• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.119-124
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• 2001

This study was performed to investigate the effects of the peptide to carrier ratio on the immune and biological functions to inhibin immunization in Hanwoo. A peptide sequence kom the alpha -subunit (19~32 peptide) of porcine inhibin was synthesized for antigen and conjugated to human serum albumin(HSA) for carrier protein. Anti-inhibin sera(AI) were produced 52 day later from rabbit after injection of inhibin-$\alpha$ -subunit peptide conjugator for antigen with the interval of 2 weeks. Immune-blotting analysis using antibody specific fur inhibin-$\alpha$ subunits revealed that the inhibin was detected at 1.0 cm bovine follicular fluid(bFF). However, each stage of corpus lutea and 0.1 cm of follicular fluid were not detected. The maximal contents of estradiol-17 $\beta$ in Hanwoo ovarian follicular fluid were detected at 2.0 cm of follicular size(diameter), but the mean total contents of these hormone decreased significantly with decreasing diameter of follicles. However, progesterone contents of follicular fluid were high at 1.0 cm of follicle. Progesterone secretion by Hanwoo granulosa cell cultured for 48 hr in vitro was significantly (p＜0.05) inhibited in 5% bFF and 5% bFF + 5% AI addition group compared with control group. Estradiol-17 $\beta$ secretion by Hanwoo granulosa cell cultured for 48 hr in vitro was significantly (p＜0.05) increased in 5% AI and 5% AI + 5% bFF addtion group compared with control group. However, the groups added 5% AI were not changed compared to control groups in progesterone and estradiol-17 $\beta$. Taken together, we suggested that inhibin in the mature FF plays a pivotal role on the biosynthesis of steroid hormone of follicular cells during follicular development.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.71-81
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• 1994

These experiments were carried out to investigate the effects of human follicular fluid (hFF) as a protein supplement on development of mammalian embryo as well as to find out ways toward effective use of hFF. The developmental rates of mouse embryos to the blastocyst and implantation stages were significantly higher in T6 ＋hFF than T6＋hFCS. Classified hFF according to the maturity of contained oocytes (M-hFF and Im-hFF), and compared the rates of development of mouse embryo cultured in M-hFF or Im-hFF to culture medium T6. Total protein, albumin and estradiol concentrations were higher in M-hFF than Im-hFF (P<0.05). The developmental rates of mouse embryos to the blastocyst and hatching blastocyst stages cultured in Im-hFF were significantly lower than those in M-hFF and the basic medium. In accordance of the results of human IVF, hFF has been divided into 4 groups. The developmental rates of mouse embryos to the blastocyst stage in presense of hFF from pregnant patients, who have good grade embryos, were significantly higher than those in hFF from patients who have poor grade embryos or were not pregnant. In addition, the rates of development of human embryo were compared in presense of BSA, hFF or hFCS. The developmental rates of human embryos cultured in Ham's F10＋hFF were significantly higher than those in the Ham's F10＋BSA. These results suggests that the culture system using hFF could improve the development ability of mammalian embryos and the viability of blastocysts cultured in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.4
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• pp.210-215
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• 2011

Objective: This study was performed to identify whether growth and differentiation factor-9 (GDF-9) and transforming growth factor-${\beta}1$ (TGF-${\beta}1$) expressions would be lower in the follicular fluid (FF) of those over age 35 who underwent IVF than under age 35. Methods: A total of 24 IVF cycles (20 patients) were included in this study. All of patients were stimulated for IVF by the GnRH short protocol and divided into two groups for analysis, according to their age: <35 group (14 cycles, 11 patients) vs. ${\geq}35$ group (10 cycles, 9 patients). The expression levels of GDF-9 and TGF-${\beta}1$ were determined by western blotting and quantitative enzyme-linked immunosorbent assay. Results: The numbers of retrieved oocytes and metaphase II oocytes were significantly lower in the ${\geq}35$ group. Lower expression of GDF-9 and TGF-${\beta}1$ by western blotting in the ${\geq}35$ group were observed as well. The mean GDF-9 and TGF-${\beta}1$ levels by enzyme-linked immunosorbent assay were lower in the ${\geq}35$ group. The values were $6,850.5{\pm}928.4$ ng/L vs. $3,333.3{\pm}1,089.2$ ng/L of GDF-9 ($p$ <0.05) and $3,844.1{\pm}571.1$ ng/L vs. $2,187.7{\pm}754.0$ ng/L of TGF-${\beta}1$ ($p$ <0.05). A negative correlation between GDF-9 and age was observed (r=-0.546, $p$=0.006). Conclusion: GDF-9 and TGF-${\beta}1$ production from stimulated ovaries during IVF appears to decrease with age.

