Cryopreservation is able to store the surplus pre-embryos for freezing and furthermore thawing and transfer in a subsequent cycle. Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps, freezing media and embryonic stages on the rates of viability and development of cryopreserved mouse embryos. Female ICR mice ($6{\sim}8$ weeks old) were induced to superovulate by sequential intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48h apart. Mouse embryos were collected according to its developmental stage after the injection of hCG. Embryos were cryopreserved not only by cryoprotectant step (1 step${\sim}$4 step) but also in a variety of media (HTF, IVF medium, D-PBS) and cell stage. The results were as follows: There is no clear advantage in these freezing media of rapid method, but 4 cell and 8 cell of slow method (2, 3, 4 step) have advantage in D-PBS. The development of embryos according to cell stage become greater in 8 cell stage. In the treatment steps of cryopreservation, the development of embryo to blastocyst was similar among rapid method, but the development of 4 cell and 8 cell embryos to blastocyst according to slow method was better than rapid method.

Purpose of the present study was to find the optimal ovulation induction medicine for the maturation and development of immature oocytes and culture media for 2-cell embryos in the mouse model. ICR female mouse aged 6 to 8 weeks, were stimulated with 5 IU PMSG injection. At 47 to 50 hour post-PMSG injection, ovaries were dissected out and oocytes-cumulus complexes were punctured. The oocyte-cumulus complexes were cultured in media containing various ovulation induction medicine, CC, HMG and Metrodin for 18 hours. Female ICR mice were stimulated with 5 IU PMSG and 48 hours later were injected 5 IU of hCG, then female and male mice were mated. At 48 hour post-hCG injection, oviducts were dissected out and 2-cell embryos were flushed. The 2-cell embryos were cultured in various media, Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatography, Baxter) water, Medicult media, HTF (human tubal fluid) media for 96 hours. The results were as follows. 1. When the oocytes-cumulus complexes were cultured in $10^{-9}{\mu}g/ml{\sim}10^{-8}{\mu}g/ml$ of CC, those were suppressed in meiotic maturation $(28.2{\sim}33.7%)$. Whereas the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$, these were not effected in meiotic maturation $(54.5{\sim}72.7%)$. 2. When the oocytes-cumulus complexes were cultured in $10^{-4}{\mu}g/ml{\sim}10^{-1}{\mu}g/ml$ of Metrodin, those were suppressed in meiotic maturation $(35.7{\sim}41.5%)$. Meanwhile the oocytes-cumulus complexes were cultured in $10^{-7}{\mu}g/ml{\sim}10^{-5}{\mu}g/ml$, those were not effected in meiotic maturation $(54.2{\sim}70.3%)$. 3. When the oocytes-cumulus complexes were cultured in $10^{-5}{\mu}g/ml{\sim}10^{-4}{\mu}g/ml$ of HMG, those were suppressed in meiotic maturation $(48.2{\sim}50.4%)$. As being cultured in $10^{-7}{\mu}g/ml{\sim}10^{-6}{\mu}g/ml$, increased in meiotic maturation $(75.8{\sim}80.7%)$. 4. When the 2-cell embryos were cultured in Ham's F-10 media of milli-Q water $(3^{\circ})$, Ham's F-10 media of HPLC (high performance liquid chromatograpy, Baxter) water, Medicult media, HTF (human tubal fluid) media, developmental rates to blastocyst and hatching for 96 hour were 50.0%, 45.2%, 71.5% and 95.6%, respectively.

포유류의 자궁조직은 발정주기를 통하여 역동적으로 변화하고 있으며 이러한 자궁조직의 분화는 시상하부-뇌하수체-생식소를 잇는 축에 의해 조절되는 스테로이드 호르몬에 의해 이루어진다. 그러나 에스트로겐 (E)이 어떤 유전자를 발현하여 자궁 내의 변화를 일으키는지는 아직 자세히 알려지지 않고 있다. 본 연구는 난소를 절제한 성숙한 흰쥐에 E을 처리한 후 자궁조직 내에서 c-fos, c-jun 및 hsp25 mRNA의 발현 변화를 Northern blot analysis방법을 사용하여 연구한 것이다. c-fos및 c-jun 암원유전자의 mRNA발현은 E처리 후 1시간 이전부터 증가하기 시작하며, 3시간 이내에 최고치에 도달한 후 급격히 감소하여 기저수준으로 되돌아갔다. 반면에 hsp25 mRNA수준은 E처리 후 3시간 대에서 최고치를 나타내나 증가된 발현량이 서서히 감소하며 12시간이 지난 후 까지도 정상대조군에 비해 높은 수준으로 유지되었다. 이러한 E의 영향이 선별적인지를 검증하기 위하여 E의 길항제인 tamoxifen을 사전처리하고 E을 추가로 처리하여 c-fos, c-jun및 hsp25 mRNA의 발현이 최고치에 이르렀던 3시간대에 자궁조직을 얻어 각각의 유전자 발현량을 조사한 결과 E에 의해 증가되었던 c-fos, c-jun 및 hsp25 mRNA의 수준이 억제됨을 확인하였다. 이러한 결과는 E이 자궁조직에 영향을 미치는데 초기의 일시적인 변화를 보이는 암원유전자인 c-fos 및 c-jun이 중요한 역할을 하리라는 것을 시사하며 hsp25의 경우는 좀더 늦은 반응에 관여하거나 c-fos및 c-jun에 의하여 간접적으로 조절을 받을 수도 있음을 보여 주는 것으로 사료된다.

