Effects of Autologous Platelet-Rich Plasma on Regeneration of Damaged Endometrium in Female Rats

PURPOSE: To investigate whether autologous platelet-rich plasma (PRP) treatment can improve regeneration of the endometrium in an experimental model of ethanol-induced damage. MATERIALS AND METHODS: Sixty female Sprague-Dawley rats were randomly assigned into three groups: control group, ethanol group, and PRP-treated group (administration of 0.25 mL of PRP into both uterine cavities 72 hours after ethanol injection). After 15 days of endometrial damage, all the animals were sacrificed during the estrous cycle, and samples were taken from the mid-uterine horn. Functional and structural recovery of the endometrium was analyzed by hematoxylin-eosin (H&E) and Masson trichrome (MT) staining, real-time polymerase chain reaction (PCR) assay, and immuno-histochemical (IHC) analyses. RESULTS: H&E and MT staining confirmed significantly decreased fibrosis and increased cellular proliferation in the PRP-treated group, compared to the ethanol group. The endometrial areas in the ethanol and PRP-treated groups were 212.83±15.84 µm² and 262.34±12.33 µm² (p=0.065). Significantly stronger IHC expression of cytokeratin, homeobox A10 (HOXA10), vascular endothelial growth factor (VEGF), and Ki-67 was found in the PRP-treated group, compared to the ethanol group. In real-time PCR analyses, interleukin-1β mRNA was down-regulated, while c-Kit mRNA was up-regulated, in the PRP-treated group, compared to the ethanol group. CONCLUSION: Intrauterine administration of autologous PRP stimulated and accelerated regeneration of the endometrium and also decreased fibrosis in a murine model of damaged endometrium.

Salt-sensitive genes and their relation to obesity / 한국영양학회지

PURPOSE: Although it is well known thatmortality and morbidity due to cardiovascular diseases are higher in salt-sensitive subjects than in salt-resistant subjects, their underlying mechanisms related to obesity remain unclear. Here, we focused on salt-sensitive gene variants unrelated to monogenic obesity that interacted with sodium intake in humans. METHODS: This review was written based on the modified 3(rd) step of Khans' systematic review. Instead of the literature, subject genes were based on candidate genes screened from our preliminary Genome-Wide Association Study (GWAS). Finally, literature related to five genes strongly associated with salt sensitivity were analyzed to elucidate the mechanism of obesity. RESULTS: Salt sensitivity is a measure of how blood pressure responds to salt intake, and people are either salt-sensitive or salt-resistant. Otherwise, dietary sodium restriction may not be beneficial for everyone since salt sensitivity may be associated with inherited susceptibility. According to our previous GWAS studies, 10 candidate genes and 11 single nucleotide polymorphisms (SNPs) associated with salt sensitivity were suggested, including angiotensin converting enzyme (ACE), α-adducin1 (ADD1), angiotensinogen (AGT), cytochrome P450 family 11-subfamily β-2 (CYP11β-2), epithelial sodium channel (ENaC), G-protein b3 subunit (GNB3), G protein-coupled receptor kinases type 4 (GRK4 A142V, GRK4 A486V), 11β-hydroxysteroid dehydrogenase type-2 (HSD 11β-2), neural precursor cell-expressed developmentally down regulated 4 like (NEDD4L), and solute carrier family 12(sodium/chloride transporters)-member 3 (SLC 12A3). We found that polymorphisms of salt-sensitive genes such as ACE, CYP11β-2, GRK4, SLC12A3, and GNB3 may be positively associated with human obesity. CONCLUSION: Despite gender, ethnic, and age differences in genetics studies, hypertensive obese children and adults who are carriers of specific salt-sensitive genes are recommended to reduce their sodium intake. We believe that our findings can contribute to the prevention of early-onset of chronic diseases in obese children by facilitating personalized diet-management of obesity from childhood to adulthood.

