In order to study the implantation mechanism various methods for culture of endometrial cells in vitro have been attempted. However, a disadvantage is that primary cultures of stromal and epithelial cells do not have the ability to differentiate, and therefore cannot be reproduced in the same manner as in vivo endometrium. The object of this study is to establish a three dimensional culture of endometrial cells which are both morphologically and functionally identical to in vivo endometrium. Endometrial tissues obtained after hysterectomies were cut into thin slices and treated with collagenase and trypsin-EDTA. The stromal cells and the epithelial cells were separated by centrifugation and cultured for 24 hours in DMEM media containing 10% FCS, 100 nM progesterone, and 1 nM estradiol. The cultured stromal cells were mixed with collagen gel and solidified, after which it was covered with matrigel. Epithelial cells were inoculated on the top and then cultured for 3 days. The three dimensionally cultured endometrial cells were stained for integrin ${\alpha}1,\;{\alpha}4,\;{\beta}3$, and cyclooxygenase-l, -2 by immunohistochemistry, which all showed strong expression. The cultured epithelial cells showed the formation of microvilli, tight junctions and pinopodes by electron microscopy. Studies are currently under way utilizing this three dimensional culture model to ascertain the interaction between the embryo and human endometrial cells at the time of implantation, and it is thought that further studies into a new culture environment which would allow longer periods of culture will be necessary.

The genetic defects in human gametes and embryos can cause adverse effects on overall reproductive events. Biopsy of embryos for preimplantation genetic diagnosis (PGD) offers a new possibility of having children free of the genetic disease. In addition, advanced embryo culture method may enhance the effectiveness of embryo biopsy for the practical application of PGD. This experimental study was undertaken to evaluate the effects of coculture on the development in vitro of biopsied mouse embryos as a preclinical model for PGD of human embryos. Embryos were obtained after in vitro fertilization (IVF) from F1 hybrid mice (C57BLfemale/CBAmale). Using micromanipulation, 1, 2, 3 or 4 blastomeres of 8-cell stage embryos were aspirated through a hole made in the zona pellucida by zona drilling (ZD) with acidic Tyrode's solution (ATS). After biopsy of blastomeres, embryos were cultured in vitro for 110 hours in Ham's F-10 supplemented with 0.4% BSA or cocultured on the monolayer of Vero cells in the same medium. The frequence of blastocyst formation were recorded, and the embryos beyond blastocyst stage were stained with 10% Giemsa to count the total number of nuclei in each embryo. There was no significant difference in the blastocyst formation between the zona intact control group and the zona drilling (ZD) only, or biopsied groups. The hatching rate of all the treatment groups except 4/8 group was significantly higher than that of control group. In all the treatment groups, there was a significant reduction in the mean cell number of embryos beyond blastocyst stage ($50.2{\pm}14.0$ in control group vs. $41.2{\pm}7.9$ in ZD, $39.3{\pm}8.8$ in 7/8, $29.7{\pm}6.4$ in 6/8, $25.1{\pm}5.7$ in 5/8, and $22.1{\pm}4.3$ in 4/8 groups, p<0.05). When the same treatments were followed by coculture with Vero cells, a similar pattern was seen in the blastocyst formation and the hatching rate. However, in all the treatment groups, there was a significant increase in the mean cell number of embryos beyond blastocyst stage with coculture, compared with the parallel groups without coculture. In the cleavage rate of biopsied blastomeres cultured for 110 hours after IVF, there was no significant difference between coculture and non-coculture groups (87.2% vs. 78.7%). However, the mean cell number of embryos developed from the biopsied blastomeres was significantly higher in coculture group ($11.5{\pm}4.7\;vs.\;5.9{\pm}1.9$, p<0.05). In conclusion, biopsy of mouse embryos after ZD with ATS is a safe and highly efficient method for PGD, and coculture with Vero cells showed a positive effect on the development in vitro of biopsied mouse embryos and blastomeres as a preclinical model for PGD of human embryos.