Recent studies clearly demonstrated that the novel expression of LH gene in the rat testis, and suggested the local action of the LH-like molecule. The present study was performed to analyze the expression of LH genes in the rat accessory reproductive organs. Expression of LH subunit genes in the rat uterus and epididymis was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) and specific LH radioimmunoassay (RIA). The $LH_{beta}$ transcripts in these organs contained the published cDNA structure, the pituitary type exons 1-3, which encoded the entire $LH_{beta}$ polypeptide. Presence of the transcripts for the ${\alpha}$-subunit in the rat reproductive tissues were also confirmed by RT-PCR. In the LH RIA, significant levels of LH were detected in crude extracts from the rat ovary, uterus and epididymis. The competition curves with increasing amount of tissue extracts were parallel with those of standard peptide, indicating that the immunoreactive LH-like materials in these tissues are similar to authentic pituitary LH molecule. In rat epididymis, the highest amount of immunoreactive LH was detected in corpus area. Our findings demonstrated that the genes for LH subunits are expressed in the rat accessory reproductive organs, and suggested that these extrapituitary LH may act as a local regulator with auto and/or paracrine manner.

Objectives: The aims of this study are 1) to determine if GAST is a better indicator in predicting ovarian response to COH compared with patient's age or basal FSH level and 2) to evaluate its role in detecting abnormal ovarian response. Design: Prospective study in 118 patients undergoing IVF-ET using GnRH-a short protocol during May-September 1995. Materials and Methods: After blood sampling for basal FSH and estradiol $(E_2)$ on cycle day two, 0.5ml (0.525mg) GnRH agonist ($Suprefact^{(r)}$, Hoechst) was injected subcutaneously. Serum $E_2$ was measured 24 hours later. Initial $E_2$ difference $({\Delta}E_2)$ was defined as the change in $E_2$ on day 3 over the baseline day 2 value. Sixteen patients with ovarian cyst or single ovary or incorrect blood collection time were excluded from the analysis. The patients were divided into three groups by ${\Delta}E_2$; group A (n=30):${\Delta}E_2$<40 pg/ml, group B (n=52): 40 pg/ml${\leq}{\Delta}E_2$<100 pg/ml, group C (n=20): ${\Delta}E_2{\leq}100$ pg/ml. COH was done by GnRH agonist/HMG/hCG and IVF-ET was followed. Ratio of $E_2$ on day of hCG injection over the number of ampules of gonadotropins used ($E_2hCGday$/Amp) was regarded as ovarian responsiveness. Poor ovarian response and overstimulation were defined as $E_2$ hCGday less than 600 pg/ml and greater than 5000 pg/ml, respectively. Results: Mean age $({\pm}SEM)$ in group A, B and C were $33.7{\pm}0.8^*,\;31.5{\pm}0.6\;and\;30.6{\pm}0.5^*$, respectively ($^*$: p<0.05). Mean basal FSH level of group $A(11.1{\pm}1.1mlU/ml)$ was significantly higher than those of $B(7.4{\pm}0.2mIU/ml)$ and C $(6.8{\pm}0.4mIU/ml)$ (p<0.001). Mean $E_2hCGday$ of group A was significantly lower than those of group B or C, i.e., $1402.1{\pm}187.7pg/ml,\;3153.2{\pm}240.0pg/ml,\;4078.8{\pm}306.4pg/ml$ respectively (p<0.0001). The number of ampules of gonadotropins used in group A was significantly greater than those in group B or C: $38.6{\pm}2.3,\;24.2{\pm}1.1\;and\;18.5{\pm}1.0$ (p<0.0001). The number of oocytes retrieved in group A was significantly smaller than those in group B or C: $6.4{\pm}1.1,\;15.5{\pm}1.1\;and\;18.6{\pm}1.6$, respectively (p<0.0001). By stepwise multiple regression, only ${\Delta}E_2$ showed a significant correlation (r=0.68, p<0.0001) with $E_2HCGday$/Amp, while age or basal FSH level were not significant. Likewise, only ${\Delta}E_2$ correlated significantly with the number of oocytes retrieved (r=0.57, p<0.001). All four patients whose COH was canceled due to poor ovarian response belonged to group A only (Fisher's exact test, p<0.01). Whereas none of 30 patients in group A (0%) had overstimulation, 14 patients among 72 patients (19.4%) in group B and C had overstimulation (Fisher's exact test, p<0.01). Conclusions: These data suggest that initial $E_2$ difference after GAST may be a better prognostic indicator of ovarian response to COH than age or basal FSH level. Since initial $E_2$ difference demonstrates significant association with abnormal ovarian response such as poor ovarian response necessitating cycle cancellation or overstimulation, GAST may be helpful in monitoring and consultation of patients during COH in IVF-ET cycle.