Impact of glycosylation on the unimpaired functions of the sperm / 대한생식의학회지

One of the key factors of early development is the specification of competence between the oocyte and the sperm, which occurs during gametogenesis. However, the starting point, growth, and maturation for acquiring competence during spermatogenesis and oogenesis in mammals are very different. Spermatogenesis includes spermiogenesis, but such a metamorphosis is not observed during oogenesis. Glycosylation, a ubiquitous modification, is a preliminary requisite for distribution of the structural and functional components of spermatids for metamorphosis. In addition, glycosylation using epididymal or female genital secretory glycans is an important process for the sperm maturation, the acquisition of the potential for fertilization, and the acceleration of early embryo development. However, nonemzymatic unexpected covalent bonding of a carbohydrate and malglycosylation can result in falling fertility rates as shown in the diabetic male. So far, glycosylation during spermatogenesis and the dynamics of the plasma membrane in the process of capacitation and fertilization have been evaluated, and a powerful role of glycosylation in spermatogenesis and early development is also suggested by structural bioinformatics, functional genomics, and functional proteomics. Further understanding of glycosylation is needed to provide a better understanding of fertilization and embryo development and for the development of new diagnostic and therapeutic tools for infertility.

Regulation and 3 dimensional culture of tertiary follicle growth / 대한생식의학회지

It has been revealed that multiple cohorts of tertiary follicles develop during some animal estrous cycle and the human menstrual cycle. To reach developmental competence, oocytes need the support of somatic cells. During embryogenesis, the primordial germ cells appear, travel to the gonadal rudiments, and form follicles. The female germ cells develop within the somatic cells of the ovary, granulosa cells, and theca cells. How the oocyte and follicle cells support each other has been seriously studied. The latest technologies in genes and proteins and genetic engineering have allowed us to collect a great deal of information about folliculogenesis. For example, a few web pages (http://www.ncbi.nlm.nih.gov; http://mrg.genetics.washington.edu) provide access to databases of genomes, sequences of transcriptomes, and various tools for analyzing and discovering genes important in ovarian development. Formation of the antrum (tertiary follicle) is the final phase of folliculogenesis and the transition from intraovarian to extraovian regulation. This final step coordinates with the hypothalamic-pituitary-ovarian axis. On the other hand, currently, follicle physiology is under intense investigation, as little is known about how to overcome women's ovarian problems or how to develop competent oocytes from in vitro follicle culture or transplantation. In this review, some of the known roles of hormones and some of the genes involved in tertiary follicle growth and the general characteristics of tertiary follicles are summarized. In addition, in vitro culture of tertiary follicles is also discussed as a study model and an assisted reproductive technology model.

Concanavalin A Mediated Calcium Changes on Expansion and Hatching of the Mouse Blastocyst / 대한불임학회지

OBJECTIVE: The oligosaccharide moieties of glycoproteins and proteoglycans have a vital function in blastocyst differentiation. Concanavalin (ConA), a lectin, is known to bind on the preimplantation embryos, especially on blastocyst. In this study, we investigated whether ConA can modulate the trophoblast development and about the regulating mediator. Also, we investigated whether expansion is enough for hatching procession of the mouse blastocyst. METHOD: Embryos were collected at 72 h post hCG injection and chemicals were treated after 24 h (96 hr post hCG injection). ConA or calcium ionophore A23187 were exposed to blastocyst and than analysis the developmental process for 48 hr. Intracellular free-Ca2+ concentration in trophectoderm was measured with confocal laser microscope after exposing to ConA or calcium ionophore A23187. ConA-pretreated blastocyst exposed to the calcium ionophore A23187 and then analyzed the developmental process. Otherwise ouabain was treated to the blastocyst to block the Na+/K+-ATPase activity. RESULTS: In contrast to the control blastocyst, the ConA-exposed blastocysts developed beyond the expansion stage with significantly high rate (90.4%) at 12 h post administration. ConA induced an increase the intracellular Ca2+ concentration in trophectoderm. Calcium ionophore A23187 also stimulated expansion of blastocyst. Most of the control blastocysts developed to the hatching stage at 144 h post hCG injection. However, strongly 65% of the ConA-exposed embryos were arrested at expanded stage at same time point. The developmental progression rates to hatching stage of both ConA- and calcium ionophore A23187-expose blastocysts were significantly lower than that of the control. However ConA-pretreated embryos developed to the hatching stage like control embryos. Ouabain showed a tendency to delayed the progress to expansion stage but did not inhibit the development to the hatching stage. CONCLUSION: ConA-mediated expansion is the result of the increase of intracellular free-calcium in blastocyst stage embryo. It is suspected that expansion of the blasocyst is a essential indirect factor in hatching and the calcium may triggering the cellular mechanisms for the both expansion and hatching progression.

