Further evidence supporting the Diff-Quik stain for sperm chromatin integrity testing.

OBJECTIVE: To correlate intracytoplasmic sperm injection (ICSI) fertilization with chromatin status assessed by the Diff-Quik procedure modified with a one-minute soak step, and to determine the association of chromatin status with in vitro fertilization (IVF) pregnancy. STUDY DESIGN: This was a retrospective study of 81 IVF patients. Gradient-centrifuge washed sperm remaining after ICSI were fixed, stained by Diff-Quik, immersed in water for 1 minute, and analyzed under oil immersion light microscopy. Sperm nuclear coloration (types A-D), strict morphology, fertilization, and pregnancy status were determined. RESULTS: Sperm with light purple staining (type A) were correlated (R = 0.48, p < 0.05) with ICSI fertilization. The intraassay and interassay coefficients of variation were 5.9% and 4.1%, respectively. Sperm strict normal morphology was correlated neither with ICSI fertilization (R = 0.24, p > 0.05) nor with type A sperm (R = 0.35, p > 0.05). Sperm incubated in Fenton reagent that damaged DNA showed a time-dependent decrease in percent type A sperm. However, there was no correlation with IVF pregnancy status. CONCLUSION: This retrospective study showed that the inclusion of a one-minute soak step post-Diff-Quik staining enhanced the detection of sperm chromatin abnormalities related to ICSI fertilization. Fenton reagent-treated sperm suggested that the staining patterns correlated with DNA damage. A large prospective trial should be undertaken to confirm these findings.

Pentoxifylline attenuates HPV-16 associated necrosis in placental trophoblasts.

PURPOSE: Human papillomaviruses (HPV) have been associated with placental inflammation resulting in high-risk preterm birth. The premise for this study was that treatment with anti-inflammatory pentoxifylline would inhibit HPV-mediated placental cell death. The objectives were: (1) to study the effects of HPV-16 exposure on trophoblast cells and (2) to evaluate pentoxifylline in preventing the damaging effects of HPV-16. METHODS: Mouse embryos (1-cell) were cultured (G1plus medium, 72 h, 37 °C, 5 % CO2 in air), divided into four groups at blastocyst-stage and incubated to implantation stage. Implanted trophoblasts were exposed to either HPV-16 (group 1), concomitant HPV-16 and 3 mM pentoxifylline (group 2), 3 mM pentoxifylline only (group 3) or control medium (group 4) and further incubated for 24 h. HPV-16 were from SiHa cell lysates. Trophoblast structural integrity was assessed by morphometric analysis while apoptosis and necrosis were detected using dual-stain fluorescence assay. RESULTS: Trophoblast outgrowth was reduced by 90% (P < 0.05) in HPV-16 presence (629 ± 265 µ(2), mean + SEM) when compared with controls (6,456 ± 795 µ(2)). Pentoxifylline attenuated the effects of HPV-16 (4,308 ± 362 µ(2)). Nuclear size of HPV-16 infected trophoblasts was smallest among the groups (P < 0.005). HPV-16 decreased cell viability and increased necrosis but not apoptosis. Pentoxifylline prevented HPV-16 associated necrosis in trophoblasts. CONCLUSIONS: HPV-16 decreased nuclear size and trophoblast outgrowth but these effects were attenuated by the phosphodiesterase inhibitor, pentoxifylline. The action of HPV-16 on trophoblasts was increased cell necrosis suggesting that HPV-16 pathogenesis involved either cAMP inhibition and/or activated TNF pathways.

UVA-photoactivated riboflavin treatment of vaginal cells derived from pelvic organ prolapse cases.

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.

HPV-16 exposed mouse embryos: a potential model for pregnancy wastage.

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.

What Is the Best Lens Angle for Rigid Diagnostic Cystoscopy? A Comparison of 30-Degree and 70-Degree Lenses.

