Chromatin intact human sperm recovery is higher following glass wool column filtration as compared with density gradient centrifugation.

The sperm DNA quality as determined by chromatin integrity has been reported to be associated with in vivo and in vitro fertility. However, previous studies have evaluated preparation procedures to select motile, morphologically normal and mature spermatozoa, but not the spermatozoa with intact sperm chromatin. To determine which technique yields a population of spermatozoa with improved DNA quality, split ejaculate was processed with density gradient centrifugation (DGC) procedure and glass wool column filtration (GWF) procedure. The processed samples were analysed for sperm DNA quality using the acridine orange staining method on flow cytometry. The GWF procedure decreases the percentage of spermatozoa with DNA fragmentation resulting in more intact chromatin in the processed sample. There is a need to design a clinical study with GWF for assisted reproductive technology.

Correlation between neutral alpha-glucosidase activity and sperm DNA fragmentation.

To evaluate the association between neutral alpha-glucosidase (NAG) activity and sperm DNA fragmentation (DFI), ejaculates from 24 men undergoing evaluation for sperm DNA damage as a part of infertility assessment were analysed. The mean +/- SD and range for the semen quality of the 24 ejaculates are as follows: volume (3.1 +/- 1.3, 1.8-6.0 ml); sperm concentration (45.6 +/- 41.1, 1.3-151.2 x 10(6) ml(-1)); sperm motility (52.8 +/- 28.8, 1-95%); sperm with fragmented DNA (17.6 +/- 15.4, 1.7-56.0%); sperm with immature chromatin (9.6 +/- 3.8, 2.5-19.1%); NAG activity (37.9 +/- 18.3, 4.4-75.3 mU ml(-1)). The only sperm parameter significantly correlated with neutral alpha-glucosidase is the percentage of sperm DFI [correlation coefficient (r) = 0.4376, P = 0.03].

Vascular endothelial growth factor gene polymorphism and implantation failure.

Implantation failure is the most frequent cause of lack of pregnancy after IVF and embryo transfer. Successful implantation requires the invading blastocyst to stimulate its own blood supply through angiogenesis. Vascular endothelial growth factor (VEGF) is the best-characterized regulator of angiogenesis. Since one polymorphism of the VEGF gene, -1154 G/A, has been previously suggested to be associated with recurrent spontaneous abortion, the present study was undertaken to determine whether VEGF -1154 G/A genotype is also related to recurrent implantation failure. Buccal swabs were obtained from 70 women with a history of recurrent implantation failure after IVF-embryo transfer and from 73 control women. DNA was extracted from the buccal swabs and analysed for the presence of the VEGF -1154 A/A gene. The frequency of homozygosity of the VEGF -1154 A/A gene was significantly higher among women experiencing recurrent implantation failure compared with fertile control women (19% versus 5%, P = 0.02). It is concluded that homozygosity of the VEGF -1154 A/A gene may serve as a susceptibility factor affecting the chances of recurrent implantation failure.

The straw swim-up: a simple method to evaluate the effects of additives, media, or toxicants on sperm motility.

We describe a new method for evaluating potential toxicants, spermicides, and motility enhancers. Cryopreservation straws filled with media plus additive are emersed below the surface of an unprocessed donor ejaculate. After incubation, the sperm found within the first 4 cm of the straw are expelled and analyzed. The effects of media containing glycerol and albumin were examined. Survivability and sperm migration into the media occur independently and can be differentiated in this assay. This method allows for the use of unprocessed semen and includes an evaluation of the semen and media interaction.

Effects of urine on the functional quality of human spermatozoa.

