data show that the cryoprotecting efficacy and optimum cryoprotecting concentrations of dextrans are inversely related to their molecular mass. Dextran also served as a significant cryoprotectant in the presence of glycerol (0.87 mol l(-1)) and dimethyl sulphoxide (0.76 mol l(-1)), which are well known cryoprotectants; the action of the combined cryoprotectants was almost additive. The presence of glycerol or glycerol plus dimethyl sulphoxide caused a significant reduction (from 8.42 to 6.27 mmol l(-1)) in the optimum concentration of dextran. In the presence of the three cryoprotectants, recovery of sperm motility was as high as 58% (forward motility) and 60% (total motility).

Effect of dextrans on cryopreservation of goat cauda epididymal spermatozoa using a chemically defined medium

Experiments were carried out to investigate the cryoprotecting potential of dextrans (ranging from 10 to 2000 kDa) using a synthetic model system developed recently in this laboratory. Goat spermatozoa from the cauda epididymidis were extracted in a chemically defined medium (modified Ringer's solution) and assayed for motility using a phase-contrast microscope. The sperm cells were subjected to a freezing protocol in a computer-controlled biofreezer (cooling at 0.25 degrees C min(-1) to 5 degrees C; 5 degrees C min(-1) to -20 degrees C; 20 degrees C min(-1) to -100 degrees C) and plunged into liquid nitrogen. The frozen cells were thawed rapidly at 37 degrees C in a thermostatic waterbath. In the absence of dextran, all the spermatozoa lost their motility. The cryoprotecting efficacy of each dextran was found to be biphasic in nature. Initially, as the concentration of dextran was increased, the recovery of sperm motility also increased and reached an optimum value; however, with further increases in dextran concentration, the recovery of motility decreased sharply. Of all the sugar polymers tested, 10 kDa dextran showed the highest cryoprotecting efficacy, whereas the 2000 kDa sugar polymer was the least active. Dextrans of 10, 40, 73, 173, 252, 500 and 2000 kDa offered maximum cryorecovery of forward motility to the extent of approximately 23%, 21%, 19%, 18%, 16%, 15% and 8%, respectively. Optimum concentrations of these dextrans for cryoprotection of sperm motility were 8.42, 2.50, 1.09, 0.37, 0.31, 0.10 and 0.04 mmol l(-1), respectively. It thus appears that each dextran has a characteristic cryoprotection profile. The data show that the cryoprotecting efficacy and optimum cryoprotecting concentrations of dextrans are inversely related to their molecular mass. Dextran also served as a significant cryoprotectant in the presence of glycerol (0.87 mol l(-1)) and dimethyl sulphoxide (0.76 mol l(-1)), which are well known cryoprotectants; the action of the combined cryoprotectants was almost additive. The presence of glycerol or glycerol plus dimethyl sulphoxide caused a significant reduction (from 8.42 to 6.27 mmol l(-1)) in the optimum concentration of dextran. In the presence of the three cryoprotectants, recovery of sperm motility was as high as 58% (forward motility) and 60% (total motility).

Evaluation of human sperm function after repeated freezing and thawing.

Sperm storage via freezing has been useful for men who have difficulty masturbating during assisted reproductive technology (ART) programs and before impotency caused by chemotherapy, vasectomy, and other procedures. Studies were undertaken to evaluate the extent of cryoinjury to sperm after repeated freezing and thawing. The results showed that normozoospermic and oligozoospermic sperm survived after 3 repeated freeze-thaw cycles. The inclusion of seminal plasma did not seem to protect human sperm during freezing and thawing. There were no significant differences in recovery percentages for motile, vital, and morphologically normal sperm between slow and rapid freezing methods in thaws 1, 2, and 3 of normozoospermic and oligozoospermic unwashed (u), washed (w), and washed + seminal plasma (ws) samples. However, there were significant percentage drops in the recovery of motile and vital sperm between each thaw (ie, first to second thaw, and second to third thaw) using both slow and rapid freezing for u, w, and ws samples (P < .01). There were also no significant differences in percentage recovery of motile, vital, and morphologically normal sperm between u, w, and ws samples during thaws 1 to 3 in the normozoospermic and oligozoospermic groups. Sperm were capable of fertilizing hamster oocytes microinjected with single sperms after 3 freeze-thaw cycles as evidenced by the formation of 2 distinct pronuclei and 2 polar bodies in 22.2% and 17.2% of normozoospermic and oligozoospermic samples, respectively. The numbers of normal vital motile sperm after 3 serial freeze-thaw cycles are adequate for bringing about fertilization via intracytoplasmic sperm injection in ART programs. Thus, leftover washed sperm in laboratories that perform in vitro fertilization can be frozen, thawed, and refrozen several times without loss of the sperms' ability to fertilize. This approach has tremendous benefits for men who have difficulty producing sperm and for those with low and declining sperm counts.