• Clinical and Experimental Reproductive Medicine
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• v.38 no.2
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• pp.98-102
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• 2011

Objective: To investigate the effects of pioglitazone on controlled ovarian stimulation (COS), IVF outcomes, and follicular fluid (FF) cytokine concentrations in patients with polycystic ovary syndrome (PCOS). Methods: Eighty-six infertile patients with PCOS resistant to clomiphene citrate were randomized to receive pioglitazone (30 mg/day) or placebo on the starting day of oral contraceptive (OC) pretreatment, followed by an IVF protocol using a GnRH antagonist. Pioglitazone or placebo was administered once daily from the starting day of OC to the day of hCG injection. Results: Total dose and days of recombinant follicle-stimulating hormone administered, and the numbers of retrieved and mature oocytes, were significantly lower in the pioglitazone group than in the control group. FF tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$) and interleukin-6 (IL-6) concentrations at oocyte retrieval were also significantly lower in the pioglitazone group. The clinical pregnancy rate was higher and the incidence of severe ovarian hyperstimulation syndrome was lower in the pioglitazone group, but the differences were not statistically significant. Conclusion: Pioglitazone reduces FF TNF-${\alpha}$ and IL-6 levels, and may improve ovarian response to COS in patients with PCOS.

• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.273-280
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• 2011

It was conducted the experiment, divided into three groups as normal, poor and polycystic ovary syndrome, to detect the change of protein patterns in follicular fluid on ovarian response following controlled ovarian hyperstimulation for human IVF outcome. In the normal group, it was confirmed reproducible 57 spots in the detected total 81 spots. Then 1 spot was not found in the other groups. In the poor responder group, it was found reproducible 53 spots in the detected total 98 spots. 6 spots were down-regulation and 7 spots were up-regulation comparable with normal group. There were not 5 spots in poor responder group comparable with other groups. In the polycystic ovary syndrome group, it was expressed reproducible 53 spots in the detected total 80 spots and 3 spots were just expressed in this group. However, 4 spots were not found in polycystic ovary syndrome. 9 spots were up-regulation comparable with normal group. Significant up and down-regulation spots among the each groups were identified. The results were a cytosolic carboxypeptidase, a signal-induced proliferation-associated protein 1, a ceruloplasmin, a keratin(type II cytoskeletal 1), a polypeptide N-acetylgalactosantinyltransferase 2, a serine/threonine-protein phosphatase 4 regulatory subunit 4. It was identified that 8 spots, 6 kinds of protein are corresponded with NCBInr database research, but 10 spots were failed in the identification. In conclusion, it has been confirmed change and expression of protein on the ovarian response following COH of human.

• 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.

• Development and Reproduction
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• v.7 no.2
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• pp.105-112
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• 2003

Previously, we discovered a new MMP-2 isoform GA110, of which appearance in human follicular fluid(FF) and serum was increased by EDTA. The present study was conducted to investigate how GAI 10 can appear by EDTA. To examine possible involvement of protein disulfide isomerase(PDI), an enzyme responsible for the dimerization of protein via disulfide formation, effect of PDI inhibitor on the appearance of GA110 by EDTA was investigated. When PDI inhibitor added to FF before EDTA treatment, the gelatinolytic activity of GA110 was abolished in a concentration dependent manner. By contrast, the activity of 72 kDa gelatinase increased. However, the PDI inhibitor added to FF after EDTA treatment, the gelatinolytic activity of GA110 was unaffected. To find out the nature of the enzyme which converts 72 kDa gelatinase into GAI 10, chromatographic separation method of FF proteins was done. Using hydroxyapatite column, fractions rich in 72 kDa gelatinase were isolated and pooled. By using this pool as substrate for the 72 kDa converting enzyme, protein fractions containing the converting activity were obtained from chromatographic separation of FF onto glutathione sepharose fast flow column. When immunoblotting was performed on this enzymatically active protein fractions against polyclonal anti-PDI antibody, distinct immunoreactivity was observed, although appeared in smaller molecular weight region. Based on these observations, it is suggested that the appearance of GAI 10 in FF by EDTA treatment could be due to an activation of PDI-like enzyme, which dimerizes 72 kDa gelatinase into GAI 10 via the formation of disulfide bond between molecules.