OBJECTIVE: To determine the effect of urine on sperm function and to elucidate the reason by which it may be deleterious. DESIGN: Prospective controlled in vitro study. INTERACTIONS: Sperm were exposed to various concentrations of pooled urine in Ham's F-10 Medium supplemented with 3.5 g% bovine serum albumin (GIBCO, Grand Island, NY) and sperm exposed to Ham's F-10 Medium with pH and osmolality adjusted to mimic that of the urine (pH 5.4; 520 mOsm). MAIN OUTCOME MEASURE: Sperm motility and the ability of sperm to penetrate and migrate into mucus and to penetrate hamster oocytes were determined. RESULTS: All sperm variables studied were significantly decreased when the sperm were exposed to > or = 40% urine. Potential of hydrogen- and osmolality-adjusted Ham's F-10 did not significantly affect the sperm function. CONCLUSION: Urine is deleterious to sperm function and may involve other biochemical factors that are unrelated to pH and osmolality of urine.

Glass wool column filtration versus mini-Percoll gradient for processing poor quality semen samples.

OBJECTIVE: To compare the quality and number of spermatozoa recovered from laboratory-induced severe oligozoospermic specimens processed by mini-Percoll gradient and glass wool column filtration. DESIGN: Both sperm-processing procedures were compared in similar sperm samples adjusted to contain equal low numbers of motile spermatozoa using either dilution (oligozoospermia) or with the addition of killed sperm (oligoasthenozoospermia). The spermatozoa processed by both procedures samples were evaluated for motility, response to hypo-osmotic swelling test, and the hemizona assay. PATIENTS: Five healthy fertile sperm donors. SETTING: Private Andrology Laboratory and University Hospital. MAIN OUTCOME MEASURE: Sperm motility, hypo-osmotic swelling test, and hemizona assay results determined the efficacy of the sperm-processing procedures. RESULTS: The concentration of sperm recovered after both procedures was not affected by either preparation or processing methods. Glass wool-processed sperm had higher motility in oligoasthenozoospermic samples, bound tightly to hemizonae in higher mean numbers, and demonstrated a higher percentage of membrane-intact spermatozoa in oligozoospermic samples. CONCLUSION: Laboratory-prepared oligozoospermic samples subjected to glass wool filtration yielded more functionally intact spermatozoa than mini-Percoll gradient processing.

Increased recovery of viable spermatozoa through oscillating centrifugation.

OBJECTIVE: To compare the number and quality of spermatozoa recovered following the standard centrifugation method with those recovered following oscillation of the relative centrifugal field during the centrifugation method. DESIGN: Prospectively controlled in vitro study. SETTING: Private andrology laboratory. PATIENT(S): Seventeen healthy sperm donors. INTERVENTION(S): Equal halves of the same semen sample were evaluated following the standard centrifugation or following oscillation of the relative centrifugal field during centrifugation. MAIN OUTCOME MEASURE(S): Number of sperm, sperm motility, progressive sperm motility at 0 hours and after 3-hour of incubation at 5 degrees C in TES and Tris (buffer) (TEST)-yolk. RESULT(S): The number of sperm, sperm motility, progressive sperm motility at 0 hours and after 3-hour of incubation at 5 degrees C in TEST-yolk were significantly higher for the sperm recovered following oscillating centrifugation compared with standard centrifugation. CONCLUSION(S): Oscillating the relative centrifugal field during centrifugation yields higher number of viable spermatozoa.

The effect of preincubation of human spermatozoa in milk on sperm penetration into zona-free hamster oocytes and on sperm binding to the human zona pellucida.

OBJECTIVE: To determine whether preincubation of sperm in milk enhances the outcome of sperm penetration assay (SPA) and hemizona assay (HZA) when compared with preincubation in TES and Tris (TEST)-yolk. DESIGN: Sperm penetration assay and HZA were performed on milk and TEST-yolk preincubated spermatozoa. INTERVENTIONS: Ejaculates were washed and divided into two equal aliquots. An equal volume of heat-inactivated cow's milk (95 degrees C, 10 minutes) was added to one aliquot and an equal volume of TEST-yolk was added to the other aliquot. Both sperm mixtures were cooled slowly to 5 degrees C and incubated for 2 hours. They were then washed twice (400 x g for 8 minutes) with culture medium at 37 degrees C and processed for SPA and HZA. MAIN OUTCOME MEASURES: Percentage of oocytes penetrated and the penetration index for the SPA. The number of sperm bound to hemizona and hemizona index for the HZA. RESULTS: Sperm preincubated in milk yielded significantly higher SPA results and hemizona index when compared with TEST-yolk. The penetration index and the number of sperm bound were high but were not statistically significant. CONCLUSION: Preincubation of sperm in milk enhances the SPA and HZA outcome when compared with TEST-yolk.