The follicular fluid (FF) of ovary contains various biological active products which affected on the growth of follicles and the fertilization of oocyte in physiological reproductive process of mammals. This study was designed to determine the effects of human FF on fertilization of oocyte and embryonal development in vitro culture. The FF was prepared as clear without blood contamination by needle aspiration from mature follicles of human at the time of oocytes retrieval for in vitro fertilization (IVF). As the medium for culture in vitro of embryonal cells, human tubal fluid (HTF) supplemented with follicular fluids at concentrations of 10%, 40% and pure FF were used. These effects were compared to control group of cultured embryos in HTF supplemented with 0.4% BSA (bovine serum albumin). For IVF, 64 eggs in control group, 67 eggs in 10% FF, 57 eggs in 40% FF and 64 eggs in pure FF were respectively allocated. And the rates of fertilization were almost similar in all groups as resulting 82.81% in control, 85.07% in 10% FF, 87.71% in 40% FF and 81.25% in pure FF. On the examination for embryonal cleavage from fertilized eggs, the rates of developing to 4 cell stage was similar in all groups, as results 98.11% in control, 98.27% in 10% FF and 98% in 40% FF but 78.84% in pure FF. And the rates of developing to 8-16 cell stage were significantly reduced as 44% in 40% FF and 44.23% in pure FF (p<0.05) compare to 71.69% in control media. As likewise, the rates of developing to morular stage were also significantly reduced to 36% (p<0.05) and 21.15% (p<0.01) respectively in 40% FF and pure FF. And the rates to blastocystic stage of embryo was lowest as 7.69% in pure FF (Table 1). The quality of embryonal cells on cleavage to the 8-16 cell stage was poorer, higher concentrations of FF. The rates of grade 1 in pure FF, as 23.07%, was lowest compare to those of other groups, in which the rates of grade 1 in control, 10% FF and 40% FF were 58.49%, 47.36% and 34% respectively. And on the contrary, the rate of grade 4 in pure FF was highest as 23.07%, while those were 5.66% in control, 8.77% in 10% FF and 20% in 40% FF (Table 2). On the viability of embryos, the rate of embryonal cell death was more rise, at the higher concentrations as well as longer exposure in the follicular fluid. At 48 hours after in vitro culture of embryos, the rate of survival embryos in pure FF was markedly lowered as 44.23%, compare to that of control (p<0.05). But there was not significant difference between the rates of survival embryos in each group beside the pure FF, which the rates were 77.35% in control, 70.17% in 10% FF and 60% in 40% FF respectively. And at 72 hours after in vitro culture, the rates of survival embryos were also significantly dropped to 21.15% in pure and 36% in 40% at concentration of FF compare to 62.26% in control (p<0.05, p<0.01). Finally, the rate of embryonal death at 96 hours after in vitro culture was highest as 82.69% in pure FF among all groups which those were 35.84 in control, 56.14% in 10% FF and 64% in 40% FF respectively (Fig. 1, 2, 3). In conclusion, this study suggests that the FF has no effects, in particular, to the in vitro fertilization of oocytes but exerted a bad effect to the cleavage, quality and viability of the embryonal cells during in vitro culture. However, the FF is harmful on embryonal development at conditions in higher concentration and especially on the embryos after $8{\sim}16$ cell stage.

This study was performed to determine the significance of a baseline ovarian cyst on the response to controlled ovarian hyperstimulation and the outcome of IVF-ET. One hundred one patients who underwent IVF-ET were enrolled in this study. The outcome of 31 patients, who had an ovarian cyst of >10mm detected at ultrasound examination performed on day 3, was compared with that of 70 patients who underwent a similar protocol and did not have an ovarian cyst. E2 level on the day of hCG administration, the number of follicles, the number of oocytes retrieved, the number of embryo transferred and the pregnancy rate were evaulated. The E2 level on the day of hCG adminstration and the number of mature oocytes retrieved were lower in the group with a baseline cyst. The pregnancy rate also was significantly lower in the group with a cyst (21% versus 38%). Therefore a baseline ovarian cyst on cycle day 3 was associated with a poorer outcome after IVF-ET.

