Serum biomarkers for predicting pregnancy outcome in women undergoing IVF: human chorionic gonadotropin, progesterone, and inhibin A level at 11 days post-ET.

OBJECTIVE: This study was performed to assess the prognostic value of serum hCG, progesterone, and inhibin A levels measured at 11 days post-ET for predicting pregnancy outcome in women participating in IVF. METHODS: Between May 2005 and April 2008, sera were obtained from 70 infertile women who underwent IVF-ET at 11 days post-ET and stored. HCG, progesterone, and inhibin A levels were measured by commercial enzyme-linked immunosorbent assay kits. The predictive accuracy of hCG, progesterone, and inhibin A levels for establishment of intrauterine pregnancy and ongoing pregnancy was calculated by receiver-operating characteristic curve analysis. RESULTS: For the prediction of intrauterine and ongoing pregnancy, serum hCG was better than progesterone and inhibin A. The predictive performance of progesterone and inhibin A was similar. The serum progesterone and inhibin A levels were significantly correlated each other (r=0.915, p=0.010). CONCLUSION: A single measurement of the serum hCG level is sufficient to predict pregnancy outcome in IVF-ET patients.

Sperm nuclear DNA fragmentation and chromatin structure in one-day-old ejaculated sperm.

OBJECTIVE: To evaluate sperm nuclear DNA fragmentation and chromatin structure after 18 hours'incubation at room temperature. METHODS: Twenty-eight male partners who participating IVF treatment were prospectively included in this study. Ejaculated sperm count and motility were assessed. The sperm was then immediately processed by the conventional swim-up method. After utilization of some of the sample for routine clinical use, the remainder of each of the samples was divided into two aliquots. One aliquot was immediately assessed for sperm nuclear DNA fragmentation (TUNEL assay) and chromatin structure (toluidine blue [TB] staining). The other aliquot was incubated at room temperature for 18 hours and then assessed by two methods. Only dark-TB sperms were considered as having abnormal chromatin structure. Data before and after extended incubation were compared using a paired Student's t-test. RESULTS: Before and after extended culture, nuclear DNA fragmentation assessed by TUNEL was 4.9±4.7% and 7.0±6.4%, respectively (p=0.008). The proportion of abnormal chromatin structure (dark-TB sperm) was 8.2±5.6% and 10.3±6.5% (p<0.001), before and after incubation, respectively. CONCLUSION: After 18 hours' incubation at room temperature, sperm nuclear DNA and chromatin structure were significantly affected. The IVF practitioner should bear this information in mind when performing delayed insemination, especially for in vitro maturation cycles.

Gene expression in proliferating cells of the dinoflagellate Alexandrium catenella (Dinophyceae).

Understanding the conditions leading to harmful algal blooms, especially those produced by toxic dinoflagellate species, is important for environmental and health safety. In addition to investigations into the environmental conditions necessary for the formation of toxic blooms, we postulate that investigating gene expression in proliferating cells is essential for understanding bloom dynamics. Expressed sequence tags were produced from cultured cells of the toxic dinoflagellate Alexandrium catenella sampled during the initiation phase of growth using Sanger's method and by 454 pyrosequencing. A significant proportion of identified genes (ca. 25%) represented enzymes and proteins that participate in a variety of cellular regulatory mechanisms that may characterize proliferating cells, e.g., control of the cell cycle and division, regulation of transcription, translation and posttranslational protein modifications, signaling, intracellular trafficking, and transport. All of the several genes selected for gene expression assays due to their involvement in metabolism and the cell cycle were overexpressed during exponential growth. These data will be useful for investigating the mechanisms underlying growth and toxin production in toxic Alexandrium species and for studying and monitoring the development of toxic blooms.

The change of cytokines in tear and blood after different pterygium operation.

Pterygium is an invasion of altered ocular tissue into the cornea. Bone marrow-derived stem cells have been reported to be involved in wound healing under chemotactic factors after pterygium removal and pain may act as a trigger signal. We evaluated the change of systemic and local chemotactic factors that could affect the mobilization and migration of BMSCs to the wound bed after conventional bare sclera pterygium excision. We also applied temporary amniotic membrane patch after pterygium removal, and compared the changes of cytokines with those of conventional bare sclera excision group. Substance-P (SP), vascular endothelial growth factor (VEGF), and stem cell factor (SCF) were measured in plasma and tear using ELISA and migrating CD34(+) cells by flow cytometry. The results showed that post-operative pain was much reduced (p<0.05), and SP, VEGF and SCF kept consistently lower levels in plasma after temporary amniotic membrane application. Circulating CD34(+) cells increased slightly in the temporary amniotic membrane patch group compared with marked increase in the bare sclera group. Thus, the application of a temporary amniotic membrane after pterygium removal might be an effective therapeutic means by controlling pain and excessive infiltration of bone marrow-derived stem cells.