Expression of doc-1 in Pregnant Uterus of the Mouse / 대한불임학회지

Uterine cells carry out proliferation and differentiation for preparation the embryonic implantation during pregnancy. Therefore regulation of the cell proliferation is an essential step for uterine preparation, but there is not much information about the proliferation related genes in pregnant uterus. To identify these implantation specific genes, a PCR-select cDNA subtraction method was employed and got a few genes. One of the identified genes is a novel gene encoding oral tumor suppressor doc-1. To detect the doc-1 expression on the pregnant uterus, dot blotting, RT-PCR, and in situ hybridization were employed. Dot blotting revealed that doc-1 mRNA expression increase after implantation. During normal pregnancy, doc-1 mRNA expression was detected as early as day 1 of pregnancy with RT-PCR. Its expression was increased about 15 times after embryonic implantation. doc-1 transcript was localized in luminal epithelial cells but it was very faint during preimplantation. After starting the implantation, it localized in the stromal cells; heightened expression of doc-1 correlates with intense stromal cell proliferation surrounding the implanting blastocyst on day 6 morning. However in the decidualized cells, the intensity of localized doc-1 mRNA was weak. From those results, it is revealed that doc-1 express at pregnant uterus of the mouse. In addition it is suggested that doc-1 is the gene regulating the proliferation of the luminal epithelial cells and stromal cells during early implantation and decidualization.

Expression of doc-1 in Pregnant Uterus of the Mouse / 대한불임학회지

Uterine cells carry out proliferation and differentiation for preparation the embryonic implantation during pregnancy. Therefore regulation of the cell proliferation is an essential step for uterine preparation, but there is not much information about the proliferation related genes in pregnant uterus. To identify these implantation specific genes, a PCR-select cDNA subtraction method was employed and got a few genes. One of the identified genes is a novel gene encoding oral tumor suppressor doc-1. To detect the doc-1 expression on the pregnant uterus, dot blotting, RT-PCR, and in situ hybridization were employed. Dot blotting revealed that doc-1 mRNA expression increase after implantation. During normal pregnancy, doc-1 mRNA expression was detected as early as day 1 of pregnancy with RT-PCR. Its expression was increased about 15 times after embryonic implantation. doc-1 transcript was localized in luminal epithelial cells but it was very faint during preimplantation. After starting the implantation, it localized in the stromal cells; heightened expression of doc-1 correlates with intense stromal cell proliferation surrounding the implanting blastocyst on day 6 morning. However in the decidualized cells, the intensity of localized doc-1 mRNA was weak. From those results, it is revealed that doc-1 express at pregnant uterus of the mouse. In addition it is suggested that doc-1 is the gene regulating the proliferation of the luminal epithelial cells and stromal cells during early implantation and decidualization.

Mouse embryo culture and implantation by Type I Collagen / 대한산부인과학회잡지

OBJECTIVES: To examine the in vitro interactions of blastocyst attachment using type I collagen. MATERIALS AND METHODS: ICR mice were used and follicular growth was stimulated by pregnant mare serum gonadotropin and human chorionic gonadotropin. On day 4 of pregnancy, the uteri were removed and blastocysts were flushed. Mixtures of 1mL sterile water, 0.5mL DMEM, 2mL type collagen solution and 0.5mL 0.1M NaOH were prepared and transferred to an incubator where the collagen solution polymerized. Blastocysts were transferred to dishes previously coated with type I collagen. CMRL 1066 was used as the basic culture medium. It was supplemented with 1mM glutamine and 1mM sodium pyruvate plus 50 IU/ml penicillin and 50 mg/ml streptomycin. During the first 4 days the culture medium was supplemented with 20% fetal calf serum and thereafter with 20% heat inactivated human cord serum. All blastocysts were initially cultured for 2 days without media change. After 2 days, fresh medium was renewed daily. The stages of embryo growth were examined and recorded everyday under a dissecting microscope and classified according to the standard in vivo criteria set forth by Witschi. RESULTS: By 48h, nearly all blastocysts had attached to the surface of collagen pad. Following adhesion to the collagen pad, the blastocysts maintained their 3-dimensional integrity in contrast to control. The embryos in collagen pad were not flattening and kept polarity and spherical shape during culture. The polar trophoblast invaded the type I collagen downward unlike the horizontal growth in control. In the developmental stage of mouse blastocyst, there were significant differences between control and type I collagen group during day 4 and 5 culture. CONCLUSION: Blastocyst development was better in type I collagen group than control. Therefore, in vitro culture study using type I collagen could provide improved model for the establishment of blastocyst implantation study.