The present study was performed to investigate the efficacy and safety of laser assisted hatching (AH) on mouse embryos. Non-contact $1.48{\mu}m$ diode laser system used to create a precise hole on zona pellucida. 2-cell embryos were collected from the mice (ICR) that had the coitus vaginal plug confirmed at 48 hours after hCG injection. Collected 2-cell embryos were cultured in the HTF medium supplemented with 0.4% BSA. For experiments, embryos at 8-cell stage were used after 18-22 hours in culture. After assisted hatching, the embryos were further cultured in HTF medium containing 0.1% PVP (anti-hatching system) for 3 days. For evaluate efficiency of laser on mouse embryo hatching, the effect of AH methods (acidic tyrode, pronase and laser), the number of artificial holes (1, 2 and 3 hole) and the irradiation time of laser (2, 4, 6, 8 and 10 ms) were examined. Hatching rates of laser AH group (95.2%) was significantly higher than that of control group (50.8%), but there was no differences among the laser (95.2%), acidic tyrode (100%) and pronase (98.5%) groups. Hatching rates of the number of zona pellucida opening by laser, there were no differences among the 1 hole (87.5%),2 hole (92.1%) and 3 hole (85.9%) groups. Developmental and hatching rates of embryos according to laser irradiation time were similar in the treatment groups. Therefore, these results suggest that laser AH using non-contact $1.48{\mu}m$ diode laser is a simple and accurate and effective procedure for AH. Based on these results, laser AH could be use safely for human ART program.

본 연구에서는 소 난자 세포질내 원형정자 및 원형정자 핵을 미세주입 후 수정과정과 체외 배 발달을 조사하였다. 원형정자 주입 3시간 전에 인위적 자극을 주고 원형정자를 주입했을 때 난자들의 수정률이 주입 직후에 자극을 준 것들과 자극을 주지 않은 것들 보다 높았다. 원형정자 주입과 원형 정자핵을 주입한 후 전핵 형성률과 전핵 이동률을 조사하였으나 유의차를 발견할 수 없었다. 전핵의 형성과 이동중에 미세소관의 움직임을 간접형광면역법 및 공촛점 현미경을 사용하여 조사해 본 결과, 난활성 직후 난자의 표층에서 미세소관이 발생해서 이것에 의해 웅성 및 자성전핵이 난자 중심부로 이전됨을 볼 수 있었다. 수정 후 원형정자의 세포질내 Mitochondria의 분포상태를 알아보기 위해 MitoTracker에 노출시킨 원형정자를 주입한 후 형광현미경에서 관찰해 보았다. 그 결과 원형정자의 Mitochondria가 주입 후 4-cell 이 전까지 는 관찰되었으나 그 이 후에는 발견 할 수 없었다. 원형정자를 미세주입한 후 소 난자를 체외에서 배양했을 때 2.5%의 난자가 배반포로 발달하였다. 이상의 결과는 체외 배양한 소 난자 내에 원형정자 혹은 원형정자 핵을 미세 주입하여 정상적인 수정과 배발달이 가능한 것을 보여 주는 것이다.

목적: 일산화질소 (nitric oxide; NO)는 생식계를 비롯한 여러 생체내 기관에서 다양하고도 중요한 작용을 하는 것으로 알려져 있으며, 복강액은 난관내강과 연결되어 복강액 내의 세포 성분의 변화는 난관의 미세환경을 변화시켜 수정과 초기 배아 발생에 영향을 줄 수 있다. 본 연구는 배아 발생에 있어서 일산화질소의 역할을 이해하고 복강액 내의 NO농도 변화가 배아 발생에 미치는 역할을 조사하기 위해 수행되었다. 방법: 과배란시킨 1세대 잡종 암컷 생쥐 (C57BL${\times}$CBA/Ca)로부터 1세포기 배아를 얻어 10% synthetic serum substitute가 첨가된 modified human tubal fluid 배양액에서 4일 동안 체외배양하였다(대조군). 실험을 위해 이러한 배양조건에 sodium nitroprusside (SNP)를 $0{\sim}1mM$의 다양한 농도로 배양초기부터 첨가하거나, $200{\mu}M$ SNP를 2-, 4-, 8-세포기의 각기 다른 배아시기에 첨가하였으며, 복강경수술을 받는 42명의 여성으로부터 채취한 복강액을 SSS대신 단백질원으로 사용하여 포배아까지의 배아 발달율을 관찰하였으며, 복강액 내의 NO농도를 Griess방법에 의해 측정하였다. 배아의 apoptotic body는 H33342 염색법으로 조사하였으며 배아 발달율은 3회 이상 반복 실험한 결과의 mean${\pm}$SEM으로 나타내었다. 결과: SNP는 농도에 의존적으로 배발생을 억제하였으나 배아 단계에 대한 특이성은 관찰할 수 없었으며, 특히 $100{\mu}M$ 이상의 고농도의 SNP는 2-세포기 단계에서 배아 발생을 정지시켰다. 또한 단백질원으로 복강액 이용시 배 발생율은 복강액 내의 NO 농도에 따라 현저한 차이가 발견되었으며, $2.5{\mu}M$이상의 NO를 함유한 복강액에서 배양한 배아의 발생율은 현저하게 감소하였다. cGMP analogue인 8-bromo-cGMP를 배양액에 첨가시 배아 발생에는 변화가 없었으며, SNP에 의해 배발생이 정지된 2-세포기 배아에서 apoptotic body를 발견할 수 없었다. 결론: 이상의 결과로 보아 NO는 고농도에서 배아 발생을 저해하며, 복강액 내의 NO와 같은 성분의 변화는 배아 발생에 유해한 효과를 유발할 것으로 사료된다. 이러한 NO의 배아 발생 억제효과는 cGMP로 중재되는 경로나 apoptosis유발과는 관계가 없는 것 같다.