Cytokine secretion by human mesenchymal stem cells cocultured with damaged corneal epithelial cells.

We have previously shown that mesenchymal stem cells (MSCs) reduced corneal inflammation and neovascularization in chemically-burned rat corneas in part through paracrine action. In order to identify the molecule(s) involved, we cocultured human MSCs in the following conditions, and examined the alterations in the cytokine secretion profile; (1) human peripheral blood mononuclear cells (hPBMCs), (2) chemically-damaged human corneal epithelial cells (hCECs), (3) hPBMCs/hCECs, (4) hMSCs, (5) hMSCs/hPBMCs, (6) hMSCs/hCECs, (7) hMSCs/hPBMCs/hCECs. We found that the levels of interleukin (IL)-6 and vascular endothelial growth factor (VEGF) by hMSCs markedly increased for hMSC/hCEC cocultures, and this elevation was further remarkable by the addition of hPBMCs. The hMSCs constitutively expressed transforming growth factor (TGF)-beta1, matrix metalloproteinase (MMP)-2, and thrombospondin-1. The secretion of MMP-9 by hCECs was significantly suppressed by hMSCs. Tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and IL-10 were not detectable in any cultures. The data presented herein provide the candidate molecules mediating the MSC-mediated modulation of inflammation and angiogenesis in cornea.

The anti-inflammatory effect of subconjunctival bevacizumab on chemically burned rat corneas.

PURPOSE: To investigate the effect of bevacizumab in a model of corneal inflammation. MATERIALS AND METHODS: Epithelium from the cornea and limbus was completely removed using 100% alcohol in rats. Bevacizumab (1.25 mg/0.05 ml) or normal saline was subconjunctivally injected. One week later, corneas were examined and subjected to hematoxylin-eosin and immunofluorescent staining for VEGF. Expression of IL-2, IFN-gamma, IL-6, and TGF-beta 1 were analyzed by ELISA. RESULTS: Bevacizumab showed a borderline reduction in corneal neovascularization (11.0 +/- 4.8% and 18.0 +/- 10.0% in the bevacizumab and control groups, respectively; p = 0.054), while the extent of epithelialization was not affected (5.0 +/- 3.4% and 6.1 +/- 5.2% in the bevacizumab and control groups, respectively; p = 0.715). The infiltration of inflammatory cells was reduced (99.3 +/- 22.3 cells/x 400 in the bevacizumab-injected corneas and 182.3 +/- 58.0 cells/x 400 in the controls; p = 0.013). The levels of IL-2, IFN-gamma, and IL-6 were decreased in the rats with the bevacizumab injections (44 +/- 6 and 79 +/- 9 pg/ml for IL-2 in the bevacizumab-injected group and control, respectively, p = 0.025; 45 +/- 5 and 67 +/- 6 pg/ml for IFN-gamma in the bevacizumab-injected group and controls, respectively, p = 0.043; 45 +/- 6 and 75 +/- 8 pg/ml for IL-6 in the bevacizumab-injected group and controls, respectively, p = 0.030). CONCLUSIONS: Bevacizumab showed a reduction in inflammatory cell infiltration and cytokines in chemically burned rat corneas.

Endoscopic vitrectomy improves outcomes of Seoul-type keratoprosthesis exchange in rabbit model.

PURPOSE: To investigate the efficacy of an endoscopic vitrectomy in Seoul-type keratoprosthesis (S-Kpro) exchange procedures. METHODS: Nine S-Kpro-implanted rabbit eyes were enrolled in the S-Kpro exchange. Six eyes underwent an antecedent vitrectomy by an endoscopic system and then the S-Kpro exchange (endoscopy group). In the other three eyes, previously placed S-Kpros were removed, and a conventional vitrectomy was performed using the Eckardt keratoprosthesis, followed by an implantation of new S-Kpros (Eckardt group). All eyes were evaluated with slit lamp biomicroscopy and ultrasonography weekly to evaluate the time up to the development of the total retinal detachment (RD). RESULTS: Vitreous traction membranes were found around the prolene haptics of the fixation sites in all the S-Kpro implanted rabbits; they were excised precisely through an endoscopic view in the endoscopy group. The mean survival time up to the RD development was 9.75 +/- 4.70 weeks in the endoscopy group. In contrast, total retinal detachment or dialysis over 180 degrees developed during surgery in all three eyes in the Eckardt group. CONCLUSIONS: Antecedent endoscopic vitrectomy was safe and effective for the S-Kpro exchange in a rabbit model by removing the vitreous traction near the haptics before the exchange procedures.

