Loma Linda University

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Philip Chan, PhD
Professor, Gynecology & Obstetrics
School of Medicine
Professor, Basic Sciences
School of Medicine
Publications    Scholarly Journals--Published
  • Schultz KJ, Siddighi S, Hardesty JS, Waggonner DB, Yune JJ, Chan PJ.  UVA-Photoactivated Riboflavin Treatment of Vaginal Cells Derived from Pelvic Organ Prolapse Cases. Gynecol Obstet Invest. 2014;77(2):100-3. ( 2/2014 ) Link...
    Background: The pathophysiology of pelvic organ prolapse (POP) involves vaginal collagen degradation. Strengthening collagen by UVA-photoactivated cross-linking has been demonstrated and suggested target applications include the vaginal wall. Aim: To identify UVA irradiation and riboflavin effects on vaginal cells.

    Materials and Methods: Vaginal cells were incubated for 24 h (DMEM/F-12 Ham's media) and were exposed to riboflavin (0, 0.1 and 10%) for 30 min before UVA photoactivation. Percentages of live, apoptotic and necrotic cells were determined by propidium iodide/Hoechst 33342 stains.

    Results: UVA decreased vaginal cell viability [mean ± standard error of the mean: 26.2 ± 0.5% vs. control (43.9 ± 3.8%)], but riboflavin blocked UVA-induced damage (57.9 ± 2.7 and 56.7 ± 2.1% at 0.1 and 10% riboflavin, respectively). Cells treated with low- and high-dose riboflavin had lower apoptosis (32.9 ± 1.0 and 35.5 ± 0.9%, respectively). Furthermore, riboflavin-treated cells had reduced necrosis (9.3 ± 1.7, 7.8 ± 3.0%) versus UVA-only (32.4 ± 5.5%) or control (17.1 ± 2.8%). Viability was similar for cells from the cervical and hymenal segments.

    Conclusion: The results demonstrated that riboflavin attenuated UVA damage in vaginal cells by inhibiting necrosis. Cervical and hymenal end vaginal cells were equally affected by UVA. UVA phototoxicity was reduced by the presence of riboflavin. © 2014 S. Karger AG, Basel.
  • Hong LJ, Oshiro BT, Chan PJ. HPV-16 exposed mouse embryos: a potential model for pregnancy wastage. Arch Gynecol Obstet. Jun;287(6):1093-1097, 2013. PMID: 23307167. ( 6/2013 ) Link...
    PURPOSE: Placentas from spontaneous abortions and preterm deliveries have a higher prevalence of Human papillomavirus (HPV) compared to placentas from elective abortions and term births. The objective was to determine the effects of HPV-16 on the adhesion and implantation properties of early embryo trophoblasts.

    METHODS: Two-cell mouse embryos were cultured (medium G2, 5 % CO2, 37 °C) for 72-96 h and exposed to either HPV-16 rich SiHa cell lysates which were refrigerated after mechanical lysis, thawed lysates which had been frozen for freeze/thaw lysis method, or control medium, incubated (4-5 days) and evaluated by microscopy (N = 96 embryos, 3 repeated experiments). Trophoblasts were stained and images were digitized. Adhesion and dimension data were analyzed by Chi-square and t test, respectively.

    RESULTS: HPV-16 exposed embryos exhibited less adhesion through reduced implantation compared with the control (combined lysates 53.8 vs. 85.7 %, P < 0.05). Refrigerated and thawed lysate groups had similar reduced implantations (58.3 vs. 50.0 %). Of the embryos with implantation, 100 % in the refrigerated lysates were noted to have loose or abnormal adhesion. This was measured when embryos were noted to be lost after washes with HTF. There was no difference in trophoblast viability among the groups. Total trophoblast area was greater in the HPV-16 exposed frozen lysate group (1,881.8 ± 605.3 vs. control 848.8 ± 298.0 square units, mean ± SEM).