Comparison of centrifugation- and noncentrifugation-based techniques for recovery of motile human sperm in assisted reproduction.

To compare standard density gradient centrifugation sperm preparation with a novel non-centrifugation-based dual-chamber capillary dish in efficiency for motile human sperm separation, approximately 3 mL fresh ejaculate specimens was obtained from 21 men (median age = 32 years. range 26-42 years) undergoing infertility evaluation. For each specimen, half of the sample was processed with a standard 45%/90% density gradient preparation (PureSperm. Nidacon International, Gothenburg, Sweden) followed by semen analysis. The other half was incubated in the Zech glass capillary dish (Astromedtec, Salzburg, Austria) consisting of 2 concentric wells overlaid by a U-ring and coverglass. After approximately 3 h, a 1-mL sample was taken from the central chamber and analyzed. Percentage motile sperm recovery, absolute (motile) cell number, and path velocities were compared for spermatozoa obtained from both methods. Both techniques reduced overall sperm concentration while enriching specimens with more motile spermatozoa. A trend towards higher % recovery of motile spermatozoa (p = .264) was observed with the Zech device, but at a cost of fewer absolute numbers of higher velocity cells (p = .004). The Zech device, therefore, localized a very small population of motile sperm without exposure to centrifugation stress, which has been considered potentially harmful to spermatozoa. This technique could theoretically improve efficiency by reducing time required to identify motile cells in in vitro fertilization where intracytoplasmic sperm injection is planned. However, refinements in incubation interval and suspension volumes are needed before this technique can be considered comparable to the density gradient method in recovering sperm for use in intrauterine insemination.

Relationship between motility and membrane integrity of boar spermatozoa in media varying in osmolality.

A study was conducted to evaluate the relationship between boar sperm motility and membrane integrity following exposure to media with 150-1120 mOsm. Total sperm motility was defined as the percentage of spermatozoa that had any form of motility was subjectively assessed under a light microscope. Sperm cell damage was expressed as a loss of membrane integrity as measured by a combination of fluorescent stains, carboxyfluorescein diacetate (CFDA) and propidium iodide (PI), and Hoechst 33258 (H33258). There were no significant differences between sperm motility and membrane-intact spermatozoa. as measured by CFDA-PI and H33258, in media with 250 and 300 mOsm. In anisosmotic conditions, a higher amount of membrane-intact spermatozoa than motile spermatozoa was observed. In hypo-osmotic conditions (150 mOsm), a high proportion of spermatozoa had curled or coiled tails and most of them retained their entire membrane integrity, as detected by CFDA-PI. In media with 350-1120 mOsm, some spermatozoa accumulated PI in the head region and CFDA in the mid-piece. These spermatozoa fluoresced blue at the lower region of the head, as detected by H33258. The ATP content in spermatozoa exposed to hypo- and hyperosmotic conditions was markedly reduced. There was no recovery of sperm motility on returning the spermatozoa to isosmotic conditions after 10 min incubation in anisosmotic conditions, indicating that the spermatozoa suffered an almost complete and irreversible loss of motility. This irreversible loss of motility may be a consequence of reduced ATP production in spermatozoa subjected to anisosmotic conditions. The results of this study demonstrate that plasma membrane integrity assessment in combination with sperm motility, using a range of media varying in osmolality, can give valuable information about the status and function of different sperm membranes, which might be relevant for semen preservation.

A new CentriSwim procedure to increase the recovery of motile spermatozoa.

A prospectively controlled in vitro study was performed to compare sperm concentration, sperm motility and progressive sperm motility recovered following the standard swim-up procedure and a new CentriSwim procedure. The CentriSwim procedure involves creating a centrifugal force to counteract the force of gravity during sperm swim-up procedure. Two aliquots of semen from 12 normozoospermic ejaculates and 12 laboratory-induced oligoasthenozoospermic specimens were diluted, centrifuged, and 1.0 ml of media layered over the sperm pellet. One aliquant was processed by standard swim-up technique. The other aliquant was processed by CentriSwim procedure involving centrifugation at 200 rpm on a 2-cm radius upward-directing arm, at an angle of 60 degrees for 10 min, creating roughly 0.8 g centrifugal force at room temperature (22-24 degrees C) to counteract the force of gravity. The numbers of spermatozoa recovered from the upper 0.5 ml of the medium following CentriSwim from the normozoospermic ejaculates and laboratory-induced oligoasthenozoospermic specimens were significantly higher than following standard swim-up procedure. No statistical differences in the recovery of percentage sperm motility and progressive sperm motility between the two techniques were observed. In conclusion, the CentriSwim procedure yields higher numbers of motile spermatozoa than the standard swim-up technique.

