• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.53-60
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• 2006

Objective: We tested the usefulness of swim-down technique using human follicular fluid (hFF) in sperm preparation. Methods: Semen samples were obtained from twelve male partners showing asthenozoospermia (sperm motility < 50%) at the time of routine andrologic evaluation in Seoul National University Bundang Hospital. After dividing into two aliquots, each samples were processed either by swim-down using 100% hFF or density gradient using SpermGrad. Sperm quality was assessed by computer-assisted semen analyzer (CASA). Results: Motility, Rapid motility, VCL (curvilinear velocity), ALH (amplitude of lateral head displacement), and hyperactivated sperms were significantly increased, and LIN (mean linearity) was decreased significantly after sperm preparation in both groups. Motility was significantly higher after swim-down using 100% hFF when compared with density gradient using SpermGrad ($81.2{\pm}4.7$ vs. $67.6{\pm}2.3$, p=0.02) The other parameters assessed by CASA were not different between the two methods. Conclusion: Swim-down method with 100% hFF may be a useful method in preparation of sperm from asthenozoospermia.

• 대한생식의학회:학술대회논문집
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• 1995.12a
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• pp.69-70
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• 1995

• Clinical and Experimental Reproductive Medicine
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• v.27 no.2
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• pp.151-157
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• 2000

Objective: The purpose of this study was to evaluate the effect of polycystic ovarian follicular fluid on sperm motility in human in vitro fertilization (IVF). Methods: From May, 1998 to July, 1999, 55 patients who complained of infertility were involved in this study. We obtained ovarian follicular fluids from the patients by ultrasono-guided aspiration. Subjects were divided into two groups. 20 patients who had polycystic ovarian disease were belong to study group, and 25 patients who had normal ovarian follicular fluid were belong to control group. The follicular fluid dilution was done with Ham's fluid as 10%, 20%, 50%, 100%. The sperm motility was analyzed by CASA at 6hr and 12hr after incubation in follicular fluids. Results: The levels of average path velocity (VAP) in all concentration fluid didn't show significant difference between study and control group. The other parameters including curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and linerity (LIN) were didn't show any significant difference between both groups. Conclusion: PCOD fluid had seemed to have an adverse effect on the sperm biological function. But, this study showed that PCOD fluid had no different effect on sperm motility with normal follicular fluid.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.145-150
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• 2009

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) ${\sim}4$ h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to $6{\sim}8$ cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.87-94
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• 1996

For evaluating the suitability of human follicular fluid(HFF) as protein supplement in ART, this preliminary study was performed to examine the maturation promoting activity of HFF on mouse follicular oocytes in vitro. Mouse follicular oocytes were collected from ovaries of 21-28 day old ICR mice by puncturing the antral follicles with fine needle at 48 hours after PMSG injection. The oocytes were rinsed and cultured in modified Whittingham's $T_6$ medium containing purines or dbcAMP to maintain meiotic arrest, and different concentrations of HFF were added into the culture medium to examine the effect of HFF on releasing the oocytes from the suppressive influence of the meiotic inhibitors. As a control for HFF, the maturation promoting activity of human fetal cord serum(HFCS) was investigated and compared with the activity of HFF. While HFF was separated, by molecular weight(M.W), into high M.W. fraction(M.W>30,000) and low M.W. fraction(M.W<30,000) and the effects of the fractions on meiotic resumption were investigated in the presence of the meiotic inhibitors. Also hormone analysis was performed to compare the content of hormones in HFF with that in HFCS. Same concentrations of HFF and HFCS induced similar germinal vesicle break down(GVBD) rates of the oocytes meiotic arrested by purines(4mM hypoxanthine+0.75mM adenosine), but the extrusion rate of 1st polar body(PB) of the oocytes cultured in HFF(65.0%, P<0.05) was significantly higher than that(51.6%) in HFCS. While, in the presence of 200 M dbcAMP, the maturation promoting activity of HFF (GVBD: 70.5%, $p<10^{-6}$; 1st PB extrusion: 67.1%, $p<10^{-3}$) was significantly greater than that of HFCS(GVBD: 35.2%; 1st PB extrusion: 41.1%). The oocytes cultured in the fraction of HFF containing high M.W. components showed higher meiotic maturation rates than the oocytes cultured in the low M.W. fraction of HFF. Gonadotropins and $E_2$ were known to improve the completion of maturation changes, and the levels of these hormones were higher in HFF than in HFCS. Therefore, HFF was more effective than HFCS to use for promoting meiotic resumption of mouse oocytes in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.95-102
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• 1996

This study was performed to investigate the effect of human follicular fluid (HFF) on development of mouse embryos, for evaluating the suitability of HFF as a substitutive material of human fetal cord serum in ART program. The various concentrations of HFF were added into the culture medium and the effects of HFF concentrations were examined to identify the optimal concentration of HFF for embryo development. The potency of HFF in improving embryo development was compared to that of other protein supplement. Collected HFFs were classified with the maturity of the containing oocytes; mature, immature, atretic, and then the effects of the classified HFFs on embryo development were examined. Also, HFF was separated into the low (<30,000 Da) and high (>30,000 Da) molecular weight fractions and the effects of the fractions on embryo development were investigated. The highest development rate was found in culture medium supplemented with 20% HFF, bnt this rate was reversely reduced at the concentrations of HFF higher than 20%. The development rates to the blastocyst, hatching blastocyst, attachment and outgrowth cultured in mature HFF was significantly higher than those in immature and atretic HFF, and mean cell number in blastocyst was higher in mature HFF than in immature and atretic HFF. The development rates of mouse embryos according to protein sources were significantly higher in HFF than in fetal cord serum (FCS), maternal serum (MS) and bovine serum albumin (BSA), and mean cell number in blastocyst cultured in HFF was higher than that in FCS, MS and BSA. The development rates of embryo and mean cell number in blastocyst cultured in high molecular weight fraction of HFF were higher than those in low molecular weight fraction, but the results of high molecular weight fraction were lower than those of whole HFF. Therefore, these results indicated that human mature follicular fluid was useful for improving the development of mouse embryos, which suggests a possibility that HFF also may be used efficiently for improving the culture condition in human ART program as a protein supplement.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.103-108
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• 1996