This study was performed to identify the effect of the hydrosalpinx fluid on sperm motility. It has been reported that the patients with hydrosalpinx show the outstandingly lower success rate than other patients having infertility by different factors. It is unclear that the cause of it is influenced by hydrosalpinx fluid directly or by secondary chronic inflammation of endometrium. We wanted to know if the hydrosalpinx fluid influences sperm motility parameters directly such that it is related to the development of infertility. Therefore, using computer assisted semen analyzer (CASA), we observed, from February to July, 1997, how sperm motility, sperm progressive motility, sperm curvilinear velocity, sperm lateral head displacement, sperm straightness and sperm linearity change after treating normal sperm with hydrosalpinx fluid to evaluate sperm function on infertility. The result was that the study group (n=32) has the tendency to differ from the control group (n=32) on sperm motility, progressive motility, curvilinear velocity, lateral head displacement, straightness and linearity. We concluded that the hydrosalpinx fluid, with varying degree, directly has the harmful effects on sperm motility parameters, that is, curvilinear velocity, lateral head displacement and linearity of sperm which are related to the hyperactivation, hence decreased capacitation.

To evaluate the effectiveness of intrauterine insemination (IUI) in the treatment of infertility, timed-intercourse and intrauterine insemination by husband in stimulated cycles with clomiphene citrate and gonadotropins were compared in a total of 105 cycles. Patients received 100mg of clomiphene citrate daily for 5 days starting on day 3 of the menstrual cycle followed by hMG or FSH. Doses of exogenous gonadotropins were adjusted by the follicular development and concentrations of serum estradiol $(E_2)$. More than 3 follicles reaching >16 mm were present in the ovary, 5,000 IU of hCG was administered intramusculary. Patients received a maximum of three intercourse or IUI cycles for the treatment. Severe male (<$10{\times}10^6$ 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. The overall clinical pregnancy rates were 17.1% per cycle (18/105) and 21.2% per patient (18/85). The pregnancy rates (per cycle) were 17.5% (11/63) in intercourse and 16.7% (7/42) in IUI groups, respectively. IUI had no significant improvement in pregnancy rate compared with timed-intercourse. The multiple pregnancy rates were 11.1% (1 twin and 1 triplet). No patient developed ovarian hyperstimulation. Abortion rate was 28.6% (2/7) in IUI group only. The delivery and ongoing pregnancy rates were 15.2% per cycle (16/105) and 18.8% per patient (16/85). There were no differences in age, duration of infertility, follicle size and level of estradiol $(E_2)$ on the day of hCG injection in pregnant and non-pregnant groups. However, total doses of gonadotropins were higher in pregnant group than in non-pregnant group (p<0.01). Pregnancy rate was not affected by ovulatory status at the time of insemination. These results indicate that well timed-intercourse in stimulated cycles is as effective as IUI for infertile couples.

Klinefelter's syndrome is a very important disease in gynecologic endocrinologic fields, because the patients with this karyotype complain of infertility, azospermia and ambiguous genitalia. Y chromosome is an important chromosome which determine genetic sex and the structure of gonad and genitalia. In this study, to elucidate the cytogenetic characteristics and clinical features of Klinefelter's syndrome and Y chromosomal abnormalities in Korea, we studied 303 cases of Klinefelter's syndrome and 11 cases of Y chromosomal abnormalities which were diagnosed by chromosomal analysis at the Cytogenetic Laboratory, Institute of Reproductive Medicine and Population, Seoul National University for 12 years from January 1984 to December 1996. The results of this study showed as follows: 1. In a total of 9275 cases, there were 303 cases (3.3%) of Klinefelter's syndromes, 11 cases (0.1%) of Y chromosomal abnormalities. 2. In 102 cases of patients showed typical clinical features of Klinefelter's syndrome, 101 cases (99%) of them were diagnosed to Klinefelter's syndrome in karyotyping. 3. In 303 cases of Klinefelter's syndrome, there were 277 cases (91.4%) of 47,XXY complement, 16 cases (5.3%) of mosaicism, 2 cases (0.7%) of 48,XXXY, 5 cases (1.7%) of 48,XXYY and 3 cases (1.0%) of 49,XXXXY. 4. In 303 cases of Klinefelter's syndrome, 284 cases (93.7%) of them were diagnosed after puberty and only 19 cases (6.3%) of them were diagnosed before puberty. 5. In 303 cases of Klinefelter's syndrome, there were 146 cases (48.2%) of patients with infertility-associated chief complaints, 101 cases (33.3%) of patients with typical clinical features of Klinefelter's syndrome, 22 cases (7.3%) of patients with ambiguous genitalia. 6. In patients with Klinefelter's syndrome, 48,XXYY and 49,XXXXY had serious symptoms such as mental retardation, developmental delay, Down syndrome-like features, congenital anomalies, but 48,XXYY and other mosaicisms had infertility-associated symptoms or ambiguous genitalia. 7. The 8 cases of polysomy Y (XYY complement) showed several serious symptoms such as Down syndrome-like features, mental retardation, fragile X syndrome-like feature, congenital anomalies, ambiguous genitalia which could be detected before puberty.