Spermatozoa Characteristics of Streptozotocin-induced Diabetic Wistar Rat: Acrosome Reaction and Spermatozoa Concentration / 대한불임학회지

SUMMARY: Some of the information concerning sexual function in the male diabetes has been focused upon the problems of endocrine or semen parameters. However, the characteristics of acrosome reaction and spermatozoa concentration at the epididymis and vas deferens have scarcely been studied, and the causes of the infertility has not been critically identified. So, we designed to inspect the spermatozoa concentration and the characteristics of acrosome reaction at epididymis and was deferens of diabetic Wistar rat induced by streptozotocin (STZ, 70 mg/kg, ip). Experimental animal was sacrificed at 3 days and 14 days after the STZ injection. In the diabetes-induced rat, the levels of insulin and glucose had a pattern of inverse proportion. The spermatozoa concentrations in caput and corpus epididymis were significantly decreased in all diabetic condition. In cauda epididymis, however, there was significant decrease in sperm concentration at 14 days onward. In diabetic rat, the spontaneous reaction rate of spermatozoa of cauda and was deferens were significantly higher than the control group. The ARIC (acrosome reaction to ionophore challenge) value of caudal sperm was 28.7 at control, 22.1 at 3 days, and 8.3 at 14 days. In the present study the spermatozoa concentration was decreased and the spontaneous reaction rate was increased by diabetes. In ARIC-test, it is revealed that the fertility of spermatozoa of 14 days group was lower than control or 3 days group. Diabetes mellitus may be provoke the decreased fertilization rate and subsequent infertility.

Intracytoplasmic Sperm Injection Using Frozen-Thawed and Fresh Testicular Sperm in Patients with Azoospermia / 대한산부인과학회잡지

OBJECTIVE: To investigate whether frozen-thawed testicular sperm obtained from men with azoospermia could serve as an efficacious sperm source for intracytoplasmic sperm injection (ICSI) by comparing to the results of ICSI using fresh testicular sperm. METHODS: From January 1997 to March 1999, 41 patients with azoospermia who underwent ICSIs using fresh and/or frozen-thawed testicular sperm were included in the study. In 23 patients of 41, fresh testicular sperm was left after ICSI and therefore remaining testicular sperm was frozen and frozen testicular sperm was used in next ICSI cycles. The results of ICSI were compared in frozen-thawed testicular sperm (frozen-thawed group, 30 cycles) versus fresh testicular sperm (fresh group, 39 cycles). RESULTS: The number of fertilized oocytes, grade I/II embryos, fertilization rate, clinical pregnancy rate were comparable in the frozen-thawed and fresh groups. There were also no differences in the miscarriage rate and multiple pregnancy rate between the two groups. In patient group with obstructive azoospermia, there were no significant differences in the number of fertilized oocytes, grade I/II embryos, fertilization rate, clinical pregnancy rate between the two groups. In patient group with non-obstructive azoospermia, all parameters of results of ICSI were comparable in both groups. In each non-obstructive azoospermic patient group with mixed motile/immotile sperm and patient group with only immotile sperm, there were also no significant differences in the number of fertilized oocytes, grade I/II embryos, fertilization rate, clinical pregnancy rate between the frozen-thawed and fresh groups. CONCLUSION: Our data demonstrate that using frozen-thawed and fresh testicular sperm gives rise to comparable results after ICSI irrespective of the status of sperm in patients with obstructive or non-obstructive azoospermia.