In our previous study, we observed that hydrosalpingeal fluid (HSF) adversely effect mouswe embryo development and hatching. The aim of this study was to evaluate the effect of HSF as assessed by the blastocyst development rate (BDR) and by cell counting in vitro. HSF was collected from ninie patients undergoing salpingoneostomy to correct hydrosalpinx. Two-cell embryos were obtained from superovulated ICR mice. T6 medium and $T6{\pm}0.4%$ bovine serum albumin were used as control media. T6 medium containing 10% or 50% HSF and 100% HSF from each patient were used as test media. Nine to 15 embryos were cultured in micro drops prepared from each of these media. To assess the total cell number within each blastocyst, the blastocysts were fixed and stained with Hoechst 33342 to facilitate cell counting. The mean BDR in two control media were 88.89% and 85.40%. The mean BDR in media containing 10%, 50%, 100% HSF were 85.87%, 89.58% and $75.57%^*$, respectively ($^*$: p<0.05). The overall mean cell count $({\pm}SEM)$ in control media were $87.6{\pm}9.65\;and\;90.12{\pm}11.38$. The BDR was affected adversely only by 100% HSF and not in media containing 10% or 50% HSF. Mean cell counts were decreased significantly only in blastocysts cultured 100% HSF ($63.8{\pm}13.66$; p<0.01) but not in blastocysts cultured in 10% or 50% HSF ($91.3{\pm}12.44\;and\;82.9{\pm}18.27$, respectively). Thus, it is concluded that HSF has no embyotoxic effect but has a mildly negatively effect on embryonic growth and development.

Ovarian development of the vitrified neonatal ovaries after orthotopical transplantation into the ovariectomized adult recipient mouse were observed. Ovaries were collected from the neonatal females on day of birth and grouped for fresh, vitrification for 1-minute, and 3-minute. Vitrified and thawed neonatal ovaries were orthotopically transplanted into ovarian bursa of the adult mice from which endogenous ovaries have removed just prior to the transplantation (1 minute: n=25; 3 minutes n=23). Fresh ovarian tissue transplanted (n=25) mice were included as control groups. Returning of the estrus cycles and the survival and development of the transplanted ovaries were evaluated. Intact ovaries from neonatal, and four weeks old mice were used for comparison of the ovarian development as in vivo-developed control. From 2 weeks after transplantation, 64%, 36%, and 75% of the transplanted mice showed return of the estrus cycles in fresh, 1-minute, and 3-minute groups, respectively. Four weeks after transplantation, all mice were sacrificed and ovarian tissues were recovered for histological analysis. 57.1%, 33.3%, and 64.7% mice in fresh, 1-minute, and 3-minute groups, respectively, had survived ovaries with follicles at various stages of growth from primordial to preovulatory follicles. Corpus lutea were also observed. Results of the present study suggest that 1) normal folliculogenesis has initiated in vivo after vitrification, and 2) the vitrification may be used as a preservation method for ovarian tissues for establishment of ovarian tissue bank.

In mouse, the heat shock protein 70-2 (hsp70-2) is found to have special function in spermatogenesis. Based on the observation, the hypothesis that human hspA2 (human gene; 98.2% amino acid homology with hsp70-2) might have important function in spermatogenesis in human testes was proposed. To test the hypothesis, we examined the expression of hspA2 in human tissues. Expression vector pDMC4 for expression of the human hspA2 protein using pTricHisB (invitrogen, USA) was constructed and the expressed hspA2 protein was cross-reacted with antiserum 2A raised against mouse hsp70-2 protein. Based on the cross-reactivity, we determined the expression level of hspA2 protein in human tissues by western blot analysis using the antiserum 2A. We demonstrated that antiserum 2A antibodies detected human hspA2 protein with specificity which was produced in the E.coli expression system. On Western blot analyses, significant hspA2 expression was observed in testes with normal spermatogenesis, whereas a low level of hspA2 was expressed in testis with Sertoli-cell only syndrome. Also, a small amount of hspA2 was detected in breast, stomach, prostate, colon, liver, ovary, and epididymis. These results demonstrate that the hspA2 protein is highly expressed in male specific germ cells, which in turn suggests that hspA2 protein might playa specific role during meiosis in human testes as suggested in the murine model. However, further studies should be attempted to determine the function of hspA2 protein in human spermatogenesis.

