Alan King

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.

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.

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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Microinvasive squamous carcinoma of the vulva: present status and reassessment.

A retrospective analysis of 24 patients with early invasive squamous carcinoma was performed. No nodal metastases were noted in these patients. Based on a review of the literature, no absolute definition of microinvasive carcinoma could be formulated, but a treatment outline has been formulated based on depth of invasion for Stage I lesions.

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.