Penetration of zona-free hamster oocytes by ejaculated cryopreserved gorilla spermatozoa.

Semen obtained by electroejaculation from two lowland gorillas were cryopreserved in TEST yolk to evaluate the ability of spermatozoa to penetrate zona-free hamster oocytes. Thawed semen was processed through a two-layer Percoll density gradient to obtain motile spermatozoa for the SPA. The processed sperm penetrated greater than 25% of the zona-free hamster oocytes. Thus, the use of TEST yolk to cryopreserve gorilla semen and processing the thawed semen through Percoll gradient to concentrate motile spermatozoa may facilitate sperm capacitation and the ability to penetrate oocytes.

Concentration of viable spermatozoa for artificial insemination.

Glass wool microfiber code 112 (approximately 30 mg) was gently packed to a depth of 3 mm in a 3-ml disposable syringe barrel. The column was rinsed repeatedly with Tyrode solution to remove loose glass wool fibers. Approximately 0.3 to 0.5 ml of concentrated spermatozoa (after centrifugation of semen at 500 X g for 5 minutes) was layered over the wet column and allowed to filter by gravity. The filtered spermatozoa demonstrated a significantly higher (P less than 0.001) mean percentage of motility, progressive motility, sperm unstained by eosin Y dye, and sperm with functionally intact membranes. Although there was a considerable loss of sperm, the filtered specimen contained all viable spermatozoa present before filtration. Therefore, it appears that this procedure yields a high percentage of viable spermatozoa, potentially capable of fertilization, for use in in vitro fertilization or, possibly, artificial insemination.

The effect of egg yolk medium on human sperm binding in the hemizona assay.

OBJECTIVE: To evaluate, using the hemizona assay (HZA), whether egg yolk treatment of human sperm enhances binding to the human zona pellucida in vitro and to determine whether such a treatment is as efficient as the standard swim-up procedure for promoting sperm binding ability. DESIGN: Ejaculates were divided into aliquots and half incubated at 37 degrees C for 21 hours in standard culture medium or combined with buffered medium containing chicken egg yolk and stored at 4 degrees C for 21 hours. A second set of ejaculates were split and processed by a standard 1-hour rise alone or treated with egg yolk medium. SETTING: University teaching hospital. PARTICIPANTS: Two healthy sperm donors. MAIN OUTCOME MEASURE: The number of sperm tightly bound to the hemizona were measured and compared between the groups. RESULTS: A significant increase (P less than 0.0001) in hemizona binding (n = 46) for egg yolk treatment (90.1 +/- 9.8; range 7 to 258) as compared with standard culture medium (53.0 +/- 8.8; range 0 to 228) was observed. Similarly, a significant increase (P less than 0.0001) in binding (n = 37) for egg yolk treatment (74.9 +/- 8.2; range 7 to 219) as compared with samples obtained with a sperm rise (37.1 +/- 5.7; range 2 to 122). CONCLUSIONS: Treatment of human spermatozoa with an egg yolk medium at 4 degrees C overnight significantly increases sperm binding in the HZA.

Quality assurance for sperm concentration using latex beads.