본 연구는 체외생산된 소 배반포기배를 발달 단계 및 배양일에 따라 구별하여 초자화 동결 및 융해하였을 때, 그 발달능을 유지하는지 확인하고자 실시하였다. 체외수정 후 8일간 배양된 배반포기 배는 20% ethylene glycol에 3분 동안 평형시키고, EFS40 (40% ethylene glycol, 18% ficoll, 0.3M sucrose 그리고 10% FBS가 함유된 mDPBS) 동결액에 30초 동안 노출한 후, 액체질소에 침지하여 초자화 동결되었다. 체외 생존 여부는 응해 24시간 및 48시간에 재팽창 및 탈출 또는 완전탈출로써 평가하였다. 그 결과를 요약하면 다음과 같다. 1) 체외수정 후 8일간 배 양하였을 때, 난할된 배의 배반포기 배로의 발달율은 41.0%였다 (초기 ; 7.6%, 팽창; 22.9%, 탈출; 4.6%, 완전탈출; 5.9%). 2) 배반포기배를 동결액에 노출 또는 초자화 동결하였을 때, 초자화 동결된 배반포기배의 재팽창율 (73.3%)은 대조군 및 동결액에 노출된 경우 (100, 97.0%)보다 낮았다 (p<0.05). 그러나 융해 48시간 후 탈출 또는 완전탈출 배반포기배 형성율은 초자화 동결된 경우 (66.7, 46.7%)와 노출된 경우 (66.7, 39.4%)는 유의한 차이를 나타내지 않았으나, 대조군(100, 100%)과는 차이를 보였다 (p<0.01). 그러나, 완전탈출까지 발달한 배반포기배의 총 세포수를 조사하였을 때, 각 처리군간의 유의한 차이는 없었다. 3) 배반포기배의 발달 단계에 따른 체외 생존율을 비교하였을 때, 재팽창율은 실험군간에 유의한 차이를 보이지 않았다 $(64.5{\sim}75.6%)$. 그러나 융해 48시간 후, 탈출 또는 완전탈출로써 평가된 초기 배반포기배의 발달율 (25.8, 9.7%)은 팽창 (69.7, 39.4%)및 탈출 배반포기배 (53.3, 43.3%)의 발달율보다 낮게 나타났다 (p<0.05). 4) 또한, 배양 7, 8 그리고 9일의 팽창 배반포기배를 초자화 동결하였을 때, 8일 및 9일간 배양된 배반포기배의 재팽창율은 7일간 배양된 경우보다 낮게 나타났다 (7일; 93.9%, 8일; 75.8%, 9일; 87.5%) (p<0.05). 그러나 완전탈출 배반포기배로의 발달율에서는 처리군간에 유의한 차이를 보이지 않았다 (7일; 36.4%, 8일; 36.4%, 9일; 31.3%). 이러한 결과는 EFS40을 이용한 2단계 초자화 동결 방법이 체외생산 된 팽창 및 탈출 배반포기배의 동결에 유용하게 이용될 수 있음을 시사한다.

The role of amino acids in culture media for IVF-ET was examined in a total of 76 cycles. Patients received clomiphene citrate (CC) followed by hMG or GnRH-a combined with gonadotropins (FSH/hMG) for controlled ovarian hyperstimulation. Severe male (<$4{\times}10^6$ motile sperm) or age factor (>39 y) patients were excluded in this study. Pregnancy was classified as clinical if a gestational sac or fetal cardiac activity was seen on ultrasound. No significant differences were found in age, duration of infertility, follicle size, the level of $E_2$ on the day of hCG injection, the mean number of oocytes retrieved, total motile sperm count, fertilization rate and the mean number of embryos transferred between bHTF (without amino acids) and mHTF (with amino acids) groups. However, total ampules of gonadotropins were higher (p<0.01) in mHTF group than bHTF group. Significantly (p<0.05) more clinical pregnancies were recorded in mHTF group (13/30) compared with bHTF group (9/46). The multiple pregnancy rates were 11.1% in bHTF group and 7.7% in mHTF group. There were one ectopic pregnancy in mHTF group and one heterotopic pregnancy in bHTF group. Abortion rates were 22.2% in bHTF group and 7.7% in mHTF, respectively. The ongoing pregnancy or livebirth rate was significantly (p<0.05) higher in mHTF group (12/30) than bHTF group (7/46). These results suggest that the addition of amino acids in culture media is essential for culture of zygotes in vitro and adjustment of energy substrates in phosphate-free culture media appears to be beneficial for human IVF-ET procedure.