The optimal degree of cystoscope to perform diagnostic cystoscopy is dependent on the surgeon's discretion because there are no studies addressing the superiority of one degree of cystoscope over another. The objective was to determine which lens, 70-degree versus 30-degree lens, was better in identifying lesions scattered throughout the bladder. METHODS: A simulation was created using 3 different artificial bladder models complete with sutures representing lesions placed at different locations in each bladder. Gynecologists and urologists performed cystoscopy using both the 30- and 70-degree lenses on the bladder models in a randomized and single-blinded fashion. The physicians performed routine diagnostic cystoscopy and noted the number of lesions throughout the bladder. The number of lesions each physician identified and the time to complete cystoscopy were noted. A total of 16 physicians participated, and there were a total of 18 lesions among the 3 different bladder models. RESULTS: A total of 86 cystoscopy trials were obtained from all physicians and bladder models attempted. The odds of detecting a lesion with the 70-degree lens cystoscope was 1.77 times greater than the 30-degree lens cystoscope (95% confidence interval, 1.24-2.53; P = 0.002). There was also difference in the average number of lesions found between the 30- and 70-degree cystoscopes with 2.6 ± 2.7 more lesions identified using the 70-degree cystoscope compared with the 30-degree cystoscope. In terms of specific location, 2.6 ± 1.7 more lesions were found at the bladder neck using the 70-degree lens scope versus the 30-degree lens scope (95% confidence interval, 1.37-3.83; P = 0.013). CONCLUSIONS: The results suggested that the 70-degree lens was the better choice for the identification of bladder lesions when compared with the 30-degree lens in rigid diagnostic cystoscopy.

Male age and sperm necrosis in assisted reproductive technologies.

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.

Perioperative serum creatinine changes and ureteral injury.

PURPOSE: To illustrate a simple method that screens for ureteral injury in the acute postoperative period after urogynecologic surgeries. METHODS: Serum creatinine measurements in the preoperative (baseline) and postoperative periods of urogynecologic surgeries were determined and the correlation of the change to ureteral injury and/or obstruction analyzed. The sample size calculation showed 7 cases and 28 controls were sufficient to detect significant changes in creatinine. Each of the seven cases was matched for age and type of surgery with a control patient in a 1:4 ratio following standard protocol. RESULTS: Chart review of patients (273 cases) undergoing urogynecologic surgeries from October 2009 to June 2014 were undertaken. There were 7 cases of ureteral injury and 28 matching control cases. All cases had intraoperative cystoscopy confirming bilateral ureteral flow. In the ureteral injury group, blockage of ureter was confirmed by CT scan with IV contrast. There was a 59.8% increase in serum creatinine levels postoperative in the ureteral injury group versus a 3.8% decrease in controls. A difference of creatinine levels greater than or equal to 0.3 mg/dL over baseline was evident in ureteral injury cases. CONCLUSION: A small change in serum creatinine level over baseline after urogynecologic surgery alerted the possibility of ureteral injury or obstruction. A simple and inexpensive evaluation of perioperative creatinine levels can promptly diagnose ureteral damage in the acute postoperative period for gynecologic reconstructive surgeries.

Food intake diet and sperm characteristics in a blue zone: a Loma Linda Study.

OBJECTIVES: The study examined the effect the life-long vegetarian diet on male fertility and focused on vegetarians living in the Loma Linda blue zone, a demographic area known for life longevity. The objective was to compare sperm characteristics of vegetarian with non-vegetarian males. STUDY DESIGN: The cross-sectional observational study was based on semen analyses of 474 males from 2009 to 2013. Patients categorized themselves as either life-long lacto-ovo vegetarians (N=26; vegetable diet with dairy and egg products), vegans (N=5; strictly vegetables with no animal products) or non-vegetarians (N=443; no diet restrictions). Sperm quality was assessed using a computer-aided sperm analyzer and strict morphology and chromatin integrity were manually evaluated. RESULTS: Lacto-ovo vegetarians had lower sperm concentration (50.7±7.4M/mL versus non-vegetarians 69.6±3.2M/mL, mean±S.E.M.). Total motility was lower in the lacto-ovo and vegan groups (33.2±3.8% and 51.8±13.4% respectively) versus non-vegetarians (58.2±1.0%). Vegans had lowest hyperactive motility (0.8±0.7% versus lacto-ovo 5.2±1.2 and non-vegetarians 4.8±0.3%). Sperm strict morphologies were similar for the 3 groups. There were no differences in rapid progression and chromatin integrity. CONCLUSIONS: The study showed that the vegetables-based food intake decreased sperm quality. In particular, a reduction in sperm quality in male factor patients would be clinically significant and would require review. Furthermore, inadequate sperm hyperactivation in vegans suggested compromised membrane calcium selective channels. However, the study results are cautiously interpreted and more corroborative studies are needed.