Characterization of subsets of human spermatozoa at different stages of maturation: implications in the diagnosis and treatment of male infertility.

Reactive oxygen species (ROS)-induced damage of membrane phospholipids and DNA in human spermatozoa has been implicated in the pathogenesis of male infertility. In this study, variations in ROS production, DNA structure (as measured by the sperm chromatin structure assay) and lipid composition, were studied in human spermatozoa at different stages of maturation. Sperm subsets were isolated by discontinuous density gradient centrifugation of semen samples obtained from healthy donors and from infertility patients. DNA damage and ROS production were highest in immature spermatozoa with cytoplasmic retention and abnormal head morphology, and lowest in mature spermatozoa. Docosahexaenoic acid and sterol content were highest in immature germ cells and immature spermatozoa, and lowest in mature spermatozoa. The relative proportion of ROS-producing immature spermatozoa in the sample was directly correlated with DNA damage in mature spermatozoa, and inversely correlated with the recovery of motile spermatozoa. There was no correlation between DNA damage and sperm morphology in mature spermatozoa. The high levels of ROS production and DNA damage observed in immature spermatozoa may be indicative of derangements in the regulation of spermiogenesis. DNA damage in mature spermatozoa may be the result of oxidative damage by ROS-producing immature spermatozoa during sperm migration from the seminiferous tubules to the epididymis.

A prospective study of programmable freezer vs. nitrogen vapor sperm cryopreservation techniques.

Objective: Sperm cryopreservation by liquid nitrogen vapor is simpler, faster and cheaper than programmable biologic freezing (J Repro Med 1999 Feb;44(2):87–90). However, whether or not sperm recover equally well with both techniques is not well defined. We sought to examine the recoverability of both ejaculated and aspirated sperm frozen by each of these two methods to determine which method produces superior rates of motile sperm recovery after freezing and thawing.

Fine needle sperm aspiration: A favored method for sperm retrieval in obstructive azospermia.

Objective: to evaluate the outcome of intracytoplasmic sperm injection (ICSI) cycles in patients with obstructive azospermia undergoing fine needle sperm aspiration compared to open testicular biopsy. Design: Retrospective study. Materials/Methods: Between May 1994 and November 2000,a total of 245 patients underwent 329 cycles of ICSI with surgical sperm retrieval. Only patients with obstructive azospermia (n = 174 patients, 190 cycles) were included. They were divided into 2 groups based on the technique of sperm retrieval: Group 1 (n = 146) underwent fine needle sperm aspiration (72 testicular and 74 epididymal) while group 2 (n = 44) underwent open testicular biopsy. Mean female age was 33.9 ± 6.3 and 33.8 ± 5.6 years in the 2 groups respectively. Statistical analysis was done with analysis of variance or χ2 test with p < 0.05 considered statistically significant. Results: There was no significant difference among the 2 groups in age, stimulation, downregulation protocol, number of eggs retrieved, number of embryos transferred or method of embryo transfer. Outcome of ICSI following surgical sperm retrieval in the 2 groups of obstructive azospermia is shown in Table 1. Among patients with obstructive azospermia undergoing testicular sperm aspiration (n = 72 cycles), ongoing pregnancy rate per cycle and embryo transfer were 27.8% and 28.2% respectively compared to 36.5% and 37.5% in patients undergoing epididymal sperm aspiration (n = 74 cycles). The difference was also not statistically significant (p 0.05).Table 1Outcome of ICSI in patients with obstructive azospermia undergoing sperm aspiration vs testicular biopsy.Sperm aspiration (n = 146)Open biopsy (n = 44)Motile sperm recovery rate %83.681.8 ∗Difference is not statistically significant (p > 0.05).Fertilization rate %61.966.2 ∗Difference is not statistically significant (p > 0.05).Implantation rate %11.711.7 ∗Difference is not statistically significant (p > 0.05).Clinical pregnancy rate/embryo transfer %43.431.8 ∗Difference is not statistically significant (p > 0.05).Ongoing pregnancy rate/embryo transfer %32.929.5 ∗Difference is not statistically significant (p > 0.05).∗ Difference is not statistically significant (p > 0.05). Open table in a new tab Conclusions: Fine needle sperm aspiration resulted in similar fertilization, implantation and pregnancy rates compared to open testicular biopsy in patients with obstructive azospermia. Pregnancy rates were also comparable with testicular and epididymal sperm aspiration. As a less invasive and technically easier procedure, fine needle sperm aspiration is recommended for surgical sperm recovery in patients with obstructive azospermia compared to open testicular biopsy. Supported by: IVF Michigan, Michigan State University.

