William C. Patton

A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes.

OBJECTIVE To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. DESIGN The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. SETTING Clinical and academic research environment. PATIENT(S) 59 patients undergoing fertility treatment. INTERVENTION(S) Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. MAIN OUTCOME MEASURE(S) Sperm head DNA density and sperm variables. RESULT(S) A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. CONCLUSION(S) The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

An alternative medicine study of herbal effects on the penetration of zona-free hamster oocytes and the integrity of sperm deoxyribonucleic acid

Abstract Objective: To analyze the effects of certain herbs on sperm DNA and on the fertilization process. Design: Prospective comparative study. Setting: Clinical and academic research environment. Patient(s): Donor sperm specimens. Intervention(s): Zona-free hamster oocytes were incubated for 1 hour in saw palmetto ( Serenoa repens ), echinacea purpura, ginkgo biloba, St. Johns wort ( Hypericum perforatum ), or control medium before sperm-oocyte interaction. The DNA of herb-treated sperm was analyzed with denaturing gradient gel electrophoresis. Main Outcome Measure(s): Oocyte penetration and integrity of the sperm BRCA1 exon 11 gene. Result(s): Pretreatment of oocytes with 0.6 mg/mL of St. Johns wort resulted in zero penetration. A lower concentration (0.06 mg/mL) had no effect. High concentrations of echinacea and ginkgo also resulted in reduced oocyte penetration. Exposure of sperm to echinacea purpura and St. Johns wort resulted in DNA denaturation. In contrast, saw palmetto and ginkgo had no effect. Sperm exposed to 0.6 mg/mL of St. Johns wort showed mutation of the BRCA1 exon 11 gene. Conclusion(s): High concentrations of St. Johns wort, echinacea, and ginkgo had adverse effects on oocytes. Saw palmetto had no effect. The data suggested that St. Johns wort, ginkgo, and echinacea at high concentrations damage reproductive cells. St. Johns wort was mutagenic to sperm cells.

Inhibition of human sperm motility by specific herbs used in alternative medicine

Purpose:Our purpose was to analyze sperm motility parameters in the presence of herbs.Methods:Washed sperm were incubated in either saw-palmetto (Serenoa repens, Permixon Sabal serrulatum), echinacea purpura, ginkgo biloba, St. Johns wort (Hypericum perforatum), or control medium. Parameters were measured on a Hamilton-Thorn analyzer after 1, 4, 24, and 48 hr at 37°C.Results:Sperm motility was inhibited at the high concentration (0.6 mg/mL) of St. Johns wort. Curvilinear velocities and beat cross frequencies also decreased, but not hyperactivation. High-concentration saw-palmetto, echinacea, or ginkgo inhibited motility at 24 and 48 hr.Conclusions:A potent inhibition of sperm motility was seen in St. Johns wort unrelated to changes in pH. Furthermore, sperm viability was compromised in St. Johns wort, suggesting a spermicidal effect. Metabolic changes were observed in saw-palmetto–treated sperm. High-concentration echinacea purpura interfered with sperm enzymes. Ginkgo did not have an antioxidant effect on sperm motility.

Tenacity of Exogenous Human Papillomavirus DNA in Sperm Washing

Purpose:Sperm cells have been shown to take up exogenous DNA readily. The hypothesis was that sperm washing would remove exogenous viral DNA infecting sperm cells. The objective was to compare three types of sperm washing procedures for their capacity to remove exogenous human papillomavirus (HPV) DNA from infected sperm.Methods:Prewashed sperm were equally divided and sperm in one portion were exposed to L1 HPV DNA fragments for 30 min at 37°C. Untreated washed sperm served as the control. After transfection, the sperm were washed by either centrifuge, two-layer Isolate colloid wash, or test-yolk buffer procedures. Sperm parameters were measured on a Hamilton Thorn HTM-C analyzer. Sperm DNA were extracted and polymerase chain reaction (PCR) was carried out targeting the L1 consensus gene of HPV and the designated sentinel gene, 17q21 spanning the D17S855 gene. Amplified products were analyzed in 2% agarose gel electrophoresis.Results:PCR analyses detected the consensus L1 HPV gene in sperm after they were processed through either of the three procedures. Controls were negative for the L1 gene. Extracted DNA were verified by PCR amplification of 17q21 spanning the D17S855 gene. Transfected sperm had higher percentages of total motility and progression compared with the control. Centrifuged, washed, transfected sperm exhibited a greater curvilinear velocity and hyperactivation.Conclusions:The data showed that washing would not remove exogenous HPV DNA from sperm cells. The viral DNA was tenaciously bound to the sperm, suggesting an internalization into the sperm. The viral DNA also increased the motility of the sperm by affecting the velocity and progression of the sperm, which suggested either an increase in metabolism, an enhancement of the calcium-regulated motility mechanism, or an artifact of PCR reagents. More studies are needed to elucidate the mechanism of DNA stimulated sperm motility.

Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones

OBJECTIVES To compare the aniline blue assay with and without eosin, and to correlate the results with pregnancy outcome after intracytoplasmic sperm injection (ICSI) procedure. DESIGN A retrospective study. SETTING University-based fertility center. PATIENT(S) One hundred thirty infertile patients. INTERVENTION(S) Left-over washed sperm after each ICSI procedure were fixed on glass slides and stained with aniline blue with and without eosin. MAIN OUTCOME MEASURE(S) Chromatin condensation, pregnancy, and age. RESULT(S) Percentage chromatin condensation assessed by aniline blue-eosin was higher compared with standard aniline blue (72.4 +/- 2.4% vs. 64.0 +/- 2.4% [mean +/- SEM]). Chromatin condensation was higher in pregnant (86.6 +/- 0.9%) versus nonpregnant (80.9 +/- 2.1%) women age 35 years or more. In younger women, chromatin condensation was not correlated with pregnancy outcome. There was no correlation between chromatin condensation and ICSI fertilization or male age. CONCLUSION(S) Adding eosin counterstain to aniline blue improved assessment of chromatin condensation, suggesting that the standard assay underestimated chromatin condensation. The association between chromatin condensation and pregnancy in older but not younger women suggests that oocytes of younger women had the capacity to compensate for the immature sperm shortcomings.

Antibiotics: effect on cryopreserved-thawed human sperm motility in vitro

OBJECTIVE To analyze the motility and fertilizing capacity of sperm treated with different antibiotics. DESIGN Prospective comparative study. SETTING Clinical and academic research environment. PATIENT(S) Pooled cryopreserved donor sperm (n = 14). INTERVENTION(S) Sperm were washed with Percoll and resuspended in HEPES-buffered human tubal fluid medium containing either amoxicillin, ofloxacin, ciprofloxacin hydrochloride, nitrofurantoin monohydrate, doxycycline hyclate, cefuroxime axetil, or control medium. MAIN OUTCOME MEASURE(S) Sperm kinematic and fertilizing parameters. RESULT(S) Sperm hyperactivation was decreased in physiologic concentrations of ciprofloxacin hydrochloride and doxycycline hyclate over the course of 48 hours. At pharmacologic concentrations, ciprofloxacin hydrochloride, cefuroxime axetil, and nitrofurantoin monohydrate adversely affected motility with decreased rapid progression. Cessation of motility occurred in cefuroxime axetil and nitrofurantoin monohydrate. Sperm hyperactivation was also absent. Cefuroxime axetil decreased the percentage of intact acrosomes. In contrast, physiologic doses of ciprofloxacin hydrochloride or ofloxacin enhanced sperm fertilizing capacity. CONCLUSION(S) Ciprofloxacin affected hyperactivation by altering membrane properties, whereas doxycycline inhibited the capacitation process. Cessation of motility in cefuroxime axetil was linked to disrupted sperm head membranes. Sperm motility and fertilizing capacity were decreased in nitrofurantoin because of decreased metabolism. The positive effect of ofloxacin on fertilizing capacity did not involve changes in acrosome.

Spermac stain analysis of human sperm acrosomes

OBJECTIVE To study the association between low percentages of intact sperm acrosomes and fertilization failures in conventional IVF procedures. DESIGN A retrospective study. SETTING Clinical and academic research environment. PATIENT(S) Patients undergoing treatment of infertility. INTERVENTION(S) Sperm cells were fixed and stained using the Spermac stain. MAIN OUTCOME MEASURE(S) Percentages of intact acrosomes and fertilization. RESULT(S) There was a significant association between specimens with <40% intact acrosomes and failed conventional IVF procedures. Among the 29 cases with <40% intact acrosomes, 9 cases (31%) resulted in zero penetration of the oocytes. The mean (+/-SEM) percentage of fertilization was lower in the abnormal acrosome group (43.3% +/- 6.5%) than in the normal acrosome group (64.1% +/- 5.6%). The status of the sperm acrosome was not correlated with the results of fertilization in intracytoplasmic sperm injection procedures. CONCLUSION(S) Sperm with low percentages of intact acrosomes were associated with failed fertilization. The Spermac stain was useful for assessing acrosomes and identifying possible male factor infertility problems. The results suggested that a minimum percentage of sperm with intact acrosomes are needed for fertilization to occur in vitro.

