Jung Ryeol Lee

Successful vitrification of human amnion-derived mesenchymal stem cells.

BACKGROUNDnA cryopreservation protocol for human amnion-derived mesenchymal stem cells (HAMs) is required because these cells cannot survive for long periods in culture. The aim of this study was to determine whether vitrification is a useful freezing method for storage of HAMs.nnnMETHODSnHAMs were cryopreserved using vitrification method. The morphology and viability of thawed HAMs was evaluated by Trypan Blue staining. The expression of several embryonic stem cell (ESC) markers was evaluated using flow cytometry, RT-PCR and immunocytochemistry. Von Kossa, Oil Red O and Alcian Blue staining were used to asses the differentiation potential of thawed HAMs.nnnRESULTSnThe post-thawing viability of HAMs was 84.3 +/- 3.2% (Mean +/- SD, n = 10). The thawed HAMs showed morphological characteristics indistinguishable from the non-vitrified fresh HAMs. The expression of surface antigens (strong positive for CD44, CD49d, CD59, CD90, CD105 and HLA-ABC; weak positive for HLA-G; negative for CD31, CD34, CD45, CD106, CD117 and HLA-DR) and the expression of ESC markers [CK18, fibroblast growth factor-5, GATA-4, neural cell adhesion molecule, Nestin, Oct-4, stem cell factor, HLA-ABC, Vimentin, bone morphogenetic protein (BMP) 4, hepatocyte nuclear factor 4 alpha (HNF-4 alpha), Pax-6, alpha-fetoprotein, Brachyury, BMP-2, TRA-1-60, stage-specific embryonic antigen (SSEA-3, SSEA-4)] were maintained in the vitrified-thawed HAMs. The thawed HAMs retained ability to differentiate into osteoblasts, adipocytes and chondrocytes under appropriate culture conditions.nnnCONCLUSIONSnOur results suggest that vitrification is a reliable and effective method for cryopreservation of HAMs.

Effect of antifreeze protein supplementation in vitrification medium on mouse oocyte developmental competence

OBJECTIVEnTo investigate the effect of antifreeze protein (AFP) supplementation during mouse oocyte vitrification on the survival, fertilization and embryonic development.nnnDESIGNnAnimal study.nnnSETTINGnUniversity laboratory.nnnANIMAL(S)nBDF-1 mice.nnnINTERVENTION(S)nInxa0vivo-matured metaphase II oocytes were vitrified with the use of CryoTop by two-step exposure to equilibrium and vitrification solution supplemented or not with 500 ng/mL AFP III.nnnMAIN OUTCOME MEASURE(S)nPostwarming survival, fertilization, embryonic development up to blastocyst inxa0vitro, morphology of spindle and chromosome, membrane integrity, adenosine triphosphate (ATP) contents, and several gene expressions.nnnRESULT(S)nIn the AFP-treated group, blastocyst formation rate was significantly higher and blastomere count with positive caspase was significantly lower compared with the nontreated group. Rate of intact spindle/chromosome, stable membrane, and ATP contents were significantly higher in AFP group. AFP group showed higher Mad2 and lower Eg5 gene expression. Both vitrification groups showed increased Hsf1, Zar1, and Zp1/Zp2 expression and decreased Hook1 and Zp3 expression compared with fresh control samples.nnnCONCLUSION(S)nSupplementation of AFP in vitrification medium has a protective effect on mouse oocytes for chilling injury; it can preserve spindle/membrane integrity and intracellular ATP contents. More stable spindle integrity in the AFP group may be associated with higher Mad2 and lower Eg5 gene expression.

Optimal condition of vitrification method for cryopreservation of human ovarian cortical tissues

Aim:u2002 In order to find the optimal exposure time of cryoprotectant, we performed a comparison of vitrification versus slow freezing according to the degree of normal morphology and apoptosis of human ovarian follicles.

Age‐related distributions of anti‐Müllerian hormone level and anti‐Müllerian hormone models

Objective. To determine age‐specific reference values for anti‐Müllerian hormone (AMH) and to set up an optimal model for AMH changes by age for infertility investigations. Design. Prospective study. Setting. Several infertility clinics and two university hospitals. Sample. A total of 21 226 AMH samples were obtained. Methods. Data on patients’ age, race/ethnicity, and AMH levels were available from the laboratory center data registry between November 2008 and January 2011. Main outcome measures. The distribution of AMH levels by age. From 16 972 women aged between 25 and 45 years, we established and validated five AMH‐age regression models. Results. The overall mean AMH level was 4.09 ± 3.71 ng/mL (median: 3.13 ng/mL). There was an inverse relation between AMH level and age. Among multiple regression models, the quadratic model was most appropriate to describe AMH‐age relation (log AMH = 0.205 × age – 0.005 × age2– 0.047). Conclusions. AMH levels show a progressive decline with increasing age. Age‐specific AMH values may provide more specific information useful for patients and clinicians. AMH‐age models could play a role as a basic step to approach more accurate ovarian reserve estimation.

Effect of sphingosine-1-phosphate supplementation on follicular integrity of vitrified-warmed mouse ovarian grafts.

OBJECTIVEnTo investigates the effect of sphingosine-1-phosphate (S1P) supplementation on follicular integrity and apoptosis in vitrified-warmed mouse ovarian grafts.nnnSTUDY DESIGNnOvaries from 4-week-aged ICR mice were vitrified using a vitrification solution with or without 2 μM S1P. After warming, follicular normality was assessed by histological analysis and TUNEL assay. A part of ovaries vitrified with or without 2 μM S1P was transplanted, and 2 weeks later, gross and microscopic follicular morphology was assessed.nnnRESULTSnDuring vitrification and warming, inclusion of 2 μM S1P into the vitrification solution significantly raised the rate of morphologically intact follicles compared to controls (36.6% vs. 30.8%, p=0.047). This protective effect was profound especially in primordial follicles (45.5% vs. 34.6%, p=0.034). After transplantation of vitrified-warmed ovaries, the morphological integrity of primordial follicles was superior in the S1P-treated group (55.0% vs. 39.4%, p=0.035). The rates of non-apoptotic follicles (TUNEL-negative) were similar in the two groups in either non-transplanted or transplanted ovaries.nnnCONCLUSIONnInclusion of S1P in the vitrification solution during transplantation of vitrified-warmed ovary had a beneficial effect on preservation of the primordial follicular pool.