The Effect of Granulocyte Colony Stimulating Factor and Granulocyte Macrophage Colony Stimulating Factor on Expression of Matrix Metalloproteinase-2, 9 in Mouse Embryos / 대한산부인과학회잡지

OBJECTIVES: To investigate the effect of granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) on expression of matrix metalloproteinase-2, 9 (MMP-2, 9) mRNA in mouse embryos. Materials and METHOD: From October 1997 to December 1998, morula stage mouse embryos were cultured for 48 hours with G-CSF and GM-CSF at concentrations of 0.1 pg/ml, 1 pg/ml, 10 pg/ml, 100 pg/ml, 1 ng/ml and 10 ng/ml, respectively. Embryos not treated with G-CSF or GM-CSF were served as control. Reverse transcription-polymerase chain reaction (RT-PCR) has been used to examine the expression of MMP-2, 9 mRNA in developed blastocysts. Following reverse transcription, strategically designed nested primers, optimized for specificity, were used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. Results were analyzed with Kolmogorov-Smirnov test and analysis of variance (ANOVA). The statistical significance was defined as p< 0.05. RESULTS: The relative quantities (relative volume x intensity) of MMP-2 mRNA expressed in embryos of all G-CSF treatment groups were significantly increased than in the control, especially in 10, 100 pg/ml and 1 ng/ml treatment groups. The relative quantities of MMP-2 mRNA in all GM-CSF treatment groups were also significantly increased than in the control, especially in 100 pg/ml treatment group. The relative quantities of MMP-9 mRNA of all GM-CSF treatment groups except 10 ng/ml group were significantly increased than in the control, especially 10, 100 pg/ml and 1 ng/ml treatment group. However, the relative quantity of MMP-9 mRNA was significantly increased in only 10 ng/ml G-CSF treatment group than in the control and other treatment groups. CONCLUSION: This study suggests that G-CSF and GM-CSF may increase the m-RNA expression of MMP-2 or 9 in mouse blastocysts with the concentration-specific manner.

A Case of Successful Pregnancy in Patient with Recurrent Spontaneous Abortion by Preimplantation Genetic Diagnosis Following IVF-ET / 대한불임학회지

It was reported that the etiologies of recurrent spontaneous abortion are immunologic factors, endocrinologic problems, anatomical abnormalities, genetic abnormalities, infection, and unexplained factors. Among those etiologic factors, genetic abnormalities occur in about 5% of the couples who experience recurrent spontaneous abortions, and most common parental chromosomal abnormality contributing to recurrent abortion is balanced translocation. The advent of in vitro fertilization (IVF), the development of skills associated with the handling of human embryo, and an explosion of knowledge in molecular biology have opened the possibility of early diagnosis of genetic disease in preimplantation embryos. Therefore preimplantation genetic diagnosis (PGD) is indicated for couples, infertile or not, at risk of transmitting a genetic disease. A case of successful pregnancy and term delivery by PGD using fluorescence in situ hybridization (FISH) technique in patient with recurrent spontaneous abortion due to balanced translocation is presented with brief review of literatures.

A Stimulated Acrosome Reaction Test as a Prognostic Factor in In Vitro Fertilization / 대한불임학회지

It is well known that the clinical test for responsibility of accurate fertilization capacity in male partners is very important to diagnose and treat the infertility. However, it has been reported that the traditional semen analysis cannot accurately predict fertilization and pregnancy potential. The present study was performed to evaluate the acrosomal reaction to ionophore challenge (ARIC) test as a prognostic indicator for fertilization of sperm and oocyte in an in vitro fertilization and embryo transfer (IVF-ET) program. From March 1996 to Februry 1997, 30 couples undergoing IVF program were allocated to this study group. All female partners in the study group were 35 years old or less and their serum level of basal follicle stimulating hormone (FSH) and estradiol (E2) were normal. All the male partners have normal parameters of semen analysis. The ARIC tests were performed on the day of ovum pick up and in vitro insemination in all the male partners. The controlled ovarian hyperstimulation (COH) using luteal long protocol of gonadotropin releasing hormone (GnRH) agonist was used in all couples for IVF-ET. The acrosomal reaction with 10microliter of 10% DMSO was induced spontaneously in 10.1+/-9.8%, and acrosomal reaction with calcium ionophore A 23187 was induced in 27.4+/-18.1%, and the ARIC value was 17.4+/-16.2%. There were no significant correlation between the ARIC value and the fertilization rate (r2=0.044, p=0.268). There were also no significant correlation between the ARIC value and the percentage of the grade I, II embryos (r2=0.046, p=0.261). On the basis of above results, it was suggested that ARIC test might not be a useful prognostic indicator for fertilization in IVF-ET in male partners with normal parameters of conventional semen analysis. We guessed that IVF-ET could be performed to the patients primarily without universal appilcation of ARIC test to all male partenrs, and if fertilization failure occurs, the microassisted fertilization (MAF) such as intracytoplsmic sperm injection (ICSI) might be used as an alternative mode of treatment with acceptable success rate.

