• Clinical and Experimental Reproductive Medicine
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• 제30권1호
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• pp.47-55
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• 2003

Objectives: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. Methods: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at $21{\sim}23$ days of age. Ovaries were collected $1{\sim}3$ days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for $1{\sim}2$ days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 IU PMSG. Some mice received a single intraperitoneal injection of 10 IU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. Results: Treatment of immature mice with diethylstilbestrol (DES) for $24{\sim}48$ h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for $24{\sim}48$ h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. Interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at $6{\sim}9$ h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. Conclusion: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.

• Clinical and Experimental Reproductive Medicine
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• 제31권4호
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• pp.217-223
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• 2004

Objective: To evaluate the effects of recombinant FSH (rFSH) and urinary FSH (uFSH) on the gene expressions of human endometrial stromal cells in vitro. Methods: Endometrial tissue was obtained from a pre-menopausal women undergoing hysterectomy. Primary endometrial stromal cells were isolated and in vitro cultured with FBS-free DMEM/F-12 containing 0, 10, 100, and 1, 000 mIU/ml of rFSH and uFSH for 48 hours, respectively. Total RNA was extracted from the cultured cells and subjected to real time RT-PCR for the quantitative analysis of progesterone receptor (PR), estrogen receptor $\alpha/\beta$ (ER-$\alpha/\beta$), cyclooxygenase 2 (Cox-2), leukemia inhibitory factor (LIF), homeobox A10-1 and -2 (HoxA10-1/-2). Results: Both hormone treatments slightly increased (< 3 folds) the expressions of PR, ER-$\beta$ and HoxA10-1/-2 gene. However, ER-$\alpha$ expression was increased up to five folds by treatments of both FSH for 48 hours. The LIF expression by the 10 mIU/ml of uFSH for 12 hours was significantly higher than that of rFSH (p<0.01). After 24 hours treatment of two kinds of hormones, the expression patterns of LIF were similar. The 100 and 1, 000 mIU/ml of rFSH induced significantly higher amount of Cox-2 expression than those of uFSH, respectively (p<0.05). Conclusion: This study represents no adversely effect of exogeneous gonadotropins, rFSH and uFSH, on the expression of implantation related genes. We suggest that rFSH is applicable for the assisted reproductive technology without any concern on the endometrial receptivity.

• 한국발생생물학회지:발생과생식
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• 제16권4호
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• pp.295-300
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• 2012

Manganese ($Mn^{2+}$) is a trace element that is essential for normal physiology, and is predominantly obtained from food. Several lines of evidence, however, demonstrated that overexposure to $MnCl_2$ exerts serious neurotoxicity, immunotoxicity and developmental toxicity, particularly in male. The present study aimed to evaluate the effect of 0, 1.0, 3.3, and 10 mg/kg/day doses of $MnCl_2$ on the reproductive organs in the immature female rats. Rats (PND 22; S.D. strain) were exposed to $MnCl_2$ ($MnCl_2{\cdot}4H_2O$) dissolved in drinking water for 2 weeks. The animals were sacrificed on PND 35, then the tissues were immediately removed and weighed. Histological studies were performed using the uteri tissue samples. Serum LH and FSH levels were measured with the specific ELISA kits. Body weights of the experimental group animals were not significantly different from those of control group animals. However, ovarian tissue weights in 1 mg and 3.3 mg $MnCl_2$ dose groups were significantly lower than those of control animals (p<0.05 and p<0.01, respectively). Uterine tissue weights of 3.3 mg dose $MnCl_2$ groups were significantly lower than those of control animals (p<0.01), while the 1 mg $MnCl_2$ dose and 10 mg $MnCl_2$ dose failed to induce any change in uterine weight. Similarly, only 3.3 mg $MnCl_2$ dose could induce the significant decrease in the oviduct weight compared to the control group (p<0.05). Non-reproductive tissues such as adrenal and kidney failed to respond to all doses of $MnCl_2$ exposure. The uterine histology revealed that the $MnCl_2$ exposure could affect the myometrial cell proliferation particularly in 3.3 mg dose and 10mg dose group. Serum FSH levels were significantly decreased in 1mg $MnCl_2$ dose and 10 $MnCl_2$ mg groups (p<0.05 and p<0.01, respectively). In contrast, treatment with 1 mg $MnCl_2$ dose induced a significant increment of serum LH level (p<0.05). The present study demonstrated that $MnCl_2$ exposure is capable of inducing abnormal development of reproductive tissues, at least to some extent, and altered gonadotropin secretions in immature female rats. Combined with the well-defined actions of this metal on GnRH and prolactin secretion, one can suggest the $Mn^{2+}$ might be a potential environmental mediator which is involved in the female pubertal process.