Enhanced fertility after heat-induced hyperactivation

OBJECTIVE To determine sperm hyperactivation, kinematic parameters, and fertilizing capacity after pretreating sperm at 40 degrees C for 4 hours. DESIGN Prospective study involving pooled donor sperm that were colloid washed and incubated at either 23 degrees C (control) or 40 degrees C (heat-treated) for 4 hours as pretreatment. After incubation, analyses were performed with a computer-assisted sperm analyzer, whereas separate portions of sperm were evaluated with the sperm penetration assay at 37 degrees C. SETTING Clinical and academic research environment. PATIENT(S) Cryopreserved-thawed sperm from different donors (n = 5). MAIN OUTCOME MEASURE(S) Sperm kinematic and fertilizing parameters. RESULT(S) Heat pretreatment of sperm resulted in over 22 times higher hyperactive motility at hour 4 compared with the control. The other kinematic parameters were also different. The heat-pretreated sperm group had a significantly higher percent penetration of zona-free oocytes with more swollen sperm heads per oocyte and enhanced sperm binding. CONCLUSION(S) The results showed that hyperactivation was induced by pretreatment of sperm with 40 degrees C heat, suggesting the involvement of heat factors in hyperactivation. The fertilizing capacity of sperm may be improved by the mild heat pretreatment when marked by the presence of heat-induced hyperactivation.

Silane-coated silica particle colloid processing of human sperm

Purpose: The purpose of this study was to determine differences in the quality of human sperm processed through different lots of silane-coated silica particle colloid solutions. The objectives were to compare (a) sperm kinematic parameters, (b) the sperm acrosome status, (c) the membrane integrity of the head and tail regions, (d) the DNA normality, and (e) the heat-inducible hyperactivation motility after processing sperm through either a Silane-coated silica particle colloid solution, a Percoll solution, or a simple centrifuge sperm wash (control).Methods: Sperm cells were derived from pooled cryopreserved-thawed specimens of several donors (n=10). The pooled sperm were divided and processed through either the centrifuge wash, the 90:47% two-layer Percoll, or one of three lots of silane-coated silica particle colloidal solutions from three vendors. Aliquots of sperm cells were analyzed using the Hamilton-Thorn HTM-C motility analyzer for differences in kinematics and hyperactivation. Sperm were also analyzed for membrane integrity at both head and tail regions, normal morphology, acrosome status, and viability. Sperm undergoing apoptosis were determined using the acridine orange stain. Processed sperm were also incubated at 40°C for 4 hr and the quality of the sperm was assessed using the heat-induced hyperactivation and motility parameter.Results: The data showed that after sperm processing, the number of sperm recovered was higher for the three lots of colloids (silane-coated silica particle colloid solutions) compared with Percoll processing. Total sperm motility was higher in the colloidal washes compared with the control. There were no differences in motility between Percoll- and colloid-processed sperm. In contrast, the percentages of sperm exhibiting progressive motility or hyperactivation varied among the different lots of colloid solutions. The Percoll wash solution yielded the highest percentage of sperm with intact tail membranes, whereas some lots of colloid solutions disrupted sperm head membranes. The percentages of sperm undergoing apoptosis varied for the different lots of colloid solutions. There was a marked increase in hyperactivation associated with one colloid solution after heat induction.Conclusions: The results demonstrated variability in the different lots of silane-coated silica particle colloid solutions for processing sperm. Each lot of colloid solution excelled at improving different sperm parameters. The silane-coated silica particle colloid solutions were shown to be effective in recovering motile sperm compared with Percoll but the types of motility and sperm quality varied for the different lots of colloid solutions. Due to the variability in lots of silane-coated silica colloid solutions, reported studies based on only one lot or one source of colloid solution may be difficult to interpret. Furthermore, it may be advantageous to select the best lot of silane-coated silica particle colloid solution to produce the highest number of sperm exhibiting the ideal parameters for use in assisted reproduction technologies.

Correlation Between Intact Sperm Acrosome Assessed Using the Spermac Stain and Sperm Fertilizing Capacity

Accurate determination of sperm acrosomal status is important in fertility studies. The objective was to correlate the percentage of intact acrosome assessed using the new Spermac stain with the capacity of sperm to fertilize oocytes. Sperm specimens were processed either by centrifuge wash, 48:95 Percoll gradient or test yolk buffer (TYB) wash, and tested using the zona-free hamster oocyte assay. The results indicated a correlation between the percentage of sperm with intact acrosome reaction and the percentage of sperm penetrating the oocytes in the TYB-washed group. The data suggest the usefulness of the Spermac stain for assessing the acrosomal status and in predicting the fertilizing capacity of the sperm.