The Effect of Epidermal Growth Factor on the Preimplantation Development, Implantation and Its Receptor Expression in Mouse Embryos / 대한산부인과학회잡지

This study was performed to investigate the influence of epidermal growth factor (EGF) on preimplantation development, implantation, and expression of epidermal growth factor receptor (EGFR) in mouse embryos. Riverse transcription-polymerase chain reaction (RT-PCR) has been used to examine the presence of transcripts. Following reverse transcription, strategically designed nested primers, optimised for specificity, were used for amplification from the cDNA equivalent of a single embryo. The products were then verified by restriction enzyme digestion and sequence analysis. Eight-cell stage mouse embryos were cultured for 48hrs with EGF at concentrations of 0.1, 1.0, 10 and 100 ng/ml. Embryos not treated with EGF were served as control. The percentages of embryos which developed to the expanded, hatched blastocyst stage and in vitro implantation at 48hrs were determined. The percentages of fully expanded murine blastocysts at 48hrs in all EGF treated group were not significantly different from the control. The percentages of hatched blastocysts were significantly higher in EGF treatment group at 0.1ng/ml (90.7%), 10 ng/ml (89.3%) compared to the control (82.1%; p < 0.05, p < 0.05). The percentages of implanted blastocyst in vitro were significantly higher following incubation with EGF at concentrations of O.lng/ml (38.1%; p < 0.05), 1.0ng/ml (33.3%; p < 0.05), 10ng/ml (22.2%; p < 0.05) compared to the control (10.7%). Embryo development and implantation in vitro were not significantly inhibited or enhanced in cultures supplemented with 100ng/ml EGF compared to the control. The mRNA concentration of EGFR in embryos treated with 0.1ng/ml of EGF were significantly higher than those of the control and other EGF treatment groups. The implantation rate and mRNA concentration of EGFR in embryos treated with 0.1ng/ml of EGF group were significantly higher than those of other treatd groups. In conclusion, EGF may have a stimulatory role in embryonic development, implantation and expression of EGFR in embryo itself with concentration-specific manner. These results suggest that EGF may act directly on the mouse embryo and favor its implantaion, irtespective of the presence ar absence of the endometrium.

Effects of Indomethacin on Development and Hatching of Mouse Embryo / 대한불임학회지

The present study was designed to define the role of prostaglandin in the development and hatching of mouse embryo. The effects of indomethacin, an inhibitor of prostaglandin synthesis, on the development and hatching of morula and blastocyst were examined. In early morula stage, embryos were degenerated significantly at 100 muM and 200 muM indomethacin. However, :he viability of embryos was not influenced by concentration in any other embryonic stages. In all embryonic stages, the hatching was suppressed with concentration dependent manner, but expansion was not suppressed. Particularly, in 84h embryos post hCG injection, the hatching was suppressed significantly compared with post hCG 72h or 96h embryos. When embryos were treated with 100 muM indomethacin for a specific time (12h) in according to the development stage, the hatching was suppressed all groups. These suppressional effect was decreased as embryonic development stage was progressed. However, the expansion was not affected in all treatment group. This study suggests that hatching-related metabolic substances are synthesized from morula stage and intraembryonic signaling mediated prostaglandin was important for development and hatching of mouse embryo.

Effect of Embryo Number and Incubation Volume on the Development of Pre- and Post-implantation Mouse Embryos In Vitro / 대한불임학회지