• 한국발생생물학회지:발생과생식
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• 제15권3호
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• pp.265-272
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• 2011

It is well known that adipose tissue or body fat has been proved as a crucial component of brain-peripheral axis which can modulate the activities of reproductive hormonal axis in female mammals including rodents and human. Concerning the male reproduction, however, the role of adipose tissue has not been thoroughly studied. The present study was carried out to elucidate the effect of a high-fat (HF) diet on the reproductive system of postpubertal male rats. The HF diet (45% energy from fat, HF group) was applied to male rats from week 8 after birth for 4 weeks. The blood glucose levels, body and tissue weights were measured. Histological studies were performed to assess the structural alterations in the reproductive tissues. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus and pituitary, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Body weights (p<0.01) and blood glucose levels (p<0.01) of HF group were significantly higher than those of control animals. Similarly, the weights of epididymis (p<0.05), prostate (p<0.01), seminal vesicle (p<0.01) in HF group were higher than control levels. The weights of testis were not changed. The weights of kidney (p<0.001) and spleen (p<0.01) were significantly higher than control levels while the adrenal and pancreas weights were not changed. There were only slight alterations in the microstructures of accessory sex organs; the shape of luminal epithelial cells in epididymis from HF group were relatively thicker and bigger than those from control animals. In the semi-quantitative RT-PCR studies, the mRNA levels of hypothalamic GnRH (p<0.05) in HF group were significantly higher than those from the control animals. The mRNA levels of kisspeptin in HF group tend to be higher than control levels, the difference was not significant. Unlike the hypothalamic GnRH expression, the mRNA levels of pituitary $LH{\beta}$ and $FSH{\beta}$ were significantly decreased in HF group (p<0.05). The present study indicated that the 4-weeks feeding HF diet during the postpubertal period can alter the hypothalamus-pituitary (H-P) neuroendocrine reproductive system These results suggest that the increased body fat and the altered leptin input might disturb the H-P reproductive hormonal activities in male rats, and the changed activities seem to be responsible for the changes of tissue weights in accessory sex organs.

• Clinical and Experimental Reproductive Medicine
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• 제28권2호
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• pp.79-86
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• 2001

Objective: Mouse pre-antral follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and/or phygiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Methods: Pre-antral follicles ($91{\sim}120{\mu}m$) were isolated mechanically by fine 30G needles not using enzymes from ovaries of 3-6 week-old female ICR mice. Isolated pre-antral follicles were cultured in $20{\mu}l$ droplets of TCM (n=17; follicles: $107.8{\pm}1.58{\mu}m$; oocytes: $57.9{\pm}1.2{\mu}m$) or MEM (n=12; follicles: $109.3{\pm}2.53{\mu}m$; oocytes: $55.4{\pm}1.6{\mu}m$) under mineral oil on the 60 mm culture dish. All experimental media was supplemented with 10% FBS without Gns and/or physiological factors. Pre antral follicles were individually cultured for 8 days. Antram formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using precalibrated ocular micrometer at X200 magnifications during in vitro culture. Results were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when p<0.05. Results: Antrum formation had started in two culture media on day 2. On day 8, antrum formation had occurred in 58.3% of pre-antral follicles cultured in DMEM, but only in 23.5% of those cultured in TCM (p=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day 4 and 8. On day 4, follicular diameter was similar (p=0.1338) in TCM ($119.4{\pm}2.58{\mu}m$) and MEM ($125.4{\pm}4.52{\mu}m$). However, on day 8, diameters of pre-antral follicles cultured in MEM ($168.9{\pm}17.29{\mu}m$) were significantly bigger (p=0.0248) than that in TCM ($126.7{\pm}4.28{\mu}m$). On day 4 and 8, diameters of intra-follicular oocytes were similar in TCM ($67.1{\pm}1.3$ and $72.4{\pm}0.9{\mu}m$) and MEM ($65.2{\pm}1.7$ and $73.3{\pm}1.5{\mu}m$), respectively. Conclusion: We can conform that medium without Gns and/or physiological factors can be used for in vitro antrum formation and growth of pre-antral follicles and intra-follicular oocytes in mouse. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Clinical and Experimental Reproductive Medicine
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• 제37권2호
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• pp.153-158
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• 2010