Efficient cultivation conditions for human limbal epithelial cells.

To compare the stem niche in different culture conditions of limbal epithelial cells, the suspended human limbal epithelial cells (HLECs) were seeded on the 3T3-pretreated plates and the other suspended cells were plated on amniotic membranes (AMs) which were either cryo-preserved or freeze-dried. All were cultured for 10 to 12 days. Reverse transcription-polymerase chain reaction (RT-PCR) for ATP-binding cassette, subfamily G, member 2 (ABCG2), p63, cytokeratin 12, and connexin 43 were performed in cultivated HLECs and their expression levels were compared. The mRNA expression of all markers examined showed no statistically significant differences between the cells on cryo-preserved and on freeze-dried AM. The expression of p63 and cytokeratin 12 in cultivated cells on AMs were significantly lower than those in 3T3-cocultured cells on RT-PCR and immunofluorescent staining. Cultivated HLECs on AMs showed reduced proliferation and differentiation while maintaining stem-property regardless of the preservative method of AM.

Transforming growth factor-beta expression in rat eyes with mechanical debridement of corneal epithelium or epithelial flap.

PURPOSE: To compare corneal haze and transforming growth factor (TGF)-beta expression in rat eyes with mechanical debridement of corneal epithelium or a chemically induced epithelial flap. SETTING: Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea. METHODS: Sixty corneas from 60 Sprague-Dawley rats were treated in 1 of 4 ways using a 2.0 mm trephine: corneal epithelium mechanically removed (Group 1), central cornea exposed to 20% ethanol for 30 seconds (Group 2), corneal epithelial flap made by applying 20% ethanol for 30 seconds and flap amputated (Group 3), corneal epithelial flap repositioned after ethanol-assisted detachment of epithelium as in Group 3 (Group 4). Corneal haze was graded. The TGF-beta expression was measured in corneas and lacrimal glands using reverse transcription polymerase chain reaction and immunohistochemistry until day 30. Semiquantitative analysis was done in a density ratio of TGF-beta/beta-actin with image analyzer and densitometry. RESULTS: Corneal haze was more severe in Groups 1 and 3 than in Groups 2 and 4. By day 7, mRNA expression of TGF-beta2 and type II receptor in corneas had increased more in Groups 1 and 3 than in Groups 2 and 4. In lacrimal glands, only TGF-beta1 in Group 3 increased until day 7. In corneas, staining of both TGF-beta1 and beta2 increased, more prominently in Groups 1 and 3. Lacrimal gland staining was more intense in Groups 1 and 3. CONCLUSION: Well-positioned corneal epithelial flaps may decrease corneal haze by reducing expression of TGF-beta; inadvertent removal of an epithelial flap made by ethanol seems to exacerbate haze by increasing TGF-beta.

A novel electroporation method using a capillary and wire-type electrode.

Electroporation is widely used to achieve gene transfection. A common problem in electroporation is that it has a lower viability than any other transfection method. In this study, we developed a novel electroporation device using a capillary tip and a pipette that was effective on a wide range of mammalian cells, including cell lines, primary cells, and stem cells. The capillary electroporation system considerably reduced cell death during electroporation because of its wire-type electrode, which has a small surface area. The experimental results also indicated that the cell viability was dependent on the change in pH induced by electrolysis during electroporation. Additionally, the use of a long and narrow capillary tube combined with simple pipetting shortened the overall time of the electroporation process by up to 15 min, even under different conditions with 24 samples. These results were supported by comparison with a conventional electroporation system. The transfection rate and the cell viability were enhanced by the use of the capillary system, which had a high transfection rate of more than 80% in general cell lines such as HeLa and COS-7, and more than 50% in hard-to-transfect cells such as stem or primary cells. The viability was approximately 70-80% in all cell types used in this study.

The anti-inflammatory and anti-angiogenic role of mesenchymal stem cells in corneal wound healing following chemical injury.

To investigate the anti-inflammatory and anti-angiogenic effects of mesenchymal stem cells (MSC) in the chemically burned corneas, we mechanically removed the corneal epithelium of rats after 100% alcohol instillation. The rats were then randomized into four groups: fresh media, conditioned media derived from the MSC culture (MSC-CM), MSC applied topically to the damaged corneas for 2 hours immediately after the injury or MSC-CM applied either once or 3 times per day for 3 consecutive days. Corneal surface was evaluated every week. After 3 weeks, the corneas were stained with the hematoxylin-eosin, and the expression of interleukin (IL)-2, interferon (IFN)-gamma, IL-6, IL-10, transforming growth factor (TGF)-beta1, thrombospondin-1 (TSP-1), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor (VEGF) were analyzed. CD4+ cells were assessed in the corneas. We found that both MSC and three-time applied MSC-CM (1) reduced corneal inflammation and neovascularization, (2) decreased IL-2 and IFN-gamma, although increased IL-10 and TGF-beta1 as well as IL-6, (3) reduced the infiltration of CD4+ cells, and (4) upregulated the expression of TSP-1, although downregulated that of MMP-2. Interestingly, whereas three-time application of MSC-CM was partially effective, transplantation of MSC achieved a better outcome in suppressing corneal inflammation. The results of this study suggest that the anti-inflammatory and anti-angiogenic action of MSC in the chemically burned corneas might be mediated in part through paracrine pathways involving soluble factors such as IL-10, TGF-beta1, IL-6 and TSP-1.