The effects of embryo number and incubation volume on the development of mouse embryos were evaluated. The growth rate of two-cell mouse embryos to attached blastocyst stage and the growth rate of blastocysts to early somite stage were assessed after culture in different incubation volumes and embryo densities. Embryos were collected from ICR female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by ICR males. In experiment 1, groups of one, five, ten, twenty 2-cell embryos were cultured in 10-, 50-, 500-, 1000-microliter drops of BWW media under mineral oil at 37 degrees C in a humidified atmosphere of 5% CO2 and 95% air. As the incubation volume decreased, significantly (p<0.05) higher rates of embryos reached morular and blastocyst stage on day 3 and 4 culture, respectively In experiment 2, groups of one, five, ten, twenty blastocysts were cultured in 1- and 2-ml volumes of CMRL 1066 media under same condition as in experiment 1. However the reverse was the result. Decreasing the number of embryos incubated per volume from 1 to 20 significantly (p<0.05) increased the number of blastocysts reaching the late egg cylinde. (LEC) and early somite (ES) stage on day 6 and 8 culture, respectively, regardless of incubation volume. Blastocysts cultured in 2ml had higher (p<0.05) development rates to LEC and ES stage on day 6 and 8 culture, respectively, than embryos cultured in 1ml. Our results suggest that the effects of embryo number and incubation volume on the development of mouse embryos are stage specific and the shifting point was between hatching and EEC stage.

A Comparison of Controlled Ovarian Hyperstimulation with Intrauterine Insemination to in vitro Fertilization and Embryo Transfer in the Treatment of Male Infertility Caused by Sperm Surface Antibodies / 대한산부인과학회잡지

This prospective study was performed to evaluate the effectiveness of controlled ovarian hyperstimulation(COH) with intrauterine insemination(IUI) versus in vitro fertilization and embryo transfer(IVF-ET) in the treatment of male infertility caused by sperm surface antibodies. From March 1995 to August 1996, 29 couples with male immunologic infertility entered the trial. Only men with >or=40% motile spermatozoa with bound antibodies of immunoglobulin (Ig)G, IgA or a combination of both in direct immunobead test(IBT) were included in this study. There was no evidence of other factors in infertility in any infertile couples. The couples were randomized to undergo either COH with IUI(IUI group), or IVF-ET(IVF group). IUI group and IVF group were similar with respect to female and male age, duration of infertility, and IBT results. There were no significant differences between two groups with regard to the amount of gonadotropins required, days of gonadotropins administration, serum estradiol concentration on the day of human chorionic gonadotropin(hCG) administration, the number of mature (>or=14mm) follicles, or endometrial thickness. A total of 10 clinical pregnancies were obtained in IUI group, and 12 in IVF group. In 2 of 30 IVF cycles, intracytoplasmic sperm injection(ICSI) was performed because of fertilization failure. One patient became pregnant after ICSI. There were no significant differences between two groups in the clinical pregnancy rate per cycle (31.3% vs 40.0%), miscarriage rate(20.0% vs 8.3%), and multiple pregnancy rate(20.0% vs 16.7%). There were also no significant differences in pregnancy outcome between two groups according to the Ig isotype of sperm surface antisperm antibody(ASA)(GA group, IgG ASA >or= 40%, IgA ASA>or=40%; G group, IgG ASA >or=40%, IgA or=40%). This study suggests that it could be reasonable to offer COH with IUI to the patients with infertility caused by sperm surface ASA, prior to their referral for more expensive and invasive procedure, IVF-ET.

The Effects of Retinoic Acid on Gastrulation of Hatched Mouse Embroyos / 대한산부인과학회잡지

Retinoic acid(RA), formed in vivo by oxidation of retinol, is known as morphogenic signal. RA plays an active role in normal embryonic development at physiological concentration, but excess RA can be a powerful teratogen in human and animals. The present study was designed to examine the direct effect of RA on murine embryogenesis(gastrulation) and to define the specific development processes perturbed by RA. Five to fifteen blastocysts were randomly assigned to separate culture dishes of the experimental group. Various concentrations of RA(10(-9) M, 10(-7) M, and 10(-5) M) were used in culturing blastocysts. In the effect of RA on the normal grouwth of embryo, the rates of development to the stages of attachment, early egg cylinder(EEC), late egg cylinder(LEC), and early somite(ES)were significantly(p < 0.01) decreased as the RA concentration increased. Stil in the yolk sac formation rate, there was a significant, dose-dependent difference(p < 0.01) according to the RA concentration. In the degeneration of embryos by RA, the effect was more apparent as the concentration of Ra increased. The production rates of embryos devoid of egg cylinder region and embryos with abnormal egg cylinder region were increased (p < 0.01)in a dose-dependent manner according to RA concentration. In conclusion, RA probably act as teratogen at gastrula stage embryos in high concentration and effect of teratogenesis is dose-dependent.