Through the previous studies(I,II), it was observed that human follicular fluid(HFF) was more effective than human fetal cord serum(HFCS) on promoting meiotic resumption of oocytes and improving embryonic development of mouse in vitro. On the basis of these results, we have gradually exchanged HFCS with HFF as protein supplement in human ART. This study was performed to investigate the efficiency of HFF on improving the pregnancy rate in ART. Oocytes were retrieved transvaginally from patients treated with pituitary suppression with GnRH-agonist and ovarian stimulation with human menopausal gonadotro-pin(HMG) and pure follicle stimulating hormone(FSH). Aspirated oocytes were rinsed and cultured in TCM-199 containing HFF, and the concentrations of HFF were adjusted to 10, 20, and 30% according to the use for insemination, embryo growth and embryo transfer, respectively. As possible as, we tried to do embryo transfer into fallopian tube to mimic the coincidence of the cell stage with the place of sojourn in vivo, so we performed various ART programs(IVF & ET; in vitro fertilization, ZIFT; zygote intra fallopian-tube transfer, ZIFT & ET) according to the tubal conditions of patients. On the while, intra cytoplasmic sperm injection(ICSI) was used to assist IVF of the patients who had shown poor standard IVF results by immunological or severe male factor. Of the 255 cycles of ART programs using HFF as protein supplement, 118 cycles were turn out to be succeeded in pregnancy(46.2%, per cycle, p<0.05), while 21 pregnancies were achieved in the 69 cycles using HFCS(30.4%). The 255 cycles using HFF were subdivided into cycles with the type of ART programs, and each pregnancy rate of the ART programs were 44.7% (IVF & ET, 76/170 cycles), 53.4%(ZIFT, 31/58 cycles) and 40.7% (ZIFT & ET, 11/27 cycles), respectively. In the 61 ICSI cycles using HFF, 28 cycles succeed in pregnancy(45.9%), while 7 pregnancies were obtained in the 17 ICSI cycles using HFCS. Also the ongoing pregnancy rate in the group using HFF(78.8%, 93/118 cycles) was higher than that in the group using HFCS(61.9%). Therefore, we found that the use of HFF as protein supplement was more suitable and effective than the use of HFCS to improve the pregnancy rate in ART.

• Development and Reproduction
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• v.15 no.4
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• pp.385-391
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• 2011

Human in vitro fertilization and embryo transfer (IVF-ET) program is a general procedure for infertile couples since first successful delivery on 1978 in UK and Korean first on 1985. Recently in Korea, more than 42,000 cases per year of IVF-ET were performed and showed good pregnancy rates compared worldwide data. The human IVF-ET procedure use many consumables, such as ovum pick-up (OPU) needles, centrifuge tubes, culture dish, ICSI pipette, culture media and ET catheters. Major of these materials are supported by the global companies. Thus, Korean IVF-ET program might be placed unstable situation by global economical risks. These uncertain problems could be overcome by the domestic production of IVF-ET materials and consumables. Two times questionnaires for Korean clinicians and researchers about the domestic production were performed and analyzed. Many of them requested domestic OPU needles, ET catheters, culture media and ICSI pipettes under good quality control and quality assurance system. This trial may be contributed to industrialization and to global competence of Korean IVF-ET program. The results of this survey can be provide a fundamental base for development and production of domestic materials and consumables for human IVF-ET program.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.11-18
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• 2004

The aim of this study was to evaluate the improvement of testicular sperm motility following different culture conditions such as human follicular fluid (hFF) and temperature. Testicular tissues obtained from azoospermia (n=21) were minced into small pieces by blade and recovered sperm suspension were cultured in Ham's F10 with or without 40% hFF at different temperatures (Group I: 37$^{\circ}C$/with hFF, Group II: 32$^{\circ}C$/withGroup III: 37$^{\circ}C$/without, Group IV:32$^{\circ}C$ /without The motility and viability of sperm were monitored during culture for 48 hours. Initial motility of testicular sperm was 10.9$\pm$1.9%. After 24 hours culture, sperm motility was 23.5$\pm$2.1% (Group I), 8.1$\pm$1.1% (Group II), 10.4$\pm$ 1.4% (Group III) and 4.0$\pm$0.8% (Group IV), respectively. After 48 hours, the motility had been changed as 32$\pm$2.3% (Group I), 14.3$\pm$1.7% (Group II), 5.3 $\pm$1.4% (Group III) and 4.3$\pm$0.9% (Group IV). In hFF group (I and II), sperm motility of group I cultured at 37$^{\circ}C$ was higher than those of group II at 32$^{\circ}C$. But, sperm viability of group I cultured at 37$^{\circ}C$ was lower than those of group II at 32$^{\circ}C$ (54.4$\pm$4.1% vs. 59.4$\pm$3.7%) after cultured for 48 hours. We acquired the best motility of testicular sperm when performed in vitro culture for 48 hours in hFF supplemented medium at 37$^{\circ}C$. Increase of sperm motility by in vitro culture could be useful tool fur human TESE-ICSI program.