There have been many reports to date regarding the role of GnRH as a local regulatory factor of ovarian function as studies of human and rat ovaries revealed GnRH and its receptor. In recent studies it has been shown that GnRH directly causes apoptosis in the granulosa cells of the rat ovary, and such results leads to the suggestion that the use of GnRH agonist for more stable long term ovarian hyperstimulation in human IVF-ET programs causes granulosa cell apoptosis which may lead to follicular atresia. Therefore this study attempts to determine if granulosa-luteal cell apoptosis occurs in patients during IVF-ET programs in which GnRH agonist is employed for ovarian hyperstimulation. The quality of oocyte-cumulus complexes obtained during ovum pickup procedures were assessed morphologically and then the fertilization rate and developmental rate was determined. Apoptotic cells among the granulosa-luteal cells obtained during the same procedure were observed after staining with Hematoxylin-eosin. The fragmentation degree of DNA extracted from granulosa-luteal cells was determined and comparatively analyzed. There was no difference in the average age of the patients, the number of oocytes retrieved, and fertilization and developmental rates between the FSH/hMG group and GnRH-long group. There was also no difference in the apoptosis rate and pyknosis rate in the granulosa-luteal cells between the two groups. However, when the oocyte-cumulus complexes were morphoogically divided into the healthy group and atretic group without regard for the method of hyperstimulation, the results showed that the number of oocytes obtained averaged $11.09{\pm}8.75\;and\;10.33{\pm}4.53$ per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental rate (65.96%, 41.51%, respectively, p<0.01) was significantly increased in the healthy group when compared to the atretic group. The degree of apoptosis in the granulosa-luteal cells showed that in the healthy group it was 2.25% which was not significantly different from the atretic group (2.77%), but the pyknosis rate in the atretic group (27.81%) was significantly higher compared to the healthy group (11.35%, p<0.01). The quantity of DNA fragmentation in the FSH/hMG group was 32.22%, while in the GnRH-long group it was 34.27%, showing no significant difference. On the other hand the degree of DNA fragmentation was 39.05% and 11.83% in the healthy group and atretic group, respectively, showing significantly higher increase in the atretic group (p<0.01). The above results suggest that death of granulosa-luteal cells according to the state of the oocyte-cumulus complex is more related to pyknosis rather than apoptosis. Also, the GnRH agonist used in ovarian hyperstimulation does not seem to directly affect the apoptosis of retrieved oocytes and granulosa-luteal cells, and which is thought to be due to the suppression of the apoptogenic effect of GnRH agonist as a result of the high doses of FSH administered.

The insulin-like growth factor (IGF)s are believed to one of several growth factors that play an adjunctive role in ovarian follicular development. These factors circulate bound to a family of IGF-binding protein (IGFBP)s. It is known that circulating IGFBPs are involved in the transport of IGFs to tissues and modulate IGFs actions at local tissue. The purposes of this study were to evaluate changes in serum IGFBPs profiles during normal ovulatory menstrual cylce and to compare serum IGFBPs profiles in periovulatory phase of between normal ovulatory menstrual cylce and controlled hyperstimulated cycle. Fasting blood samples were obtained from 15 normal healthy women throughout normal ovulatory menstural cyle and on the day of aspiration of oocyte from 10 patients undergoing ovarian hyperstimuation for in vito fertilization-embryo transfer. Serum IGFBP-1 - IGFBP-4 were measured by western ligand blot and immunoprecipitation. Serum $17{\beta}$-estradiol was determined by radioimmunoassay. Type and molecular weight of serum IGFBP did not changed during normal ovulatory menstural cycle. No significant variation in the relative proportion and level of each IGFBP was found throughout normal ovulatory menstural cyle. Also, the relative proportion and level of each IGFBP did not correlated with serum $17{\beta}$-estradiol level. There was no significant difference in the relative proportion and level of each serum IGFBP between on the day of ovulation in normal ovulatory menstrual cylce and on the day of aspiration of oocyte in controlled hyperstimulated cycle. Our data indicate that IGFBPs have regulatory functions in ovary through an paracrine and autocrine rather than endocrine mechanism during normal ovulatory menstural cycle.