목 적: 과배란유도 후 자궁강내 인공수정 시술시 황체기 보강으로서 경구 미분화 프로게스테론 투여법과 경구 디드로게스테론 투여법의 임상적 효용성을 비교하고자 하였다. 연구방법: 2007년 1월부터 2009년 8월까지 시행된 과배란유도 후 자궁강내 인공수정 시술 183주기를 후향적으로 분석하였다. 과배란유도는 성선자극호르몬 단독 또는 클로미펜과 성선자극호르몬의 병합요법을 사용하였다. 136주기에서는 황체기 보강으로서 경구 미분화 프로게스테론을 하루 300 mg으로 투여하였고 47주기에서는 디드로게스테론을 일일 20 mg으로 투여하였다. 결 과: 여성의 연령, 불임 인자, 성숙난포수 ($\geq$16 mm), 총운동성정자수, triggering 날의 자궁내막 두께는 두 군간 유의한 차이가 없었다. 자궁내 태낭이 확인되는 임상적 임신율은 미분화 프로게스테론 투여군에서 21.3%, 디드로게 스테론 투여군에서 19.1%로 차이가 없었다 (p=0.92). 유산율은 미분화 프로게스테론 투여군에서 다소 높은 경향을 보였으나 통계학적인 차이는 없었다 (34.5% vs. 11.1%, p=0.36). 결 론: 황체기 보강으로서 경구 디드로게스테론 투여법은 경구 미분화 프로게스테론 투여에 비하여 비슷한 임신율과 유산율을 보였다. 그러나 상대적으로 디드로게스테론 투여군의 수가 적어 좀더 많은 환자를 대상으로 한 전향적 연구가 필요하다.

• Clinical and Experimental Reproductive Medicine
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• 제28권4호
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• pp.307-315
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• 2001

Objective: To evaluate the clinical outcomes and influencing factors of in vitro fertilization and embryo transfer (IVF-ET) in patients with failed pregnancy after microsurgical reversal of tubal sterilization. Materials and Methods : From January, 1997 to December, 2000, IVF-ET was performed in two groups; the study TR (tubal reanastomosis) group consisted of 147 cycles in 66 patients with failed microsurgical reversal of tubal sterilization, and the control group of 115 cycles in 67 patients with bilateral tubal occlusion (BTO). The two groups were evaluated and compared for clinical characteristics, clinical pregnancy rates, and factors influencing the outcomes of IVF-ET. Results: Compared with the control BTO group, age and the previous parity were significantly higher ($36.3{\pm}2.7$ vs. $33.6{\pm}2.0$ years, p<0.05; $1.6{\pm}0.7$ vs. $0.2{\pm}0.4$, p<0.05), and the clinical pregnancy rate per cycle was significantly lower (23.8% (35/147) vs. 29.3% (34/115), p<0.05) in the TR group. Difference in the clinical pregnancy rates was age-related, since there was no significant difference between the two groups, except for the previous parity ($1.6{\pm}0.7$ vs. $0.1{\pm}0.3$, p<0.05), when the patients aged 37 years or older were excluded. No difference was found in terms of the following: the proportion of controlled ovarian hyperstimulation (COH) cycles with GnRH agonist ultrashort protocol, the duration of COH, the dosage of gonadotropins used, and the numbers of oocytes retrieved and of embryos transferred, irrespective of age correction. Conclusions: The outcomes of IVF-ET following the failed microsurgical reversal of tubal sterilization depend upon patient age. The previous fertility of patients does not seem to be a factor of better IVF-ET prognosis.

• Clinical and Experimental Reproductive Medicine
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• 제18권2호
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• pp.201-208
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• 1991

