Biomedical subjects

P J Chan

Publications and source records attributed to P J Chan.

At least 19 recordsLinked to original sources

PubMed2001

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.

Acridine Orange↗

PubMed2001

Updating quality control assays in the assisted reproductive technologies laboratory with a cryopreserved hamster oocyte DNA cytogenotoxic assay.

PURPOSE: Despite advances in assisted reproduction, there is no progress in quality control bioassays. The objectives were to develop a comet assay to measure DNA fragmentation in thawed cryopreserved oocytes and compare this assay with one-cell mouse embryo bioassay. METHODS: Thawed hamster oocytes from a commercial source were incubated in culture media with either 0-, 50-, or 100-microM hydrogen peroxide, or, in media exposed to different contact materials and unknown proficiency analytes. Incubation time was 1.5 h at 37 degrees C. The oocytes were dried, fixed, stained with acridine orange, embedded in a mini-agarose layer and electrophoresis was carried out. Fluorescent images were analyzed. The results were compared with standard one-cell mouse assay data. RESULTS: The 100-microM hydrogen peroxide treatment caused greatest DNA fragmentation in the hamster oocytes at Hours 1 and 2. A dose response was observed. Intraassay coefficient of variation was 5.7%. Only one of the five materials tested passed both assays. The data for the unknown proficiency analytes were similar for both assays. CONCLUSIONS: The oocyte comet assay demonstrated DNA fragmentation in the presence of toxic substances. The detection of toxicity in two materials that passed the mouse bioassay suggested increased sensitivity in the new assay. The oocyte comet assay and the mouse bioassay results matched in the proficiency test. However, more studies are still needed to determine optimal sensitivity.

Animals↗

PubMed2001

Cryopreservation of human cumulus cells for co-cultures and assessment of DNA damage after thawing using the comet assay.

PURPOSE: Cumulus cells have been shown to be beneficial for blastocysts formation in co-cultures but information on cumulus cryopreservation is lacking. The objective was to use the fixed cell comet assay to analyze for DNA damage in cumulus cells after cryopreservation. METHODS: Discarded cumulus cells from follicular aspirates obtained during assisted reproduction procedures (N = 4 cases) were pooled and cryopreserved in either 40% ethylene glycol and 0.5 M sucrose, 12:20% glycerol-egg yolk medium, 28% glycerol hypoosmolar medium or control medium. The cells were processed and stored in liquid nitrogen for 48 h. The thawed cells were smeared on glass slides, fixed, stained with acridine orange, embedded in a mini-agarose layer, and electrophoresis carried out. Fluorescent images were analyzed. RESULTS: The cumulus tail moment, a calculated index of DNA damage, was significantly lower for each of the three cryoprotectant when compared with the control. The two cryoprotectants containing glycerol were associated with higher cumulus cell viability. However, the glycerol-egg yolk combination yielded the highest cell viability. CONCLUSIONS: The cumulus comet assay demonstrated similar DNA integrity in cells frozen in each of the three cryoprotectants. The glycerol-egg yolk medium had the highest cell viability with little or no DNA damage after freeze-thaw. More studies are needed to examine the long-term effect of the cryoprotectants on thawed cumulus cell viability.

Acridine Orange↗

PubMed2001

Oocyte comet assay of luteal phase sera from nonpregnant patients after assisted reproductive procedures.