    CONCLUSIONS: HPV-16 inhibited trophoblasts adhesion needed for normal implantation, but not embryo development. Total trophoblast spread was increased after HPV-16 exposure suggesting that HPV-16 altered trophoblast migration. These results suggest that HPV-16 may induce abnormal placental growth resulting in pregnancy wastage.
  • Wong A, Chuan SS, Patton WC, Jacobson JD, Corselli J, Chan PJ.  "Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones." Fertil Steril . 2008 Nov;90(5):1999-2002. ( 11/2008 ) Link...
    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.
  • Siddighi S, Chan CA, Patton WC, Jacobson JD, Chan PJ.  "Male age and sperm necrosis in assisted reproductive technologies." Urol Int. 2007;79(3):231-234 ( 10/2007 )
    INTRODUCTION: Sperm apoptosis is well characterized but studies on the effect of male age and necrozoospermia are lacking. The objectives were: (a) to analyze percentages of apoptotic and necrotic sperm in ejaculates, and (b) to compare the results between younger and older age groups. MATERIALS AND METHODS: Routine semen analyses were carried out (n = 189 males) and sperm cells were analyzed by dual fluorescence assay Hoechst 33342 and propidium iodide, and the acridine orange test. RESULTS: The percentage of necrotic sperm in the ejaculate increased by 22% for males aged over 35. There was a positive correlation between age and necrosis (R = 0.30). Sperm apoptosis increased by 17% in males aged 45 and older. The population of DNA intact sperm declined in males aged 40 and over (R = -0.21). There were no age-related changes in strict normal morphology, sperm concentration and semen volume. A decrease in rapid progressive motility was correlated (R = -0.24) with male age and was significant after age 35. CONCLUSIONS: The study demonstrated increased necrosis, DNA damage and apoptosis while rapid progression and total motility declined with advancing age in the male beginning as early as age 35. The order of the observed changes was sequential, suggesting the involvement of different pathways in sperm necrosis after age 40.
  • Kam TL, Jacobson JD, Patton WC, Corselli JU, Chan PJ.. "Retention of membrane charge attributes by cryopreserved-thawed sperm and zeta selection." J Assist Reprod Genet. 2007;24(9):429-434. ( 9/2007 )
    PURPOSE: Mature sperm can be selected based on their negative zeta electrokinetic potential. The zeta selection of cryopreserved sperm is unknown. The objective was to study the effect of zeta processing on the morphology and kinematic parameters of cryopreserved-thawed sperm. METHODS: Colloid-washed sperm (N = 9 cases) were cryopreserved for 24 h, thawed and diluted in serum-free medium in positive-charged tubes. After centrifugation, the tubes were decanted, serum-supplemented medium was added and the resuspended sperm were analyzed. Untreated sperm and fresh sperm served as the controls. RESULTS: There were improvements in strict normal morphology in fresh (11.8 +/- 0.3 versus control 8.8 +/- 0.3 %, mean +/- SEM) and thawed (8.7 +/- 0.2 versus control 5.4 +/- 0.2%) sperm after zeta processing. Percent sperm necrosis was reduced after zeta processing (66.0 +/- 0.6 versus unprocessed 74.6 +/- 0.3%). Progression decreased by 50% but not total motility after zeta processing of thawed sperm. CONCLUSIONS: The results suggested that the cryopreservation process did not impact the sperm membrane net zeta potential and higher percentages of sperm with normal strict morphology, acrosome integrity and reduced necrosis were recovered. The zeta method was simple and improved the selection of quality sperm after cryopreservation but more studies would be needed before routine clinical application.
  • Fynewever TL, Agcaoili E, Jacobson JD, Patton WC, Chan PJ.. "In vitro tagging of embryos with nanoparticles.." Journal Assist. Reprod. and Genetics 24.2-3 (2007): 61-65. ( 2/2007 - 3/2007 )
    PURPOSE: To develop an in vitro method for tagging embryos and to compare the development of the embryos after nanoparticles injection versus externally-applied nanoparticles derived from either polystyrene or polyacrylonitrile. METHODS: Each mouse 1-cell embryo (the selected test-model) was either: (a) injected by intracytoplasmic injection or (b) co-incubated with different nanoparticles at 37 degrees C, 5% CO2 in air. The embryos were assessed after 2 and 6 days of culture. RESULTS: Embryo development was similar for externally-applied polystyrene nanoparticles and control (97.6 +/- 2.7 versus 100.0 +/- 0%) but different for polyacrylonitrile nanoparticles (90.0 +/- 2.8 %) on day 2. However, the results were similar on Day 6. Injected embryos were linked to lower percent development on Day 2. Few injected embryos reached blastocyst stage on Day 6 after a brief UV-fluorescence exposure. CONCLUSIONS: Tagging embryos by external polystyrene-based nanoparticles was the better method when compared with injected nanoparticles. Larger nanoparticles in microsphere range were easier to qualitate. Inhibited hatching limited their use beyond the blastocyst stage.