Mouse embryonic stem cells for quality control testing in assisted reproductive technology programs.

OBJECTIVE: To compare the mouse embryonic stem (ES) cell assay with the sperm motility test or 1-cell mouse embryo bioassay for embryotoxic materials. STUDY DESIGN: Cryo-preserved-thawed mouse ES-D3 cells, 1-cell mouse embryos and donor sperm were incubated for 1-4 days in culture medium exposed to a control and 4 different test materials. ES cell viability (eosin method), apoptosis (Sybr-Gold fluorescence), development of blastocysts and sperm motility parameters were measured. RESULTS: The initial viabilities of ES cell were determined to be 37.0 +/- 4.2% (n = 225) and 54.8 +/- 7.4% (n = 218) by the eosin and Sybr Gold methods, respectively. Reduced viability of ES cells in latex glove-treated medium (25.6 +/- 0.3% and 25.7 +/- 0.3% versus control, 32.8 +/- 0.2% and 33.5 +/- 1.0% by eosin or Sybr Gold, respectively, p < 0.05) was consistent with standard bioassays. However, toxicity in the syringe was detected only by the ES cell assay. The ES cell assay sensitivities were 33% and 67% (eosin and Sybr Gold methods, respectively), and specificities were 100% for both methods. CONCLUSION: Mouse ES cell assay based on Sybr-Gold asssessment was as effective as standard bioassays for detecting embryotoxicity. The results suggested that the mouse ES assay could be used for testing contact materials and DNA-modifying agents. More studies are needed to refine and enhance the sensitivity of the ES cell assay for routine use in assisted reproductive technology clinics.

Selenium Attenuates HPV-18 Associated Apoptosis in Embryo-Derived Trophoblastic Cells but Not Inner Cell Mass In Vitro.

Objectives. Human papillomaviruses (HPV) are associated with cell cycle arrest. This study focused on antioxidant selenomethionine (SeMet) inhibition of HPV-mediated necrosis. The objectives were to determine HPV-18 effects on embryonic cells and to evaluate SeMet in blocking HPV-18 effects. Methods. Fertilized mouse embryos were cultured for 5 days to implanted trophoblasts and exposed to either control medium (group 1), HPV-18 (group 2), combined HPV-18 and 0.5 µM SeMet (group 3), or combined HPV-18 and 5.0 µM SeMet (group 4). After 48 hrs, trophoblast integrity and, apoptosis/necrosis were assessed using morphometric and dual-stain fluorescence assays, respectively. Results. HPV-18 exposed trophoblasts nuclei (253.8 ± 28.5 sq·µ) were 29% smaller than controls (355.6 ± 35.9 sq·µ). Supplementation with 0.5 and 5.0 µM SeMet prevented nuclear shrinkage after HPV-18 exposure. HPV-18 infected trophoblasts remained larger with SeMet supplementation. HPV-18 decreased cell viability by 44% but SeMet supplementation sustained cell viability. Apoptosis was lower when SeMet was present. HPV-18 decreased inner cell mass (ICM) viability by over 60%. Conclusions. HPV-18 decreased nuclear size and trophoblast viability but these effects were attenuated by the antioxidant SeMet. SeMet blocked HPV-18 associated apoptosis process in trophoblasts but not ICM cells suggesting involvement of different oxidative stress pathways.

Combined ultrasound-curcumin treatment of human cervical cancer cells.