PURPOSE: Serum factors in patients with recurrent spontaneous abortions (RSA) inhibit mouse embryo development in vitro. Serum factors affecting DNA integrity remain to be tested. The null hypothesis was that patient sera do not affect DNA integrity. The objectives were (a) to use the oocyte comet assay to assess DNA damage after exposure to patient sera and (b) to determine the effect of sera from gravidity 0 parity 0 patients to induce DNA apoptosis. METHODS: Luteal phase sera were drawn 1 week after embryo transfer following assisted reproductive procedures. Frozen-thawed hamster zona intact oocytes at metaphase II were incubated in groups of eight in either control medium or medium supplemented with 50% patient serum for 1.5 h at 37 degrees C in room air. The oocytes were fixed, stained in acridine orange, embedded in agarose, lysed, and alkaline electrophoresis performed. The intensities of the digitized fluorescent images were analyzed. RESULTS: The sera of nonpregnant patients (64%) caused significant fragmentation of hamster oocyte DNA when compared with pregnant patient sera. This difference was also observed when adjusted for patient age. Sera of patients that had never been pregnant also resulted in oocyte DNA fragmentation. CONCLUSIONS: The results suggested that sera from patients that did not conceive contained factors that did not support cell growth by causing DNA fragmentation and apoptosis. The level of the apoptotic factors varied from cycle to cycle. However, more studies are needed to determine if the sera factors actually reach the uterine environment to cause the undesirable effects.

Abortion, Habitual↗

PubMed2001

A comparison of heat-induced hyperactivation in patients' sperm after colloid or pentoxifylline wash methods.

OBJECTIVE: Our purpose was to compare kinematic parameters of human sperm after processing through two different wash methods and 40 degrees C heat treatment. STUDY DESIGN: Sperm specimens (N = 169 cases) were washed by either colloid or pentoxifylline wash methods, and the motility parameters were measured at either 37 degrees C or 40 degrees C at baseline (0 hours) and after 4 hours. Five randomly selected washed specimens with matching 37 degrees C (control) or 40 degrees C heat treatments were assessed for changes in a sentinel gene. RESULTS: The percentage of sperm hyperactive motility was >5 times higher after the 40 degrees C heat treatment, in comparison with the 37 degrees C treatment, for both the colloid- and the pentoxifylline-washed sperm. The percentages of total motility and progression were equally enhanced in heated sperm for the two wash methods. No changes were detected in the sentinel gene with the heat treatment. CONCLUSION: Sperm cells mildly heated at 40 degrees C responded with greater motility, progression, and hyperactivation. The data suggest that mild heat is a stimulus for sperm function because greater sperm hyperactivation is associated with increased sperm fertilizing capacity. The absence of change in the sentinel gene in heated sperm suggests that a temperature of 40 degrees C is too low to initiate alterations in the highly condensed sperm chromatin. More studies are needed before mild heating of ejaculated sperm becomes acceptable for use in assisted reproductive technologies.

Colloids↗

PubMed2001

Human sperm deoxyribonucleic acid fragmentation by specific types of papillomavirus.

OBJECTIVE: Human papillomaviruses are present in up to 64.3% of patients' sperm. The objectives were (1) to determine human papillomavirus deoxyribonucleic acid effects on sperm deoxyribonucleic acid integrity and (2) to assess human papillomavirus differential effects on the sperm cell. STUDY DESIGN: Two-layer colloid washed sperm were exposed to E6-E7 deoxyribonucleic acid fragments generated from human papillomavirus types 16, 18, 31, 33, 6/11, or control (DQA1) for 24 hours. The motility parameters were measured and analyzed. Pilot studies were performed to develop a fixed sperm comet assay to assess deoxyribonucleic acid fragmentation. RESULTS: Significant sperm deoxyribonucleic acid fragmentation occurred after exposure to deoxyribonucleic acid of human papillomavirus types 16 and 31. Human papillomavirus deoxyribonucleic acid fragment size was not a factor. Human papillomavirus types 18, 33, and 6/11 did not compromise sperm deoxyribonucleic acid integrity. Washed sperm motility was higher in the presence of human papillomavirus deoxyribonucleic acid except for type 6/11. Amplitude of head displacement was lower for human papillomavirus types 16 and 6/11. Sperm linearity was increased for all human papillomavirus types except type 18. CONCLUSION: Human papillomavirus type 16 and 31 deoxyribonucleic acid caused deoxyribonucleic acid breakages characteristic of apoptotic but not necrotic sperm. The data suggest that these human papillomavirus types may adversely affect subsequent embryonic development after fertilization. Sperm deoxyribonucleic acid appears to resist human papillomavirus types 18, 33, and 6/11 or repairing mechanisms occurred. Although enhanced motility was found in human papillomavirus-exposed sperm, important velocity parameters were decreased, suggesting impaired sperm function.