OBJECTIVE: To provide a simple, universally applicable method of quality assurance for sperm counting, thereby reducing intercounting chamber variation. DESIGN: By using a known concentration of latex beads, the sperm:bead ratio can be used to calculate the actual sperm count. MAIN OUTCOME MEASURES: The mean sperm and bead counts were determined in both a Spot-lite hemocytometer (Baxter Diagnostics, McGaw Park, IL) and a Makler chamber (Polymedco Inc., Yorktown, NY) from 21 different ejaculates mixed with a known concentration of beads. The hemocytometer chamber was used as the standard counting chamber because it consistently yielded a low variation in sperm count. The adjusted sperm concentration of the Makler chamber was calculated using the following formula [hemocytometer beads]/[Makler beads] x [Makler sperm]. RESULTS: Observed mean +/- SD sperm counts were significantly different between the hemocytometer chamber (110.6 +/- 66.2 x 10(6)/mL) and Makler chamber (173.3 +/- 103.5 x 10(6)/mL). However, calculated Makler chamber sperm counts (118.1 +/- 76.1 x 10(6)/mL) was not statistically different from observed hemocytometer sperm counts. CONCLUSION: This novel approach to sperm counting using a known concentration of latex beads as a reference material can be used to reduce variation in sperm counting between observers, counting chambers, and possibly computerized sperm analyzers.

Comparison of the methods of artificial insemination on the incidence of conception in single unmarried women.

OBJECTIVE: To compare pregnancy rates after intrauterine insemination (IUI) versus pericervical insemination in absolute male factor infertility using each patient as her own control. DESIGN: Ovulatory women with patent fallopian tubes without male partners were alternately inseminated with cryopreserved donor semen using either IUI or pericervical insemination techniques. A total of 81 cycles, which included up to 4 cycles per patient were performed. In this manner a comparison between the efficacy of each method could be evaluated. SETTING: The donor insemination program at the Center For Assisted Reproduction at Northwestern University Medical School. PATIENTS: Twenty-six single, healthy, unmarried women with patent fallopian tubes and < 40 years of age without male partners (absolute male factor infertility). MAIN OUTCOME MEASURES: Positive quantitative serum subunit of human chorionic gonadotropin followed by the presence of an intrauterine gestational sac seen by transvaginal ultrasonography. RESULTS: Fourteen (54%) of 26 patients conceived including two (14%) miscarriages within four insemination cycles. Seven (17.5%) patients after IUI, and 7 (17.1%) patients after pericervical insemination conceived. The pregnancy rates were similar regardless of the order of insemination method. CONCLUSION: These findings reveal that there is no statistical difference in the pregnancy outcome between these two methods of insemination in absolute male factor infertility.

The relative distribution of viable sperm in the antegrade and retrograde portions of ejaculates obtained after electrostimulation.

OBJECTIVE: To determine the relative concentration, motility, and viability of spermatozoa in the antegrade and retrograde portions of the ejaculate after electroejaculation in spinal cord injured men. DESIGN: Retrospective. SETTING: University outpatient clinic providing tertiary care in reproductive rehabilitation. PATIENTS: The antegrade and retrograde portions of 22 ejaculates obtained from five spinal cord-injured men were analyzed for sperm density, mean sperm motility, and percentage of total motile and viable sperm yield. RESULTS: The number of spermatozoa were uniformly distributed between the antegrade (54.4%) and retrograde (45.6%) ejaculates. However, of the total sperm yield in both compartments, 66.3% of the motile spermatozoa and 71% of the viable sperm were found in the antegrade compartment (P less than 0.05). Additionally, mean sperm motility was significantly higher in the antegrade ejaculate (P less than 0.05). CONCLUSIONS: Significantly impaired sperm motility and viability are noted in the retrograde ejaculate. Efforts should therefore be directed to maximizing the antegrade portion of the electro-ejaculate and optimizing the technique of preserving functional sperm in the intravesical compartment.

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.

Human sperm selection by glass wool filtration and two-layer, discontinuous Percoll gradient centrifugation.