Efficacy of the device combining high-frequency transcutaneous electrical nerve stimulation and thermotherapy for relieving primary dysmenorrhea: a randomized, single-blind, placebo-controlled trial

OBJECTIVEnTo investigate the efficacy and safety of the combined therapy with high-frequency transcutaneous electrical nerve stimulation (hf-TENS) and thermotherapy in relieving primary dysmenorrheal pain.nnnSTUDY DESIGNnIn this randomized, single-blind, placebo-controlled study, 115 women with moderate or severe primary dysmenorrhea were assigned to the study or control group at a ratio of 1:1. Subjects in the study group used an integrated hf-TENS/thermotherapy device, whereas control subjects used a sham device. A visual analog scale was used to measure pain intensity. Variables related to pain relief, including reduction rate of dysmenorrheal score, were compared between the groups.nnnRESULTSnThe dysmenorrheal score was significantly reduced in the study group compared to the control group following the use of the devices. The duration of pain relief was significantly increased in the study group compared to the control group. There were no differences between the groups in the brief pain inventory scores, numbers of ibuprofen tablets taken orally, and World Health Organization quality of life-BREF scores. No adverse events were observed related to the use of the study device.nnnCONCLUSIONSnThe combination of hf-TENS and thermotherapy was effective in relieving acute pain in women with moderate or severe primary dysmenorrhea.

Effect of intravenous ascorbic acid infusion on blood loss during laparoscopic myomectomy: a randomized, double-blind, placebo-controlled trial

OBJECTIVEnMost interventions aimed at reducing bleeding during myomectomy lack sufficient evidence regarding their effectiveness. Recently, it was reported that intraoperative ascorbic acid administration effectively reduced blood loss during abdominal myomectomy. Therefore, this study aimed to investigate whether intravenous ascorbic acid infusion would affect intraoperative blood loss in women undergoing laparoscopic myomectomy.nnnSTUDY DESIGNnA randomized, double-blind, parallel-group, placebo-controlled trial including 50 women undergoing laparoscopic myomectomy was conducted. Women with ≤4 myomas, ≤9cm in maximum diameter were eligible. The study:control group ratio was 1:1. Starting 30minutes before anesthesia, 2g of ascorbic acid or a placebo were administered for 2hours intraoperatively. Intraoperative blood loss, the primary endpoint, was calculated as the difference between the volume of fluids acquired from suction and that used for irrigation of the abdominal cavity during surgery using constant values.nnnRESULTSnAmong the 50 randomized women, 1 and 3 in the study and control groups, respectively, were excluded due to withdrawal of consent, cancelation of surgery, or non-measurement of the primary endpoint. The baseline and operative characteristics were similar between the study and control groups, as was the intraoperative blood loss (193±204mL vs. 159±193mL, P=0.52). In addition, the operating time (95±29min vs. 110±52min; P=0.50) and decrease in hemoglobin level after surgery (1.9±1.31g/dL vs. 1.4±1.4g/dL; P=0.24) were similar between the study and control groups.nnnCONCLUSIONSnIntravenous ascorbic acid infusion did not reduce intraoperative blood loss in women undergoing laparoscopic myomectomy.nnnCLINICAL TRIAL REGISTRATIONnClinicalTrials.gov, www.clinicaltrials.gov, NCT01715597.

Effect of trichostatin A on fertilization and embryo development during extended culture of mouse oocyte.

We performed this study to investigate the effect of histone deacetylase inhibition during extended culture of in vitro matured mouse oocytes. In vitro matured mouse (BDF1) oocytes were cultured in vitro for 6, 12, and 24 h, respectively, and then inseminated. During in vitro culture for 6 and 12 h, two doses of trichostatin A (TSA), a histone deacetylase inhibitor, were added (100 nM and 500 nM) to the culture medium and the oocytes were then inseminated. During the 24-h in vitro culture, two doses of TSA were added (100 nM and 500 nM) to the medium and the oocytes were activated with 10 mM SrCl2. After the 6-h culture, the fertilization rate was similar to that of the control group, but the blastocyst formation rate was significantly decreased. After the 12-h culture, both the fertilization and blastocyst formation rates were significantly decreased. After the 24-h culture, total fertilization failure occurred. In the oocytes cultured for 6 and 12 h, the fertilization and blastocyst formation rates did not differ between the TSA-supplemented and control groups. Although extended culture of the mouse oocytes significantly affected their fertilization and embryo development, TSA supplementation did not overcome their decreased developmental potential.

Chapter 4 Role of Antioxidants and Antifreeze Proteins in Cryopreservation/Vitrification

In recent years, supplementation of antioxidants and antifreeze proteins during cryopreservation/vitrification has significantly improved the survival and function of oocytes and ovarian tissues (OT) in animal models. In this chapter, the experimental protocols for the use of antioxidants and antifreeze proteins in cryopreservation/vitrification are described.

Chapter 2 Utility of Animal Models for Human Ovarian Tissue Cryopreservation

Success in cryopreservation of ovarian tissue (OT) in animal models has led to develop efficient cryo-technologies for human ovarian tissue. In this chapter, cryopreservation protocols developed for animal experiments are described.