DNA Fragmentation↗

PubMed2001

Comet assay of cumulus cell DNA status and the relationship to oocyte fertilization via intracytoplasmic sperm injection.

This paper postulates that in the ovary, the close association between the cumulus cells and the oocytes permits the fertilizing ability of the oocytes to be determined indirectly through cumulus cell DNA status. The objective was to use a modified comet assay to analyse cumulus cell DNA and relate the data to oocyte fertilization after intracytoplasmic sperm injection (ICSI) procedures. Oocytes were retrieved (n = 15 cases) and free-floating cumulus cells were pooled and smeared on clear glass slides to dry. Meanwhile, the denuded oocytes were injected with spermatozoa and fertilization was assessed, followed by embryo transfer. The fixed cumulus cells were stained in acridine orange, coated with a mini-gel agarose layer, lysed in alkaline buffer and electrophoresis performed. Analyses of fluorescent cell images (n = 449) showed that the tail moment was positively correlated to the percentage of fertilization after ICSI (r = 0.567, P < 0.05). In contrast, there was no correlation between tail moment and number of oocytes retrieved, total ampoules used, endometrial thickness and age of patient. The results suggested that the competence of the oocytes was associated with the cumulus cell DNA status. A unique feature here was the comet assay for archived material with obvious advantages.

Adult↗

PubMed2000

Sperm-mediated DNA transfer to cells of the uterus and embryo.

There is a paucity of information about sperm-mediated transmission of exogenous DNA to implanting embryos and cells of the reproductive tract. Preliminary experiments established that sperm has the capacity to actively take in exogenous DNA derived from HPV. In addition, blastocysts also take up exogenous HPV DNA, but in contrast to sperm, the process appears passive. DNA-carrying sperm migrating in an artificial glass tube or excised mouse bicornuate uteri transfected the blastocysts at the remote position using a flip-flop mechanism. There were preferential transmission of the types of HPV DNA but this was not attributed to the gene sequence or the size of the DNA fragments. The internalized DNA became undetectable unless continuous sperm bombardment or pricking took place. Mycoplasma vectors offer a novel way to enhance the transfection of blastocyst with exogenous DNA.

Animals↗

PubMed2000

Electroejaculated baboon (Papio anubis) sperm requires a higher dosage of pentoxifylline to enhance motility.

PURPOSE: Sperm collected by electroejaculation often show poor motility. The objective was to determine whether the addition of the phosphodiesterase inhibitor, pentoxifylline, would stimulate electroejaculated baboon sperm motility. METHODS: Electroejaculation was performed on several occasions on a male baboon and sperm collected after familiarization. Pentoxifylline was tested at the standard concentration (1 mg/ml) and at twice the concentration. Sperm parameters were evaluated using a sperm motility analyzer, as well as acrosome and DNA integrity techniques. RESULTS: Sperm exposed to 2 mg/ml pentoxifylline had higher total motility when compared with the control and 1 mg/ml treatment. Rapid progression and velocities were higher after pentoxifylline. The acridine orange DNA normality test showed that over 90% of collected sperm had intact unfragmented DNA. About half the sperm population had normal morphology and intact acrosomes. A low percentage had cytoplasmic droplets. CONCLUSIONS: Sperm collected by rectal probe electroejaculation required a higher concentration (2 mg/ml) of pentoxifylline for enhanced total motility, rapid progression, and higher velocity. This suggested differences in membrane properties or phosphodisterase activity in electrojeaculated sperm. The electroejaculation procedure did not denature sperm DNA at the acridine orange assay level nor were the acrosomes disrupted. The present study also documented unique information on baboon kinematic parameters.