OBJECTIVES: Human papillomavirus (HPV) is associated with cervical cancer. Studies showed curcumin inhibits HPV oncogenes expression but curcumin has low bioavailability. The objectives were: (1) to study ultrasound enhancement of curcumin effects on HeLa, SiHa and C33A, (2) to compare two frequencies for sonoporation and (3) to detect cell-free DNA released by the treatment. STUDY DESIGN: HeLa, SiHa and C33A cells (non-HPV control) were processed and exposed to either: (1) 10µM curcumin only, (2) 10µM curcumin with 8s of 7.5MHz ultrasound, (3) 10µM curcumin with 8s of 5.0MHz ultrasound, (4) control medium, or (5) 8s of 7.5MHz ultrasound. The five treated groups were incubated (48h) and analyzed by dual fluorescence apoptosis/necrosis assay. DNA in spent media was analyzed by capillary analysis. RESULTS: Combined curcumin ultrasound resulted in 9-, 12- and 16-fold higher necrosis in HeLa, SiHa and C33A cells respectively. Increased necrosis correlated with higher ultrasound frequencies. There was increased apoptosis in HeLa or SiHa cells with the combined treatment. Curcumin alone resulted in a lesser 2-4-fold increase in necrosis in the groups. Cell-free DNA was detected in the spent media of HeLa and SiHa but not C33A cultures. CONCLUSIONS: The results showed enhanced necrosis in cervical carcinoma cell lines after combined treatment and confirmed the ultrasound capacity to increase effectiveness of curcumin. Cancer cells were smaller post-treatment suggesting microtubule structural disruption. Cell-free DNA was low molecular weight consistent with lysed host cell.

A simple sperm DNA toroid integrity test and risk of miscarriage.

Current methods of analyzing sperm chromatin competency overlook the inner sperm compartment which is inaccessible to probes and reagents. By breaking the molecular protamine disulfide bridges, the DNA toroids are exposed to integrity analysis. The aim was to develop a simple nuclear toroid test and determine its association with fertilization, pregnancy, and miscarriage. The approach involved treating washed sperm remaining after ICSI procedures (N=35 cases) with acidified Triton X-100 and dithiothreitol (DTT) before Diff-Quik staining. Percentages of sperm with normal chromatin indicated by light-colored nuclei were assessed. The toroid integrity test showed more sperm with normal chromatin in the pregnant group (73.6±1.7%, mean±SEM) when compared with the miscarriage (51.2±6.6%) or nonpregnant groups (60.9±4.8%). Furthermore, the toroid results were correlated with ICSI fertilization (R=0.32, P=0.04) and pregnancy outcome (pregnant cases 73.6±1.7% versus nonpregnant 58.0±3.9%, P=0.001). ROC calculated cut-off was >70.0% for normal toroid integrity (sensitivity 0.98, specificity 0.33, and diagnostic accuracy 78.3%). An association between normal sperm toroid integrity and miscarriage was evident when the staining procedure included acidified detergent DTT pretreatment.

UVA-photoactivated riboflavin effect on isolated vaginal tissues derived from pelvic organ prolapse cases.

PURPOSE: To determine the optimal riboflavin exposure time before UVA irradiation and to study the effects of exogenous collagen on the mechano-tensile stiffness of isolated vaginal tissue strips after riboflavin UVA photoactivation. METHODS: Vaginal tissue strips from pelvic organ prolapse (POP) cases were soaked in 0.1% riboflavin (0, 10, 20, 30 min), exposed to UVA photoactivation, and tensile stiffness was measured with a tensiometer. Collagen solution was injected (0.2 mL) into each strip, exposed to riboflavin with or without UVA photoactivation, and tensile stiffness was measured (n=6). RESULTS: Vaginal tissues treated with riboflavin for 10, 20 or 30 min followed by UVA irradiation displayed 21.2, 32.4 and 33.9% stronger tensile stiffness, respectively. Exogenous collagen administered before riboflavin UVA photoactivation resulted in 20% improvement in tensile stiffness. The tensile stiffness of vaginal tissues injected with collagen without the riboflavin UVA treatment was similar to control tissues. CONCLUSION: The results demonstrated increased tensile stiffness in isolated POP-derived vaginal tissues after riboflavin UVA photoactivation suggesting improved mechanical properties from collagen cross-linking. Administering exogenous collagen before riboflavin UVA treatment also improved tensile stiffness. More studies are needed to corroborate the present minimally invasive approach for strengthening vaginal tissues.

A simple DNA disc chip in a microarray design based on modified comparative genomic hybridization for sperm DNA analysis.