  • Siddighi, S., Chan, C.A., Patton, W.C., Jacobson, J.D., King, A., Chan, P.J.. "Sperm apoptosis and age of males attending a fertility center." Urol. Int. . (2007): -. ( 1/2007 )
    Siddighi, S., Chan, C.A., Patton, W.C., Jacobson, J.D., King, A., Chan, P.J. Sperm apoptosis and age of males attending a fertility center Urol. Int. 2007; (In Press).
  • Maligaya, M.L.R., Chan, C.A., Jacobson, J.D., Patton, W.C., Corselli, J., Chan, P.J.. "Follow-up expanded study of the correlation of sperm velocity in seminal plasma and offspring gender.." Archives of Andrology 52.1 (2006): 39-44. ( 12/2006 )
    A preliminary study reported finding higher sperm velocity in seminal plasma in males of partners that conceived female offsprings. The null hypothesis was that sperm velocity was not related to the offspring gender. The objectives were: (a) to expand the previous study, and (b) to correlate offspring gender results with motility parameters determined through the computer-aided sperm analyzer (CASA) system. In combined fresh and frozen cycles (N = 187), sperm from cases with all female offsprings displayed higher curvilinear (48 +/- 1.0 mu/sec versus male 46 +/- 1.0, P < 0.05) and average path velocities (36 +/- 0.7 mu/sec versus male 34 +/- 0.7, P < 0.01). A criteria of less than 30 mu/sec or over 41 mu/sec average path velocity predicted 73 or 72% of the male or female offspring cases, respectively. A curvilinear velocity of less than 49 mu/sec or over 55 mu/sec predicted 58 or 59 % of the male or female offspring cases, respectively. Semen viscosity reflected in sperm velocity was linked to predominantly male or female sperm populations. Paracrine signals from the gender-skewed sperm precursor populations controlling viscosity merit further exploration.
  • Henneberg AA, Patton WC, Jacobson JD, Chan PJ.. "Human papilloma virus DNA exposure and embryo survival is stage-specific.." J. Assist Reprod Genet. 23.6 (2006): 255-259. ( 7/2006 )
    PURPOSE: Human papillomavirus (HPV) has been shown to disrupt late-stage implanting embryos. The objectives were (a) to assess the development of early embryos exposed to HPV DNA and (b) to analyze the blastocyst hatching process after HPV exposure. METHODS: The study involved exposing two-cell and 4-8-cell mouse embryos to DNA fragments from either HPV type 16, type 18 or DQA1 (control). The embryos were incubated for 120 h and assessed. RESULTS: HPV 16 and 18 inhibited two-cell embryo development. In contrast, delaying the exposure of HPV DNA until the 4-8-cell stage resulted in further embryonic development. There was 25.9% less blastocyst formed with HPV 16 exposure. Additionally, there were 25.9-31.8% more degenerated embryos with HPV 16 exposure. CONCLUSIONS: The study demonstrated embryo stage-specific effects of HPV on early development. The results suggested HPV exposure was linked to two-cell embryo demise and delaying the exposure of HPV until later embryo stages permitted embryo development. HPV 16 was shown to decrease blastocyst formation while HPV 18 inhibited the blastocyst hatching process.
  • Kim J, Patton WC, Corselli JU, Jacobson JD, King A, Chan PJ. "Mouse Embryonic Stem Cells for Quality Control Testing in Assisted Reproductive Technology Programs." Journal of Reproductive Medicine 50.7 (2005): 533-538. ( 1/2005 )
  • Hart EA, Patton WC, Jacobson JD, King A, Corselli J, Chan PJ.. "Luteal phase serum cell-free DNA as a marker of failed pregnancy after assisted reproductive technology." Journal of Assisted Reproduction and Genetics 22.5 (2005): 213-217. ( 1/2005 )
  • Bouma CL, Patton WC, Jacobson JD, King A, Chan PJ. "Sperm apoptosis in nonpregnant luteal phase sera after in vitro fertilization as assessed by comparative genomic hybridization.." Archives of Andrology 50.1 (2004): 41-44. ( 12/2004 )
  • Chou J.S., Jacobson, J., Patton, W., King, A. Chan, P.J.. . "Modified Isocratic Capillary Electrophoresis Detection of Cell-free DNA in Semen. ." Journal of Assisted Reproduction and Genetics 21.11 (2004): 397-400. ( 11/2004 )
  • Siddighi, S., Patton W.C., Jacobson J.D., King, A., Chan, P.J. . "Correlation of sperm parameters with apoptosis assessed by dual fluorescence DNA integrity assay.." Archives of Andrology 50.4 (2004): 311-314. ( 1/2004 )
  Non-Scholarly Journals
  • Professor Philip J. Chan"Spermac stain: Strict Morphology Collection." IVF.NET 01 01 2006: 1 - 1 ( 1/2006 ) Link...
  • Philip J. Chan PhD"Male Factor testing." LLU Fertility Website 01 12 2005: ( 12/2005 )
  • Philip J. Chan, PhD"Mini Sperm Penetration Assay Protocol." LLU Fertility Website 31 12 2005: ( 12/2005 )