Efficient cryopreservative conditions for cultivated limbal and conjunctival epithelial cells.

PURPOSE: To examine the effects of cryopreservation on the viability of cultivated corneal limbal and conjunctival epithelial cells and to evaluate the optimal conditions for cryopreservation. METHODS: The cultivated human limbal epithelial cells (HLECs) were stored in media including 20%, 50%, and 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) at -196 degrees C for 1 week. The cultivated rabbit conjunctival epithelial cells were stored in 10%, 20%, and 50% FBS with 10% glycerol or DMSO as a cryoprotectant at -196 degrees C for 1 week. After thawing, cell viability was assessed using the trypan blue vital staining and 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay. Immunofluorescent staining was performed with cytokeratin 3/12 antibody. Colony-forming efficiency (CFE) was evaluated 2 weeks after culture. RESULTS: HLECs cryopreserved with 50% FBS showed the highest cell viability, whereas those with 20% FBS revealed the lowest survival rate (87.1% +/- 0.8% and 79.8% +/- 4.01%, respectively; P = 0.030). CFE of HLECs was 2.13 +/- 1.35%, 2.31 +/- 2.23%, and 1.94 +/- 0.72% in cells with 20%, 50%, and 90% FBS, respectively (P > 0.05). For conjunctival epithelial cells, the cell viability was the highest with 50% FBS and 10% glycerol (95.0% +/- 4.27%), and the lowest survival rate was observed in the condition of 10% FBS and 10% DMSO (80.0% +/- 5.49%). CFE of cryopreserved conjunctival epithelial cells was 14.1% +/- 1.9% in cells with 20% FBS and glycerol and 13.5% +/- 2.0% in those with 20% FBS and DMSO (P > 0.05). HLECs expressed CK3/12 after cryopreservation in all conditions examined. CONCLUSIONS: The best results were yielded by 50% FBS for cell viability in HLECs. Glycerol seems to be superior to DMSO in cell viability of the rabbit conjunctival epithelium after cryopreservation.

A prospective, randomized, placebo-controlled, double-blinded, and split-face clinical study on LED phototherapy for skin rejuvenation: clinical, profilometric, histologic, ultrastructural, and biochemical evaluations and comparison of three different treatment settings.

Light-emitting diodes (LEDs) are considered to be effective in skin rejuvenation. We investigated the clinical efficacy of LED phototherapy for skin rejuvenation through the comparison with three different treatment parameters and a control, and also examined the LED-induced histological, ultrastructural, and biochemical changes. Seventy-six patients with facial wrinkles were treated with quasimonochromatic LED devices on the right half of their faces. All subjects were randomly divided into four groups treated with either 830nm alone, 633nm alone, a combination of 830 and 633nm, or a sham treatment light, twice a week for four weeks. Serial photography, profilometry, and objective measurements of the skin elasticity and melanin were performed during the treatment period with a three-month follow-up period. The subject's and investigator's assessments were double-blinded. Skin specimens were evaluated for the histologic and ultrastructural changes, alteration in the status of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), and the changes in the mRNA levels of IL-1ss, TNF-alpha, ICAM-1, IL-6 and connexin 43 (Cx43), by utilizing specific stains, TEM, immunohistochemistry, and real-time RT-PCR, respectively. In the results, objectively measured data showed significant reductions of wrinkles (maximum: 36%) and increases of skin elasticity (maximum: 19%) compared to baseline on the treated face in the three treatment groups. Histologically, a marked increase in the amount of collagen and elastic fibers in all treatment groups was observed. Ultrastructural examination demonstrated highly activated fibroblasts, surrounded by abundant elastic and collagen fibers. Immunohistochemistry showed an increase of TIMP-1 and 2. RT-PCR results showed the mRNA levels of IL-1ss, TNF-alpha, ICAM-1, and Cx43 increased after LED phototherapy whereas that of IL-6 decreased. This therapy was well-tolerated by all patients with no adverse effects. We concluded that 830 and 633nm LED phototherapy is an effective approach for skin rejuvenation.