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

Since the blastocyst is broken and spreads out on a flat plastic culture dish (two dimensional culture) during in vitro development, it has been difficult to study the implantation process. It also has been difficult to analyse the interactions between endometrial epithelial and stromal cells because of the lack of a long-term in vitro model which can stimulate in vivo characteristics, as these cells eventually fail to proliferate or cease to express differentiated functions. Recently nontransformed cell lines, CUE-P and CUS-V2, derived from rat endometrial epithelium and stroma were reported. In this study, morphology of CUE-P and CUS-V2 was examined and oxytocin gene expression by CUE-P cells was demonstrated by RT-PCR. The CUE-P cells have a cuboidal morphology and CUS-V2 cells resemble fibroblast and exhibit a spindle-like morphology. In RT-PCR, same size of PCR products of oxytocin gene at hypothalamus, uterus and CUE-P cells were demonstrated. These results showed three dimensional culture system could be made by using the new cell lines.

당뇨병은 생식내분비 조직의 구조나 기능 변화를 유발하여 호르몬 합성 및 분비량의 변화를 야기하고, 정자의 운동성 등에 영향을 미치는 것으로 알려져 왔다. 그러나 부정소와 수정관내 정자의 농도 변화나 수정능력 획득 및 침체반응에 미치는 영향은 잘 알려져 있지 않다. 본 실험에서는 Wistar 쥐에 streptozotocin을 투여하여 당뇨병을 유발시킨 후 3일과 14일에 부정소 각 부위와 수정관내 정자 농도 변화를 조사하고 부정소 꼬리와 수정관내 정자의 침체반응 유도 실험 (acrosome reaction to ionophore challenge test, ARIC test)을 이용하여 정자의 수정 능력을 평가하였다. Streptozotocin을 주사한 후 혈액내 인슐린 및 포도당 농도는 당뇨병 경과 기간이 길어짐에 따라 반비례 관계를 보였다. 부정소 머리와 몸통내 정자의 농도는 3일군에서부터 감소하기 시작하나 꼬리에서는 14일군 $(15.2{\pm}2.1)$에서 대조군 $(28.1{\pm}4.0)$이나 3일군에 $(24.8{\pm}2.9)$비해 유의하게 감소하였다. 수정관내 절자 농도는 14일군이 $0.025{\pm}0.013$으로 대조군과 $(0.108{\pm}0.03)$ 3일군에 $(0.067{\pm}0.046)$ 비해 유의하게 감소하였으며, 3일군도 대조군에 비해 유의한 차이를 보였다. 자발적 첨체반응율은 대조군의 부정소 꼬리정자는 $37.1{\pm}2.4$이고 수정관내 정자는 $49.3{\pm}2.4$로 두 부위간 유의한 차이를 보였다. 3일군과 14일군의 부정소 꼬리와 수정관내 정자의 자발적 첨체반응율은 대조군에 비해 유의하게 증가하였다. 한편 14일군의 수정관내 정자의 자발적 첨체반응율은 대조군이나 3일군에 비해 유의하게 증가하였다. ARIC test 결과 대조군과 3일군에서는 20%이상 차이를 보였으나 14일군에서는 약 8.4% 차이를 보였다. 위의 결과가 부정소의 성숙 조절기능 이상 또는 정자형성 이상에 기인한 것인지는 더 연구되어야 하나 당뇨병 병력이 길어짐에 따라 정자의 수적인 감소, 자발적 침체반응의 증가나 침체반응의 약화가 유발되어 생식능력의 감소 원인으로 작용하는 것으로 사료된다.

Male genital tract inflammatory conditions may be associated with unexplained infertility. The presence of cytokine such as tumor necrosis factor-alpha (TNF-${\alpha}$) was reported in the semen of infertile men. However, the effect of these cytokines on human sperm function is still unclear. The purpose of this study was to investigate the in-vitro effects of TNF-alpha on human sperm motility with computer assisted sperm analysis. Washed sperm from 16 normal men were incubated without and with TNF-${\alpha}$ (0.1, 10, 1000 ng/ml). The changes of parameters of sperm motility were recorded at different time intervals (0, 5, 24 hour). There was no significant change of parameters of sperm motility in the incubation with TNF-${\alpha}$. It is suggested that TNF-${\alpha}$ alone does not interfere with the sperm motility and more studies are needed.