Sperm retrieval, fertilization, and pregnancy outcome in repeated testicular sperm aspiration.

To report the outcome of sperm retrieval and results after ICSI in up to six repeated testicular sperm aspiration procedures. Twenty-two men with obstructive and thirty-four men with nonobstructive azoospermia underwent 50 and 91 needle aspirations, respectively. Sufficiency of spermatozoa for ICSI and cryopreservation, fertilization rate, and pregnancy outcome was analyzed retrospectively. No major differences were found in sperm recovery or pregnancy outcome in the repeated cycles. Testicular aspirate containing motile spermatozoa with maintained fertilizing capacity was obtained in up to six repeated procedures in the nonobstructive group. No postoperative complications were reported for any of the participants. Testicular sperm aspiration is a simple and effective method of sperm retrieval, which can be performed from the same testis up to several times with good recovery of motile spermatozoa for ICSI and maintaining high fertilization and pregnancy rates, in men with both obstructive and nonobstructive azoospermia.

Comparison of protective media and freezing techniques for cryopreservation of human semen

Objective: To evaluate the influence of cryopreservation medium and freezing–thawing techniques on human sperm motility and morphology. Study design: 63 semen samples were obtained from 39 donors to the artificial insemination programme. Possible effects of the sperm dilution with cryomedium on the motility were examined 10 min after exposure of 24 high initial quality semen samples to TEST–yolk {zwitterion–citrate–egg yolk extender containing TES [N-Tris (hydroxymethyl) methylaminoethane sulfonic acid] and Tris [(hydroxymethyl) aminomethane]} and human sperm preservation medium (HSPM). Post-thaw sperm motility from 24 frozen semen samples was examined comparing the cryoprotective efficacy of TEST–yolk and HSPM following different freezing techniques (vapour freezing, fast programmable freezing and slow programmable freezing). The relationship of sperm morphology to the effects of freezing was investigated on 39 semen samples following different freezing techniques. Post-thaw sperm motility from 39 frozen semen samples was compared among three groups divided according to the percentage of morphologically normal cells (<40, 40–50 and >50%) in fresh semen. Results: Exposure of spermatozoa to cryomedia for 10 min at room temperature significantly reduced motility in TEST–yolk treatment group for 9% and in HSPM treatment group for 18% (P<0.01). The recovery of motile sperms (mean±standard deviation) was 49±15.7, 43±15.2 and 52±16.8% when TEST–yolk was used and 34±17.8, 32±18.2 and 50±13.6% when HSPM was used as a cryopreservative following vapour freezing, and fast and slow programmable freezing, respectively. Following vapour freezing and also following fast programmable freezing, the recovery of motile sperm was significantly higher (P<0.05) after addition of TEST–yolk medium than after addition of HSPM. Post-thaw motility of the sperm cryopreserved in HSPM showed significant differences (P<0.05) after three different freezing techniques. The recovery of motile sperms was 57±26.4, 38±8.6 and 38±17.3% in groups with >50, 40–50 and <40% morphologically normal cells, respectively. The percentage of morphologically normal spermatozoa was reduced 8% after vapour freezing and 6 and 3% after fast and slow programmable freezing, respectively. The results were statistically analysed using sas/stat software. Conclusions: Slow programmable freezing was superior to vapour freezing and fast programmable freezing as a method for sperm cryopreservation. However, none of these methods of freezing had discernible effects on sperm morphology. Motility of spermatozoa decreased due to the exposure of semen to cryomedium. TEST–yolk was a superior cryomedium to HSPM. Fresh semen with more than 50% of morphologically normal cells showed the best recovery of motile cells after freezing and thawing.

Bacterial contamination and sperm recovery after semen preparation by density gradient centrifugation using silane-coated silica particles at different g forces

The effects of density gradient centrifugation through silane-coated silica particles (PureSperm) using 100, 200, 300 and 500 g on bacterial contamination of sperm samples and recovery of motile spermatozoa from sperm samples were investigated with conventional culturing techniques and microscopic visual assessment. The recovery of motile spermatozoa was variable and was not improved using 500 g compared to the recommended 300 g. The bacterial contamination was highly decreased by gradient centrifugation through PureSperm and was almost abolished when strict aseptic techniques were used, with changes to sterile Pasteur pipettes and tubes prior to washing procedures.