Acridine Orange↗

PubMed2000

Analysis of papillomavirus consensus L1 gene in a closed colony of baboons (Papio anubis).

OBJECTIVE: The purpose of this study was to analyze cervical specimens and semen from a closed colony of baboons for the presence of the papillomavirus consensus L1 gene. STUDY DESIGN: Cervical swabs were collected from lightly anesthetized female baboons. Semen was collected from a male baboon by standard electroejaculation techniques. Deoxyribonucleic acid was extracted from the cells by two different methods and analyzed by polymerase chain reaction targeting the L1 consensus gene common for >25 genital papillomaviruses. RESULTS: Analyses of the polymerase chain reaction-amplified products did not reveal bands for the papillomavirus in either the cervical specimens or the semen. CONCLUSIONS: The hypothesis of a linkage between primates with papillomavirus as a common factor is not supported by the results of this study. This information is also important in assisting clinicians in setting up specific pathogen-free colonies of baboons.

Animals↗

PubMed2000

Type 5 phosphodiesterase regulation of human sperm motility.

OBJECTIVE: Inhibition of phosphodiesterases results in the buildup of intracellular cyclic nucleotides, which have been shown to affect sperm motility and acrosome reaction. The objective of this study was to determine whether the cyclic guanosine monophosphate-specific type 5 phosphodiesterase inhibitor sildenafil has an effect on sperm motility and acrosome parameters. STUDY DESIGN: Sperm cells were washed by two-layer colloid wash and resuspended in modified human tubal fluid with 5% serum albumin. They were incubated in the presence of different concentrations (0-40 nmol/L) of the type 5 phosphodiesterase inhibitor sildenafil. Aliquots of sperm were removed at hours 0, 4, 24, and 48, and motility parameters were measured on the Hamilton-Thorn HTM-C (Hamilton-Thorn Research, Danvers, Mass) motility analyzer. Sperm acrosomes were analyzed with the Spermac (Stain Enterprises, South Africa; distributed by Sage Biopharma, Bedminster, NJ) acrosome stain. RESULTS: Sperm progressive motility and hyperactivation were stimulated to greater than the control at hour 4, followed by a decrease. There was a dose-dependent effect of the type 5 phosphodiesterase inhibitor on sperm motility parameters but not on percentage of cells with acrosome reaction. The type 5 phosphodiesterase inhibitor stimulated sperm acrosome reaction by almost 50% above the control. CONCLUSION: These results suggest that inhibition of type 5 phosphodiesterase activity in human sperm resulted in enhanced progressive motility and hyperactivation. In addition, inhibition of type 5 phosphodiesterase also caused an increase in acrosome reaction. This suggests a role for type 5 phosphodiesterase in preventing premature acrosome reaction, which is associated with failed fertilization.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1999

Absence of human papillomavirus E6-E7 transforming genes from HPV 16 and 18 in malignant ovarian carcinoma.

Ovarian carcinoma is one of the frequent causes of death from malignancies in the United States. A report excited the scientific community when human papillomavirus were identified in advanced epithelial ovarian carcinoma tissues in 10 of 12 patients. A few studies also identified HPV DNA in ovarian carcinoma tissues. However, several researchers employing polymerase chain reaction techniques and using different oligonucleotide probes did not detect HPV DNA in ovarian carcinoma tissues. The objective was to determine the presence of the E6-E7 genes of HPV types 16 and 18 in archived paraffin-embedded malignant ovarian carcinoma using primers targeting. Archived human malignant ovarian cancer tissues (N = 20 cases) embedded in paraffin blocks were processed, and DNA was extracted and the presence of DNA verified by p53 amplifications. PCR analyses were performed on the extracted DNA together with appropriate controls. The results showed an absence of E6-E7 genes of HPV types 16 and 18 in ovarian carcinoma. However, the presence of other HPV types or gene regions is not ruled out and more studies are needed to resolve the question of HPV involvement in ovarian carcinogenesis.