• 대한생식의학회:학술대회논문집
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• 2002.11a
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• pp.81.1-81.1
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• 2002

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.241-245
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• 1994

Despite increasing success rate of IVF, poor response to ovarian stimulation remains a problem. So, attempts to improve ovarian responses, for example, by using combined gonadotropin-releasing hormone analogue(GnRH-a) and human menopausal gonadotropin(hMG) have shown limited success. It is reported that response of granulosa cells in vitro to FSH is stimulated by co-incubation with IGF-l, and IGF-l production can be increased by growth hormone. This suggest that combination regimen of G.H. and hMG may augment follicle recruitment. In fifteen patients who had previous history of poor ovarian response to gonadotropin stimulation after pituitary suppression with mid -luteal GnRH-a, the effectiveness of cotreatment with G.H. in IVF program was evaluated using a combination regimen of G.R. and hMG at Korea University Hospital IVF Clinic. Ovarian responses to gonadotropin stimulation in control and GH-treated cycles assessed by total dose and duration of hMG treatment, follicular development and peak $E_2$ level, number of eggs retrieved, and fertilization rates were also assessed. In each group, serum and follicular fluid IGF-1 concentrations on day of egg collection were measured by RIA after acidification and extraction by reveresed phase chromatography. Patients receiving G.H. required fewer days and ampules of gonadotropins, developed more oocytes, and more embryos transferred. But, the differences were not statistically significant, except the duration of hMG treatment. Our data showed a significantly higher concentration of IGF-l in the serum, not in the follicular fluid, of patients treated with G.H. compared with control group. These data suggest that growth hormone treatment does not improve the ovarian response in women with limited ovarian reserve to gonadotropin stimulation for IVF.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.3
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• pp.126-131
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• 2017

Objective: Oocyte degeneration often occurs after intracytoplasmic sperm injection (ICSI), and the risk factor is low-quality oocytes. The follicular fluid (FF) provides a crucial microenvironment for oocyte development. We investigated the relationships between the FF volume aspirated from individual follicles and oocyte retrieval, oocyte maturity, oolemma stretchability, fertilization, and development. Methods: This retrospective study included data obtained from 229 ICSI cycles. Ovarian stimulation was performed according to a gonadotropin-releasing hormone antagonist protocol. Each follicle was individually aspirated and divided into six groups according to FF volume ( < 1.0, 1.0 to < 2.0, 2.0 to < 3.0, 3.0 to < 4.0, 4.0 to < 5.0, and ${\geq}5.0mL$). Oolemma stretchability during ICSI was evaluated using a mechanical stimulus for oolemma penetration, that is, the stretchability was assessed by oolemma penetration with aspiration (high stretchability) or without aspiration (low stretchability). Results: Oocyte retrieval rates were significantly lower in the < 1.0 mL group than in the ${\geq}1.0mL$ groups (46.0% [86/187] vs. 67.5%-74.3% [172/255 to 124/167], respectively; p< 0.01). Low oolemma stretchability was significantly more common in the < 1.0 mL group than in the ${\geq}1.0mL$ groups during ICSI (22.0% [13/59] vs. 5.8%-9.4% [6/104 to 13/139], respectively; p= 0.018). There was a relationship between FF volume and oolemma stretchability. However, there were no significant differences in the rates of fertilization, cleavage, ${\geq}7$ cells at day 3, and blastocyst development among all groups. Conclusion: FF volume is potentially associated with the stretchability of metaphase II oolemma during ICSI. Regarding oolemma stretchability, ensuring a uniform follicular size during ovarian stimulation is crucial to obtain good-quality oocytes.