Development of a Simple Sperm Cryopreservation Model Using a Chemically Defined Medium and Goat Cauda Epididymal Spermatozoa

This investigation was carried out to develop a simple sperm cryopreservation model using a chemically defined synthetic medium (modified Ringer's solution) and mature goat cauda epididymal sperm as the model system. Rates of cooling, freezing, and maximum freezing temperature were manipulated with the help of a computer-controlled programmable biofreezer. Highly motile goat cauda sperm dispersed in a modified Ringer's solution was subjected to the freezing protocol: cooling 0.25°C min−1 to 5°C, 5°C min −1 to −20°C, 20°C min−1 to −100°C, prior to plunging into liquid nitrogen. In the absence of any cryoprotective agent, all of the spermatozoa lost their motility. Addition of glycerol (0.22 to 0.87 M) caused a dose-dependent increase of sperm motility recovery. The highest recovery of forward and total motility was (32 and 35%, respectively) at 0.87 M. Further increase of the glycerol concentration caused a marked decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery (0–18%) of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells. Like glycerol, dimethyl sulfoxide (Me2SO) and ethylene glycol also showed a dose-dependent increase in motility recovery as well as a biphasic curve of cryoprotection. At optimal concentrations, dimethyl sulfoxide (1.00 M) and ethylene glycol (1.29 M) were effective in recovering sperm motility to the extent of 20 and 13%, respectively. Thus these reagents have markedly lower cryoprotection potential than glycerol.

Assessment of two devices for in vitro preparation of human sperm.

The aim of the study was to assess and compare the efficiency of the ZSC-II versus the Sperm Select techniques in preparing human sperm from normozoospermic patients for use in intrauterine insemination and other assisted reproductive technologies. Twenty-five patients were included in the study. Semen was collected at intercourse and processed via 2 sperm preparation methods. Recovery of motile sperm and other sperm qualitative measurements were evaluated before and after ZSC-II and Sperm Select sperm preparation. Sperm qualitative measurements were not significantly different after the ZSC-II and Sperm Select procedures (p > .05). Differences (p < .05) were noted between the 2 procedures in the number of sperm recovered. A higher survival rate (longevity test, 72 h) was observed after ZSC-II sperm preparation and recovery. The ZSC-II procedure yielded higher total motile sperm than the Sperm Select. The superiority in longevity may suggest possible advantages in obtaining higher fertilization and pregnancy rates.

Comparison of two colloidal silica-based sperm separation media with a non–silica-based medium

Objective: To study and compare the effects of three sperm separation media, two silica-based (Percoll; Pharmacia Biotech AB, Uppsala, Sweden, and Isolate; Irvine Scientific, Santa Ana, CA) and one non–silica-based (Ixaprep; Medicult, Copenhagen, Denmark), on the recovery of progressive motile sperm, the percentage of sperm with normal morphology, various sperm motion characteristics determined by computer-aided sperm analysis, and the percentage of acrosome-reacted sperm. Design: Prospective study. Setting: A university-based assisted reproductive technology center. Patient(s): Male partners of couples attending our infertility clinic. Intervention(s): None. Main Outcome Measure(s): Various semen parameters. Result(s): Both Isolate and Ixaprep resulted in enhanced recovery of motile spermatozoa compared with Percoll. The percentage of sperm with forward progressive motility, the percentage of sperm with normal morphology, and various sperm motion characteristics were similar after the use of Percoll and Isolate and were significantly better than after the use of Ixaprep. The same percentage of acrosome-reacted spermatozoa was observed with all three media. Similar results were observed in both normal and subnormal semen samples. Conclusion(s): The use of Isolate and Ixaprep resulted in better recovery of motile spermatozoa. Other semen parameters were similar with the use of Isolate and Percoll, whereas the use of Ixaprep was associated with lower sperm velocities and fewer morphologically normal spermatozoa.

Increased recovery of swim-up spermatozoa by application of “antigravitational” centrifugation

Objective: To compare the quantity and quality of sperm recovered following the 10-minute application of an antigravitational force during the swim-up procedure with the standard 60-minute swim-up (SSU) procedure. Design: Prospectively controlled in vitro study. Setting: Private andrology laboratory and hospital-based infertility practice. Intervention(s): Equal aliquots of semen were evaluated following various intervals of antigravitational centrifugation swim-up (ACSU). ACSU and SSU were then compared. Patient(s): Thirty-eight men undergoing therapeutic testing. Main Outcome Measures: Sperm concentration, sperm motility, and progressive sperm motility. Results: The number of sperm recovered from the ACSU procedure was significantly higher than from the SSU procedure. No significant differences in percent motile sperm and progressive motile sperm recovery between the two procedures were observed. Conclusion: The ACSU procedure yields a higher number of motile spermatozoa in a much shorter time.