OBJECTIVE: A DNA disc chip assay, based on comparative genomic hybridization, was designed to measure changes in sperm DNA intensities. The objective was to analyze the DNA integrity of hyperactive sperm cells after mild heat treatment. DESIGN: The assay based on a multiple cell comet assay was used to analyze changes in genomic DNA. Washed sperm DNA were tested on the assay and images stored in a microarray design. SETTING: Clinical and academic research environment. PATIENT(S): Frozen-thawed washed sperm from different donors (n = 7). INTERVENTION(S): Discarded sperm leftover from trial washes carried out at 37 degrees and 40 degrees C were frozen and processed for the DNA disc chip assay. MAIN OUTCOME MEASURE(S): Fluorescent intensities of DNA disc chips and sperm variables. RESULT(S): Heat treatment resulted in more than eightfold increase in sperm hyperactive motility with little degradation in DNA integrity. Sperm with low hyperactivation was associated with alterations in DNA after heat treatment. CONCLUSION(S): The DNA disc chip assay was simple, inexpensive, and permitted assisted reproduction technologies laboratories to use comparative genomic hybridization for cytogenotoxicity testing. However, the assay required manual processing, a fluorescent microscope, and computer. The data showed an association between sperm hyperactivation and DNA integrity suggesting that the hyperactivation marker may be used for selecting quality sperm for intracytoplasmic sperm injection. More studies are needed to examine temperature effects on ejaculated human sperm.

Temperature variable and the efficiency of sperm mediated transfection of HPV16 DNA into cells.

AIM: To pretreat sperm at various temperatures before exposure to human papillomavirus (HPV) 16 DNA fragments and to assess the efficiency of HPV carrier sperm to transfect cumulus cells. METHODS: Cumulus cells from follicular aspirates were obtained, pooled and divided into culture dishes containing Sybr Gold-stained HPV DNA carrying sperm that were either pretreated at 4 degree C, 37 degree C or 40 degree C (n = 5). The cells were incubated in 5% CO(2) in air mixture at 37 degree C for 24 hours. The efficiency of sperm to take up fluorescent HPV DNA was determined at hour 0. After incubation, cumulus cell viability was assessed using the eosin method and the percentages of fluorescent cumulus cells determined. RESULTS: Over half of all the cumulus cells became fluorescent with the highest percentage in the 37 degree C group. Sperm pretreated at 4 degree C had the greatest amount of HPV DNA fragments. Total sperm motility was similar for the 3 pretreatment groups. There were no differences in cumulus viability among the groups. CONCLUSION: Sperm pretreated at 37 degree C transferred the greatest amount of fluorescent HPV DNA fragments to the cumulus cells. The HPV DNA was observed in the nuclear and cytoplasmic compartments. The data suggested the possibility of sperm as a vector for the transmission of HPV DNA to the cumulus cells surrounding ovulated oocytes, which might lead to early implantation failures.

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.

Influence of nanoparticles on morphological differentiation of mouse embryonic stem cells.

OBJECTIVE: To assess mouse embryonic stem (ES) cell viability, growth, and differentiated morphology after exposure to different concentrations of nanoparticles. DESIGN: Cell culture for 6 days. SETTING: University research laboratory. ANIMALS: Cryopreserved mouse ES-D3 (American Type Culture Collection, Manassas, VA) cells. INTERVENTION(S): ES cells were exposed to either 0 (control), 0.4, or 12.2 million/mL mixed-size fluorescent nanoparticles in culture (37 degrees C, 5% CO(2) in air) for 6 days. MAIN OUTCOME MEASURE(S): Cell viability and morphometric analysis were performed. RESULT(S): ES cells exposed to both concentrations of nanoparticles exhibited smaller cell surface area. The effect was not concentration dependent. In contrast, ES cell nucleus size was unaffected. The nanoparticles distributed into the cytoplasm, pseudopods, and the perinuclear region. ES cell viabilities were reduced 40% and 30% in the low versus high relative concentration, respectively. ES cells in low-concentration nanoparticles became mostly columnar and embryoid body shaped. However, in high-concentration nanoparticles, they differentiated toward fibroblast-like and less squamous types. CONCLUSION(S): The observed reduced ES cell surface area suggested disruption of cytoskeletal development but not nuclear organization by nanoparticles. The ring-like formation of nanoparticles around the nucleus and the resulting cell morphologies suggested nanoparticles may influence differentiation.

In vitro tagging of embryos with nanoparticles.

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.

Retention of membrane charge attributes by cryopreserved-thawed sperm and zeta selection.

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.

Human papilloma virus DNA exposure and embryo survival is stage-specific.

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.