Glass wool filtration and two-layer, discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation resulted in an average recovery of 50% to 70% of the progressively motile and about 50% of the hypoosmotic swelling (HOS)-positive spermatozoa. Glass wool filtration tended to be more successful than Percoll centrifugation when the ejaculates were asthenozoospermic or produced a suspect/abnormal HOS test. After selection, the acrosin activity increased approximately two- to threefold, but no significant improvement in the percentage of normal sperm forms occurred. Experiments with mixtures of untreated and frozen-thawed ejaculates confirmed that glass wool filtration is more effective in removing nonmotile and HOS-negative spermatozoa than the two-layer Percoll centrifugation technique when the percentage of these types of spermatozoa in the ejaculate is high. The simplicity of these techniques and the good recovery of apparently viable spermatozoa makes these methods more desirable than other, more complicated techniques or procedures that yield a lower recovery of motile spermatozoa.

Effects of peritoneal fluids from patients with endometriosis on capacitated spermatozoa.

OBJECTIVE: To determine the effects of peritoneal fluid (PF) from patients with endometriosis on capacitated sperm. DESIGN: Capacitated donor sperm were allowed to migrate into straws filled with pooled PF from patients with unexplained infertility without endometriosis (n = 4) and those with mild (n = 4) and moderate-severe (n = 4) endometriosis, respectively, for 3 hours. MAIN OUTCOME MEASURES: Sperm motility, sperm membrane integrity, and acrosomal loss were determined. RESULTS: Statistically, no significant differences were found among the various sperm parameters determined. CONCLUSION: Peritoneal fluid from patients with endometriosis does not appear to adversely affect capacitated spermatozoa.

Anaphylaxis during intrauterine insemination secondary to bovine serum albumin.

A case of anaphylaxis is reported after IUI with sperm processed in Tyrode's solution supplemented with BSA. The patient had a positive prick cutaneous test to BSA and also had specific IgE antibody against it. Repeat IUI using the patient's own blood serum instead of BSA for processing the sperm proceeded without incident. Clinicians should be aware of this rare, but not inconsequential, adverse effect of using xenogeneic proteins during IUI and other assisted reproductive techniques.

Treatment of human spermatozoa with an egg yolk medium can enhance the outcome of in vitro fertilization.

In at least 4 of 7 cases, fertilization of intact human oocytes was more successful when spermatozoa were pretreated with TEST yolk medium at 5 degrees C for 2 hours as compared with the standard treatment with Ham's F-10 only. Both pregnancies that were obtained after the transfer of the fertilized oocytes resulted from oocytes fertilized by TEST yolk-treated spermatozoa. No decrease in fertilization occurred in any of the cases after TEST yolk treatment. If these results hold true for a larger series of patients, it may be worthwhile for the standard IVF incubation system of spermatozoa to include TEST yolk.

Release, extraction, and stability of hyaluronidase associated with human spermatozoa. Comparisons with the rabbit.

In contrast to the rabbit: 1) no hyaluronidase could be detected in fresh or frozen human seminal plasma, all color formation on assay being due to a dialyzable, heat stable factor; 2) almost no hyaluronidase could be extracted from frozen-thawed human testicles; 3) the hyaluronidase from human spermatozoa was rapidly inactivated upon release, either spontaneously or on extraction; and 4) a large decrease in hyaluronidase activity occurred when human spermatozoa were stored under various conditions. Rabbit spermatozoa contained six to 13 times more hyaluronidase than human spermatozoa. These results show that distinct species differences exist in the hyaluronidase associated with spermatozoa. None of the human genital tract sources studied could be used to obtain adequate amounts of hyaluronidase for the further isolation and purification of the enzyme. For both human and rabbit spermatozoa, it was optimal to add the cells directly to the assay system for the quantitation of hyaluronidase on spermatozoa rather than extracting the spermatozoa and testing the extracts. The spermatozoa of both species appear to be capable of digesting hyaluronic acid directly. As had previously been found for acrosin, a higher amount of hyaluronidase was maintained when human spermatozoa were cryopreserved in a zwitter buffer (TESTCY) rather than in glycerol.