Recently the application of gonadotropin-releasing hormone (GnRH) agonist to superovulation in previous poor responders has resulted in the improved outcomes after in vitro fertilization (IVF) outcome. However, poor responders with poor estradiol $(E_2)$ rise or single dominant follicle are a particularly challenging group. Recent reports have also shown that patients with higher basal serum follicle stimulating hormone (FSH) level, result in poorer ovarian response and lower pregnancy rate. Analysis of the differences of superovulation outcomes according to the different protocols of GnRH agonist, long (L, n = 18) and short (S, n = 16) protocols, in patients with high basal FSH levels (>20mIU/ml) were undertaken at Seoul National University Hospital from June to October 1990. The administration of GnRH agonist was begun on day 21 of the cycle in long protocol, and on day 2 in short protocol. Ages of patients and husbands, basal FSH and luteinizing hormone (LH) levels and FSH/LH ratio did not differ significantly. Types and causes of infertility were evenly distributed. Whereas the duration of stimulation and the amounts of gonadotropins administered were significantly reduced in short protocol, the numbers of oocytes retrieved and cleaved, the cleavage rate and the number of embryos transferred were higher in long protocol without statistical signifieance. The pregnancy rate per ET was 16.7% (2/12) in short protocol, and 17.6% (3/17) in long protocol. These data suggest that both protocols result in the similar superovulation outcomes in patients with higher basal serum FSH levels.

• Clinical and Experimental Reproductive Medicine
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• 제24권1호
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• pp.135-141
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• 1997

The early studies demonstrated that the relative amount of FSH was important for stimulating normal ovarian activity and demonstrated the existence of a threshold level for FSH, above which follicular growth was activated. It was found that only a modest increase in circulating FSH level above the threshold (between 10 and 30%) was required to stimulate folliculogenesis. In addition, FSH is primary responsible for initiating estradiol production through the activation of the aromatase enzyme system in granulosa cells, follicular secretion and growth. LH on the other hand, plays a supportive role in ovarian steroidogenesis, stimulating the ovarian thecal cells to produce androgen, the precursor for estradiol synthesis. But there is now an increasing number of reports in the literature demonstrating an adverse effect of LH on fertility and miscarriage in infertile and fertile women. So HP-FSH is the drug of a highly purified FSH preparation which has a higher specific activity and far fewer impurities than FSH. This study was performed to evaluate the efficacy and safety of HP-FSH administered (SC; subcutaneous) versus FSH(IM; intramuscular) for ovulation induction. 20 candidates patients for ovulation induction were participated. All patients underwent pituitary desensitizing with a long gonadotropin-releasing hormone (GnRH) agonist protocol and ovulation induction was started with HP-FSH SC (10 patients; group I) or FSH IM (10 patients; group II). After ovulation, outcome of ovulation induction and local reaction of injection site were compared. There were no difference of outcome of ovulation in two groups except pregnancy rate/embryo transfer. Group I had a higher pregnancy rate/ embryo transfer than Group II (44.4% Vs 28.6%). Pain, redness, tenderness, bruising and itching when the injection received on the first 5 days of treated (50 SC and 50 IM injections) were assessed. There were no significant difference (P>0.05) in the incidence of tenderness, bruising and itching between the IM and SC injection. But IM injection (FSH) had a tendency of higher above incidence. The number of reports of pain, redness were significantly increased in IM injection group (P<0.05). These results indicate that SC administration of HP-FSH has been shown to be as effect for superovulation as traditional gonadotropins, with an improved safety profile due to the removal of extaneous proteins.

• 한국수정란이식학회지
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• 제28권3호
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• pp.237-241
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• 2013

It is generally accepted that chronic stress impairs female reproduction. It negatively affects ovarian function and the number of ovulated oocytes. Chronic stress lowers the number of retrieved oocytes. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. The main corticosteroids are cortisol, cortisone, 11-deoxycortisol and corticosterone, cortisol being one of the most commonly used welfare and stress physiological indicator. In this study, we investigated the effect of cortisol level on progesterone patterns and ovulation in the dog. Cortisol and progesterone level of serum were analyzed by radioimmunoassay. The day of ovulation was considered as the day when serum progesterone concentration was 6.0~8.0 ng/ml. In vivo dog oocytes were collected by flushing oviducts of mixed-breed bitches at three days after ovulation. We classified dogs as having group 1 (cortisol level, 0 ${\leq}$ or < $2{\mu}g/dl$), group 2 (corisol level, 2 ${\leq}$ or < $4{\mu}g/dl$), group 3 (cortisol level, 4 ${\leq}$ or < $6{\mu}g/dl$) and group 4 (cortisol level, $6{\mu}g/dl$ ${\leq}$). The patterns of progesterone were not different in four cortisol groups. The average numbers of retrieved oocytes was not different in four cortisol groups. These results suggest that different cortisol levels on estrus dogs do not affect ovulation, number of ovulated oocytes and progesterone changes.