Clinical correlation of basal FSH level and IVF-ET outcome / 대한산부인과학회지

OBJECTIVE: The objective of this study is to evaluate the value of basal follicular stimulating hormone level on clinical outcome in women undergoing IVF-ET. METHOD: A descriptive and retrospective study of 730 cycles of IVF-ET chosen from 2002 to 2004 in CHA fertility center. RESULTS: Basal FSH screening appeared to be a fairly informative predictor of achiving pregnancy especially in GnRH agonist long protocol in women undergoing IVF-ET. In addition, basal FSH level shows significant difference compared ongoing pregnancy with early abortion group in GnRH antagonist group. CONCLUSION: Therefore, we were able to predict the ovarian response and IVF-ET outcome using FSH level. Furthermore, this information allow more precise counseling for patients.

Rapid prenatal diagnosis of Down syndrome and Edward syndrome by fluorescence In situ hybridization:Clinical experience with 309 cases

PURPOSE: The purpose of this study was to evaluate the clinical utility of rapid detection of Down syndrome and Edward syndrome by Interphase Fluorescence in Situ Hybridization (FISH) analysis METHODS: A retrospective study in 309 cases of amniotic fluid samples, analysed by interphase FISH with DNA probes specific to chromosome 18 and 21, was performed. All FISH results were compared with conventional cytogenetic karyotypings. RESULTS: The results were considered as informative and they were obtained within 48 hrs. A case of Down syndrome and a case of Edward syndrome were diagnosed by FISH and confirmed by subsequent cytogenetic analysis. In 12 cases with normal FISH results, the cytogenetic analysis showed a case of partial trisomy 22, three cases of sex chromosomal aneuploidy, two cases of mosaicism, two cases of microdeletion, and four cases of structural rearrangement. CONCLUSION: FISH is a rapid and effective diagnostic method, which can be used as an adjunctive test to cytogenetic analysis, for prenatal identification of chromosome aneuploidies. For the more genome- wide screening with variety of probes, the technique of FISH is both expensive and labor-intensive.

Clinical evaluation of ultrasound-guided embryo transfer / 대한산부인과학회지

OBJECTIVE: Clinical evaluation of the efficacy of ultrasound-guided embryo transfer (ET) comparing with embryo transfer without ultrasound guidance (Non-US). METHODS: From January 2003 to April 2003, we examined the efficacy of ultrasound-guided embryo transfer (ET) on clinical outcome from in-vitro fertilization (IVF-ET) cycle. One hundred thirty patients were prospectively randomized into two groups: 69 patients had ultrasound-guided ET (US) and 61 patients had clinical touch embryo transfer without ultrasound guidance (Non-US). RESULTS: There were no statistically significant differences between the two groups with respect to age, cause of infertility, and the characteristics of the IVF cycle. The pregnany rate (42.0%: 40.9%, p=0.9043) and implantation rate (26.6%: 23.1%; p=0.5057) were similar in both groups. CONCLUSION: There was no significant improvement in pregnancy and implantation rates, following the use of ultrasound guidance during ET, but ultrasound assistance would suggest that decrease in cervical and uterine trauma, decrease in the total duration of ET time, and increase in the easy transfer rate can play a positive role in embryo transfer.

Mitochondrial DNA Copy Number in the Patients of Korean Polycystic Ovary Syndrome (PCOS) / 대한불임학회지

OBJECTIVE: We analyzed quantification of mitochondria DNA (mtDNA) to investigate the relationship of mitochondria and pathogenesis of PCOS. MATERIALS AND METHODS: Peripheral blood samples were collected from 28 patients with PCOS who were under the inclusion criteria for PCOS and from 28 healthy controls. Genomic DNA was used to analyze real-time PCR for mtDNA copy number quantification. The mtDNA copy number was compared between the control and PCOS groups. All data was expressed as mean +/- SD. Statistical analysis was assessed by t-test. RESULTS: In this study, the mtDNA CT was 11.67+/-0.422 in PCOS patients and 11.51+/-0.722 in control group, respectively. The mtDNA copy number was 1726410.71+/-407858.591 the patients of in PCOS and 2167887.51+/-252459.28 in control group (p=0.08), respectively. CONCLUSION: In our study, using real-time PCR, there was a tendency of lower mtDNA copy number in the patients of PCOS when comparing to the control group even though statistical difference was not significant. However, more extensive analysis is required to clarity relationship between mtDNA copy number and pathogenesis of PCOS.

Identification of Oocyte-Specific Diva-Associated Proteins using Mass Spectrometry / 대한불임학회지

OBJECTIVE: We previously described that Diva is highly expressed in matured metaphase II (MII) oocytes compared to immature germinal vesicle (GV) oocytes in mouse.1 We report here that the expression of Diva transcript as well as protein is oocyte-specific. To elucidate its physiological role in oocyte, the binding partner(s) of Diva has been identified by using immunoprecipitation (IP) followed by Mass Spectrometry. METHODS: NIH/3T3 cells were transiently transfected for 24 h with either empty vector for control or FLAG-tagged mouse Diva construct, and IP was performed with anti-FLAG antibody. The immuno-isolated complexes were resolved by SDS-PAGE on a 12% gel followed by Coomassie Blue staining. For in-gel digestion, 15 bands of interest were excised manually and digested with trypsin. All mass spectra were acquired at a positive reflector mode by a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). Proteins were identified by searching the NCBI nonredundant database using MASCOT Peptide Mass Fingerprint software (Matrixscience, London). RESULTS: Diva-associated complexes were formed in FLAG-tagged mouse Diva-overexpressed NIH/3T3 cells via IP using anti-FLAG-conjugated beads. Among the excised 15 bands, actin and actin-binding proteins such as tropomyosin, tropomodulin 3, and alpha-actinin were identified. Binding between Diva and actin or tropomyosin was confirmed by IP followed by Western blot analysis. Both bindings were also detected endogenously in mouse ovaries, indicating that Diva works with actin and tropomyosin. CONCLUSIONS: This is the first report that immuno-isolated Diva-associated complexes are related to actin filament of the cytoskeletal system. When we consider the association of Diva with actin and tropomyosin, oocyte-specific Diva may play a role in modulating the cytoskeletal system during oocyte maturation.