Objective: Heat shock protein 70-2 (Hsp70-2) gene knockout mice are found to have premeiotic arrest at the primary spermatocyte stage with a complete absence of spermatids and spermatozoa. This observation led to the hypothesis that hspA2 may be disrupted in human testes with abnormal spermatogenesis. To test this hypothesis, we studied the mRNA expression of hspA2 in infertile men with azoospermia. Design: The mRNA expression were analyzed by competitive RT-PCR among testes with normal spermatogenesis, pachytene spermatocyte arrest, and sertoli-cell only syndrome. Materials and methods: Testicular biopsy was performed in men with azoospermia (n=15). Specimens were subdivided into three groups: (group 1) normal spermatogenesis (n=5), (group 2) spermatocyte arrest (n=5), (group 3) Sertoli-cell only syndrome (n=5). Total RNA was extracted by Trizol reagent. Total extracted RNA was reverse transcribed into cDNA and amplified by PCR using specific primers for hspA2 target cDNAs. A competitive cDNA fragment was constructed by deleting a defined fragment from the target cDNA sequence, and then coamplified with the target cDNA for competitive PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal control. Results: On Competitive RT-PCR analyses for hspA2 mRNA, significant amount of hspA2 expression was observed in group 1, whereas a constitutively low level of hspA2 was expressed in groups 2 and 3. Conclusion(s): The study demonstrates that the hspA2 gene expression is down-regulated in human testes with abnormal spermatogenesis, which in turn suggests that hspA2 gene may play a specific role during meiosis in human testes.

Postpubertal mumps may result in ochitis and permanent testicular atrophy may develop following infection. This present study was initiated to evaluate the preventive effect of interferon-${\alpha}2B$ on infertilty after mumps orchitis. There were 21 patients with mumps orchitis between May 1990 and June 1997. Patients were randomly distributed into 2 groups: group 1 patients (n=13) maintained therapy with interferon-${\alpha}2B$ ($3{\times}10^6$ IU per day) and group 2 were managed by conservatively. All of the patients were evaluated with testis size measurement, mumps virus titer, hormone level, and if possible semen analysis. For group 1 patients symptoms disappeared within 2 to 3 days and the volume of testis returned to normal within 11 days and testis atrophy was not observed in all patients in follow up. But asthenospermia was continued in 4 patients (unilateral 2, bilateral 2). For group 2 patients symptoms disappeared within 5 to 6 days and the volume of testis returned to normal within 10 days and testis atrophy was observed in 3 patients (unilateral 2, bilateral 1) in floow up. Asthenospermia was continued in 4 patients (unilateral 2, bilateral 2). Sperm count and morphology were recovered all the recover in group 1, 4 patients had persistent reduced sperm count and morphology in group 2, respectively. These observations suggest that systemic interferon-${\alpha}2B$ treatment is highly effective in preventing infertilty as well as testicular atrophy after mumps orchitis.

Although anejaculation is a relatively uncommon occurrence in the general population, over 12,000 new cases are reported annually. Anejaculation may result from spinal cord injury, retroperitoneal lymph node dissection, diabetes mellitus, transverse myelitis, multiple sclerosis, or psychogenic disorders. At least 30% of men with this problem are or will be married and many will seek help to remedy their infertile state. The evolution of technique and instrumentation over the last 30 years has made electroejaculation an accessible and acceptable form of therapy. Recent successes in inducing ejaculation by means of rectal probe electrostimulation or vibratory stimulation combined with assisted reproductive techniques, such as zygote intrafallopian transfer (ZIFT), gamete intrafallopian transfer (GIFT), and in vitro fertilization (IVF), have provided these men means of producing their own biologic offspring. We have experienced a successful pregnancy with electroejaculation and in vitro fertilization in a infertile patient whose husband had an ejaculatory disturbance due to a spinal cord injury. So we report this case with a brief review of literatures.

A successful outcome of pregnancy requires an efficient uteroplacental vascular system. Since this system may be compromised by disorders of haemostasis associated with a prothrombotic state, maternal thrombophilia might be a risk factor for fetal loss. Hereditary deficiencies of the naturally occuring anticoagulants are well recognized conditions predisposing to recurrent venous thromboembolism. Since thrombotic phenomena have been implied as a cause of abortion and stillbirth, these deficiencies might increase the risk of fetal demise. We have experienced a case of antiphospholipid syndrome associated with protein C deficiency in patient with recurrent spontaneous abortion. So we report this case with a brief review of literatures.