The present study was conducted to evaluate whether vitrification could be used for ovarian tissue preservation. The important issue here is that the vitrification is very simple, easy, and economical compared to the conventional cryopreserving method that using automatic freezing instrument. Human ovarian cortical tissues were cryopreserved by vitrification with 5.5 M ethylene glycol and 1.0 M sucrose as cryoprotectant. Three points of temperature ($4^{\circ}C$, room temperature, and $37^{\circ}C$) and two points of duration (5 or 10 minutes) for cryoprotectant treatment were examined to determine the best condition for vitrification of the human ovarian cortical tissues. After thawing, viability of the isolated primordial follicles was examined by dye-exclusion method. Histological appearance of tissues before and after the cryopreservation was evaluated. There was no toxic effect of the 5.5 M ethylene glycol on the primordial follicles. However, when the tissues were treated with cryoprotectant at $37^{\circ}C$ for 10 minutes and exposed to liquid nitrogen, it seems likely that there is certain deleterious effects on the viability of the primordial follicles. The highest viability of the primordial follicles was obtained with the treatment of cryoprotectant at room temperature for 10 minutes. Follicles and oocytes survived after freezing and thawing had the similar normal shapes as was seen in the specimens before cryopreservation. It would be useful to apply vitrification in establishing ovarian tissue banking for clinical purposes.

It is well known that discard of seminal plasma from the semen and separation of motile sperm should be preceded before insemination for IUI or IVF. Till now, more than ten kinds of semen treatment methods have been developed. Of those, swim-up and Percoll methods have been used widely in ART laboratories as a routine semen treatment methods because of its advantages. However, there are reports that Percoll can make a genetic trouble because of its chemical structure and therefore the necessity has been arisen to substitute Percoll for other equivalent materials. This study was performed to evaluate the effects of three different sperm preparation methods (swim-up, Percoll and Sil-Select) on sperm motility, sperm recovery rate and fertilization rate. Also, the feasibility of using Sil-Select instead of Percoll in ART was evaluated. Each semen samples were divided into three fractions and motile sperm were recovered by swim-up, Percoll and Sil-Select gradient centrifugation methods. Normal and sub-normal criteria of fifteen semen samples and seventeen IVF cycles were included in these study. As results, no significant difference was found in sperm recovery rate in normal semen treated by a Swim-up, Percoll and Sil-Select method ($13.2{\times}10^6,\;17.5{\times}10^6\;and\;17.7{\times}10^6$ respectively). The initial sperm motility was 61.9% and this increased to 87.1%, 92.6% and 89.5% through Swim-up, Percoll and Sil-Select treatment, respectively. Higher motility was observed in Percoll and Sil-Select treated groups (81.5%, 79.2%, respectively) than swim-up group (66.8%) after incubation for 24hrs. In sub-normal group, sperm recovery rates were higher in Sil-Select group $(2.9{\times}10^6)$ than Percoll gradients group $(1.8{\times}10^6)$. In IVF cycles, the outcomes of fertilization using sperm treated by swim-up and Sil-Select group were similar (82.2%, 79.7% respectively). In conclusion, our results indicate that Sil-Select can be used as a substitute material for sperm preparation instead of Percoll.

Thrombocytopenic patients without detectable bound antiplatelet antibody should be diagnosed with idiopathic thrombocytopenic purpura (ITP) if no other cause of their decreased platelet count could be found. More recently the term "autoimmune thrombocytopenic purpura (ATP) has supplanted ITP since the disease is related to the production of autoantibodies against one's own platelets. This entity should not be confused with isoimmune thrombocytopenic purpura (also called alloimmune thrombocytopenic purpura). In this cases maternal antiplatelet antibodies directed against the PLA 1 antigen on the fetal platelets causes severe fetal and neonatal thrombocytopenia in a situation analogous to Rheusus disease. Antibodies to the negatively charged phospholipids, lupus anticoagulant, and anticardiolipin have been linked to adverse pregnancy events. Pregnant women possessing these antibodies have an increased risk of spontaneous abortion, stillbirths, intrauterine fetal growth retardation, preterm birth, and arterial and venous thrombosis. Antiphospholipid antibodies decrease or may even disappear between pregnancies only to recur with increased activity in a subsequent pregnancy and lead to loss. We have experienced a case of antiphospholipid syndrome associated with autoimmune thrombocytopenic purpura in patient with recurrent spontaneous abortion. So we report this case with a brief review of literatures.