Archives↗

PubMed1999

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

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. John's 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 BRCAI exon 11 gene. RESULT(S): Pretreatment of oocytes with 0.6 mg/mL of St. John's 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. John's wort resulted in DNA denaturation. In contrast, saw palmetto and ginkgo had no effect. Sperm exposed to 0.6 mg/mL of St. John's wort showed mutation of the BRCA1 exon 11 gene. CONCLUSION(S): High concentrations of St. John's wort, echinacea, and ginkgo had adverse effects on oocytes. Saw palmetto had no effect. The data suggested that St. John's wort, ginkgo, and echinacea at high concentrations damage reproductive cells. St. John's wort was mutagenic to sperm cells.

Animals↗

PubMed1999

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.

Acrosome↗

PubMed1999

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 degrees 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.

Chromosome Mapping↗

PubMed1999

Sperm artificially exposed to antisperm antibodies show altered deoxyribonucleic acid.

PURPOSE: Our purpose was to assess sperm deoxyribonucleic acid (DNA) integrity after exposure to antisperm antibodies. METHODS: Donor semen were divided and exposed to sera containing IgG, IgA, and IgM antisperm antibodies. Untreated portions served as the control. After incubation (1 hr, 23 degrees C), the sperm were centrifuge-washed, resuspended, and incubated (23 degrees C) for 2, 5, 7, or 9 days. Acridine orange staining and kinematic parameters were measured. The sentinel (17q21 from D17S855) and beta-globin genes were amplified and analyzed using denaturing gradient gel electrophoresis. RESULTS: Sperm preexposed to antisperm antibodies had deleted sentinel gene on days 7 and 9. The beta-globin gene was intact. There were no differences in acridine orange staining. CONCLUSIONS: Sperm artificially exposed to antisperm antibodies resulted in a subtle deletion of genetic material. The DNA alteration process was slow and was undetectable at the gross level. More studies are needed to confirm the findings and determine whether DNA repair mechanisms can reverse the damage.

Acridine Orange↗

PubMed1999

Mycoplasma-mediated uptake of the exogenous human BRCA1 gene by hatching blastocysts.

PURPOSE: Biological vectors for cell transfection are mainly viral in origin, with inherent shortcomings. Mycoplasmas are ubiquitous organisms that traverse cells easily. The objective was to determine if Ureaplasma urealyticum (T-mycoplasma) would vector exogenous BRCA1 DNA into blastocysts. METHODS: Hatching mouse blastocysts (N = 70) were incubated in the presence of either viable or dead Ureaplasma urealyticum at 37 degrees C for 1 hr. The blastocysts were exposed to human BRCA1 DNA lacking homology in the mouse genome for 2 hr, followed by DNase-1 treatment and wash. Polymerase chain reaction and agarose gel electrophoresis analysis of amplified products were performed. RESULTS: The BRCA1 gene was detected in the blastocysts only when viable Ureaplasma was present. PCR analyses of control Ureaplasma and untreated blastocysts were negative. CONCLUSION: Viable Ureaplasma organisms were shown to mediate the uptake of DNA fragments into blastocysts, resulting in transgenic mouse blastocysts with a normal human BRCA1 exon 11 gene.

Animals↗

PubMed1999

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. John's wort (Hypericum perforatum), or control medium. Parameters were measured on a Hamilton-Thorn analyzer after 1, 4, 24, and 48 hr at 37 degrees C. RESULTS: Sperm motility was inhibited at the high concentration (0.6 mg/mL) of St. John's wort. Curvilinear velocities and beat cross frequencies also decreased, but not hyperactivation. High-concentration saw-palmetto, echinacea, or gikgo inhibited motility at 24 and 48 hr. CONCLUSIONS: A potent inhibition of sperm motility was seen in St. John's wort unrelated to changes in pH. Furthermore, sperm viability was compromised in St. John's 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.

Cell Survival↗