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.
Animals ; Blastocyst ; Cell Line* ; Epithelium* ; Fibroblasts ; Gene Expression ; Hypothalamus ; Oxytocin ; Plastics ; Polymerase Chain Reaction ; Rats* ; Stromal Cells ; Uterus

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 cycle and on the day of aspiration of oocyte from 10 patients undergoing ovarian hyperstimuation for in vitro fertilization-embryo transfer. Serum IGFBP-1-IGFBP-4 were measured by western ligand blot and immunoprecipitation. Serum 17beta-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 17beta-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.
Fasting ; Female ; Humans ; Immunoprecipitation ; Insulin-Like Growth Factor Binding Proteins* ; Intercellular Signaling Peptides and Proteins ; Menstrual Cycle* ; Molecular Weight ; Oocytes ; Ovary ; Ovulation ; Radioimmunoassay

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-rosin. 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+/-8.75 and 10.33+/-4.53 per cycle, respectively, showing no significant difference, but the fertilization rate (77.05%, 56.99%, respectively, p<0.01) and developmental ,ate (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.
Animals ; Apoptosis* ; DNA ; DNA Fragmentation ; Female ; Fertilization ; Follicular Atresia ; Gonadotropin-Releasing Hormone* ; Granulosa Cells ; Hand ; Humans* ; Luteal Cells ; Oocytes ; Ovary ; Ovum ; Rats

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 analyis 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.
Adolescent ; Chromosome Aberrations ; Complement System Proteins ; Cytogenetics* ; Disorders of Sex Development ; Genitalia ; Gonads ; Humans ; Infertility ; Intellectual Disability ; Karyotype ; Karyotyping ; Klinefelter Syndrome ; Korea ; Mosaicism ; Puberty ; Reproductive Medicine ; Seoul ; Y Chromosome

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 100 mg 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 (E2). 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 (<10x106 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% (l1/63) 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 (E2) 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.
Abortion, Induced ; Age Factors ; Clomiphene* ; Estradiol ; Family Characteristics ; Female ; Gestational Sac ; Gonadotropins* ; Humans ; Infertility ; Insemination* ; Male ; Menstrual Cycle ; Ovary ; Pregnancy Rate* ; Pregnancy* ; Pregnancy, Multiple ; Spouses ; Ultrasonography

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.
Endometrium ; Female ; Head ; Humans ; Infertility ; Inflammation ; Semen ; Sperm Motility* ; Spermatozoa*

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 alphal, alpha4, beta3, and cyclooxygenase-1, -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 studied into a new culture environment which would allow longer periods of culture will be necessary.
Antigens, CD11a ; Centrifugation ; Collagen ; Collagenases ; Cyclooxygenase 1 ; Embryonic Structures ; Endometrium ; Epithelial Cells ; Estradiol ; Female ; Humans* ; Hysterectomy ; Immunohistochemistry ; Microscopy, Electron ; Microvilli ; Progesterone ; Stromal Cells ; Tight Junctions

OBJECTIVE: This study has been carried out to evaluation the effect of fertilization promoting peptide (FPP) on the kinematic parameters, capacitation and acrosome reaction of the frozen-thawed human spermatozoa. METHODS: After FPP treatment, we examined kinematic parameters, capacitation and acrosome reaction, using the methods of computer-aided sperm analysis (CASA) and chlortetracycline (CTC) fluorescence analysis. RESULTS: We have obtained the evidence that FPP can promote the capacitation and inhibit the spontaneous acrosome reaction of frozen-thawed human spermatozoa in vitro. Fpp (25~100 nM) induced a significant increase in the proportion of B-pattern capacitated spermatozoa, and a significant decrease in the proportion of F-pattern uncapacitated ones without significant stimulation of acrosomal exocytosis. In the kinematic parameters treatment, FPP treated groups maintained higher LIN, BCF and STR than those of control. The VAP, VSL, VCL and ALH were not different. Therefore it is suggested that FPP in human seminal plasma may play a positive role in promoting human sperm function.
Acrosome Reaction* ; Acrosome* ; Chlortetracycline ; Exocytosis ; Fertilization* ; Fluorescence ; Humans* ; Male ; Semen ; Spermatozoa*

OBJECTIVE: This study was carried out to compare the effects of the stepwise exposure treatments on the morphological normality, fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing and to use as a fundamental data for the cryopreservation of human oocytes. MATERIALS AND METHODS: The morphological normality and fertilization rates of the vitrified and ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were observed. After choosing the 3step exposure treatment groups, we observed the morphological normality and fertilization, blastocyst formation rate vitrified and ultra-rapid frozen mouse mature oocytes. RESULTS: The morphological normality and fertilization rates of the vitrified mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 75%, 85%, 88% and 58%, 61%, 54% respectively. There were no significant differences among treatments (p>0.05). The morphological normality and fertilization rates of the control was 92% and 65%. There were no significant differences in fertilization rate among control and treatments (p>0.05). The morphological normality and fertilization rates of the ultra-rapid frozen mouse mature oocytes after three-stepwise exposure treatments (1step, 3step and 5step) were 83%, 83%, 84% and 75%, 63%, 56% respectively. There were no significant differences among treatments (p>0.05). The morphological normality and fertilization rate of the control was 95% and 67%. There were no significant differences among control and treatments (p>0.05). The morphological normality and fertilization rate of the vitrified or ultra-rapid frozen mouse mature oocytes after 3step exposure treatment were 69% and 75%, respectively. The blastocyst formation rate was 60% and 57%. The results did not differ significantly between vitrification and ultra-rapid freezing (p>0.05). CONCLUSION: As known in the above results, there were no significant differences in the fertilization and blastocyst formation rate of the frozen-thawed mouse mature oocytes by vitrification or ultra-rapid freezing among the control and treatments. It is suggested that vitrification and ultra-rapid freezing method were effective for the cryopreservation of mouse mature oocytes.
Animals ; Blastocyst ; Cryopreservation ; Fertilization ; Freezing* ; Humans ; Mice* ; Oocytes* ; Vitrification*

No abstract available.
Animals ; Embryonic Structures* ; Gene Expression* ; Interleukin-1* ; Mice*