Philip J. Chan

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.

Effects of pentoxifylline on sperm motility and hyperactivation in normozoospermic and normokinetic semen

OBJECTIVE To determine the effects of in vitro incubation with pentoxifylline on sperm motion characteristics of spermatozoa from normozoospermic, normokinetic specimens. DESIGN Prospective, controlled experiment. SETTING Andrology laboratory, university-based fertility center. PARTICIPANTS Healthy, untreated male partners of couples attending the fertility center. INTERVENTION Each specimen was washed, pelleted by centrifugation, then resuspended in human tubal fluid medium (HTF). Two portions were incubated at 37 degrees C, one with pentoxifylline (final concentration = 1 mg/mL = 3.6 mM) and the other without pentoxifylline (control). After 1 hour, the pentoxifylline-treated portion was divided: one half was washed to remove pentoxifylline, then further incubated in HTF; the other half remained incubated in HTF with pentoxifylline. MAIN OUTCOME MEASURES Motility, hyperactivation, amplitude of lateral head displacement (ALH), curvilinear velocity (VCL), straight line velocity (VSL), linearity (LIN), beat-cross frequency. RESULTS Incubation with pentoxifylline did not increase motility, VSL, LIN, or beat-cross frequency but did significantly increase HA, VCL, and ALH at 1, 2, and 4 hours, compared with control. Incubation in control medium without pentoxifylline did not significantly increase any of the parameters measured. After 24 hours of incubation with or without pentoxifylline, all parameters measured were significantly decreased, with the exception of LIN. CONCLUSIONS Pentoxifylline does not increase percentage motility of washed spermatozoa in capacitation medium. Enhancement of sperm HA by pentoxifylline in capacitation medium occurs with normozoospermic, normokinetic semen specimens. This effect persists for up to 4 hours when pentoxifylline is removed from the medium after 1 hour of incubation.

Human papillomavirus gene sequences in washed human sperm deoxyribonucleic acid

The present study demonstrated the presence of HPV gene sequences in Percoll-washed sperm cells using polymerase chain reaction primers targeting smaller gene regions. Up to 64% of the sperm specimens were shown to contain gene sequences indicative of the presence of HPV. Human papillomavirus type 16 was detected about twice as often as HPV type 18. The results suggest the possible role of sperm as a vector for HPV.

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.

Human Papillomavirus and Blastocyst Apoptosis

AbstractPurpose: The effect of human papillomavirus (HPV) DNA from the E6-E7 region on the integrity of DNA in blastocyst stage embryonic cells was studied. The study design paralleled the event whereby HPV DNA from the infecting virus would target host cell DNA. The objectives were (a) to determine if the DNA of blastocysts were disrupted by the presence of HPV DNA and (b) to determine if the intensity of DNA damage was associated with the type of HPV. Methods: This study involved superovulating female mice, mating, collecting one-cell embryos, and culturing to the expanded blastocyst stage. The blastocysts were infected with PCR-synthesized DNA fragments from either HPV type 16, 18, 31, or 33. The blastocyst DNA were analyzed by comet assay after 24 h of incubation. The fluorescent images were digitized and the pixel intensity of each blastocyst was measured. Results: Only the DNA of HPV type 16 was associated with significant DNA fragmentation in comparison with the other HPV types. There was no relationship between HPV DNA fragment size and the intensity of DNA fragmentation. Conclusions: The data suggested that one mode of action of HPV type 16 was to initiate apoptosis of embryonic cells through DNA fragmentation. The effect of HPV 16 occurred rapidly within 24 h. The intensity of DNA damage was not linked to the specific type of HPV. However, the results do not rule out the other HPV types affecting embryos under conditions different from this study.

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.

Evidence for ease of transmission of human papillomavirus DNA from sperm to cells of the uterus and embryo

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