Activated factor XII (FXIIc: Hageman factor) is a central component of the contact activation system of blood coagulation, fibrinolysis, and kinin formation. Although patients deficient in FXIIc (up to 50% of normal) do not show increased bleeding tendency, thrombotic complications were reported in 8% to 10% among these patients. The reduced generation of bradykinin resulting in diminished release of tissue plasminogen activator is proposed as a cause of thrombosis in factor XII (FXII)-deficient patients. Similarly, in patients with elevated levels of circulating antiphospholipid antibodies, hemostasis may be impaired resulting in excessive thrombophilia. Both vascular and placental thromboses because of antiphospholipid antibodies or FXIIc deficiency have been reported to be associated with recurrent fetal loss. We have experienced a case of factor XII deficiency in woman with recurrent spontaneous abortion. So we report this case with a brief review of literatures.

Male sexual differentiation involves a cascade of events initiated by the presence on the Y chromosome of the of the SRY (sex determining region of Y chromosome) gene, which causes the indifferent gonad to develop into a testis. Hormonal products of the testis, predominantly testosterone and Mullerian inhibiting subtance (MIS), then control the sexual differentiation of the developing fetus. SRY is a transcription factor; however, target genes for its action have yet to be identified, because the DNA recognition sequence for SRY is found in many genes. Therefore the study of intersex disorders is being used to identify other genes active in the pathway of sexual differentiation. Patients with 46,XY gonadal dysgenesis, or Swyer's syndrome, have streak gonads, normal stature, and a sexually infantile phenotype with Mullerian structures present. The inheritance is usually sporadic but can be autosomal dominant or X-linked recessive. Unlike 45,X patients, stigmata of Turner syndrome are rare. As many as 20 to 30% of patients are at risk for malignant gonadal tumor formation and should undergo gonadectomy soon after the diagnosis is made. We have experienced a case of Swyer syndrome which showed a positive SRY gene in peripheral blood and gonad. So we report this case with a brief review of literatures.

Kallmann's syndrome has both a general and specific connotation in describing general condition of gonadotropin-releasing hormone (GnRH) deficiency or a particular cluster of anomalies associated with primary eunuchoidism. The familial occurrence of hypogonadotropic hypogonadism associated with anosmia, color blindness, synkinesia, and mental defect is the classic Kallmann's syndrome. Interestingly, anosmia, or lack of smell, was not found in the absence of gonadal deficiency in the original study of this disorder. This disorder was found on both sexes, but the male to female ratio was 11:1, and Kallmann's syndrome is more often listed under disorders of male hypogonadism for this reason. Gross anatomy has shown disorders of the olfactory bulbs associated with Kallmann's syndrome and it was demonstrated a failure of GnRH-containing cells to migrate from the olfactory placode to the hypothalamus and preoptic area. We have experienced a case of Kallmann's syndrome which showed a hypoplasia of olfactory bulb in MRI during the workup of primary amenorrheic patient. So we report this case with a brief review of literatures.

Dopamine agonists are commonly used in the medical treatment of prolactinomas. Bromocriptine has been the most widely used ergot derivative for two decades. Its oral administration, at a daily dose of $2.5{\sim}7.5mg$, restored normal gonadal function and normoprolactinemia in about 80% of patients. Nevertheless, a subset of patients could not achieve normal prolactin levels or resume normal gonadal function despite $15{\sim}30mg$/day bromocriptine for at least 6 months. Subsequently, these prolactinomas were consedered to be resistant to bromocriptine. The percentage of bromocriptine - resistant prolactinoma patients reported in the literature varies between 5 and 17% according to the series. Patients with bromocriptine resistance or bromocriptine intolerance have, however, been treated with other dopamine agonists, such as lysuride, pergolide, cabergoline, or quinagolide. Until cabergoline recently gained a product licence in the UK, there was no alternative dopamine agonist with a licence for this purpose. Quinagolide (CV $205{\sim}502$, Norprolac, Sandoz) is a nonergot dopamine agonist with improved selectivity for the D2 receptor, designed to retain the active pharmacophore of bromocriptine without the ergot moiety that might be responsible for side - effects. We have experienced a case of bromocriptine resistant hyperprolactinemia which was reponsive to pergolide. So we report this case with a brief review of literatures.

Different Y mutation in Yq11 occurring de novo in sterile males were first described 19 years ago. Since the phenotype of the patients was always associated with azoospermia or severe oligospermia, it was postulated that these mutations interrupt a Y spermatogenesis locus in the euchromatic Y region (Yq11) called azoospermia factor (AZF). Recently, it became possible to map AZF mutations to different subregions in Yq11by molecular deletion mapping. This indicated that azoospermia is possibly caused by more than one Y gene in Yq11 and the Yq11 chromatin structure. The frequency of AZF mutations in idiopathic sterile males $(5{\sim}20%)$ may indicate a need for a general screening programme for its analysis in infertility clinic. We have experienced a case of deletion distal to Yq11 region in azoospermic patient. So we report this case with a brief review of literatures.