An analysis of various factors affecting postpartum umbilical cord blood volume for hematopoietic stem cell transplantation / 대한산부인과학회지

OBJECTIVE: Umbilical cord blood is an effective alternative to bone marrow as a source of hematopoietic stem cells for transplantation. But the amount of collected umbilical cord blood and its contents are limited and obtaining an adequate volume of umbilical cord blood is essential for successful transplantation. The aim of this study was to identify factors that influence the volume of umbilical cord blood. METHODS: A retrospective analysis of the maternal, neonatal and placental factors that were obtained by medical record review was conducted. The variables that were evaluated for this study were mother's age, parity, gestational age, presence of maternal diabetes mellitus, route of delivery, multiple births, neonatal sex and birth weight, and placental weight. Total 484 deliveries were evaluated from March 2003 to April 2004. The statistical significance of observed differences was calculated using t-test and multiple regression analysis; p-value<0.05 was considered significant. RESULTS: Gestational age, neonatal birth weight, placental weight, parity, number of fetus and maternal diabetes mellitus were significantly associated with a greater volume of collected umbilical cord blood. Obstetric factors that influenced the total nucleated cell concentration were gestational age, neonatal birth weight, placental weight, number of fetus, and route of delivery. CONCLUSION: To prolong a gestational age as far as possible, at least beyond the 37 completed weeks of gestation, and modifying a method of vaginal delivery or cesarean section rather than conventional vaginal delivery method can increase significantly the volume of collected cord blood and the yield of the concentration of total nucleated cell.

Pregnancy outcomes in women aged 35 and older / 대한산부인과학회지

OBJECTIVE: To estimate the effect of maternal age on obstetric outcomes, a retrospective analysis was done. METHODS: Twenty six hundred and forty six women who delivered a singleton baby at our hospital from January 1, to December 31, 2004 were enrolled in this study. Subjects were divided into 3 age groups; 1) less than 35 years, 2) 35-39 years, and 3) 40 years and older. Chi-square test was used to assess the effect of age on obstetrics outcome. Then the odds ratio was calculated to represent clinically meaningful risk. RESULTS: A total of 2646 women with complete data were available; 2245 (84.9%) less than 35 years of age; 350 (13.2%) 35-39 years; and 51 (1.9%) 40 years and older. Increasing age was significantly associated with chromosomal abnormalities (OR 3.9and 8.8 for ages 35-39 years and age 40 years and older, respectively), Preterm premature rupture of membranes (OR 1.3 and 3.2) and cesarean delivery (OR 2.0 and 5.5). Patients aged 35-39 years were at increased risk for placenta previa (OR 1.8) and congenital anomaly (OR 2.8) but these were not statistically significant. The rate of the preterm delivery was increased by age (OR 1.3 and 1.9 for ages 35-39 years and age 40 years and older, respectively) but it was not statistically significant (p=0.121). We did not find advanced maternal age to be associated with a statistically increased risk for preeclampsia, congenital anomaly, gestational diabetes, placenta abruption, low birth weight, macrosomia, neonatal morbidity (NICU admission), and perinatal loss. CONCLUSION: In conclusion, although the likelihood of adverse outcomes increases with maternal age, patients and obstetric care providers can be reassured that overall maternal and fetal outcomes are favorable in this patient population.

The quantitative analysis the number of mitochondrial DNA copy using real-time PCR and mitochondrial tRNA mutation analysis at position 3243 in Korean gestational diabetes mellitus / 대한산부인과학회지

OBJECTIVE: Mitochondrial gene mutations may play a role in the development of gestational diabetes mellitus. This study has assisted to confirm the relationship between the mitochondrial DNA copy number and the GDM. METHODS: Peripheral blood samples were collected from 68 patients with GDM and from 79 controls. For the quantification of mtDNA content, a comparative analysis was performed by the amplification of endogenous control (nuclear DNA, 28S rRNA). The mitochondrial A3243G mutation analysis performed. RESULTS: The ratio of mtDNA/28S rRNA was 1.2053 +/- 0.8307 in GDM patients and 1.7975 +/- 1.1355 in control group (p=0.0004), respectively. Among 68 GDM patients, the mutation in tRNA nt 3243 was detected in only one subject. The A3243G mutation in tRNA- Leu gene, implicated in GDM was reported in 1 of 68 (1.47%) but not in controls. CONCLUSION: In this investigation, blood samples from GDM patients using the real-time polymerase chain reaction will be applied to confirm the relationship between the mitochondrial DNA copy number and the GDM. It is hypothesized that this method will help to predict GDM, and aid in developing early diagnostic methods and treatment modalities.

Clinical efficacy of GnRH antagonist multiple dose protocol and GnRH agonist long protocol in controlled ovarian hyperstimulation using rFSH in IVF-ET patients / 대한산부인과학회지

OBJECTIVE: To compare the efficacy of GnRH antagonist multi dose protocol in controlled ovarian hyperstimulation (COH) for IVF-ET or ICSI with GnRH agonist long protocol. METHODS: From January 2003 to December 2004, total of 583 cycles which underwent IVF-ET or ICSI using r-FSH were enrolled in this study. 447 cycles of the study group were performed in controlled ovarian hyperstimulation by using GnRH antagonist multi dose protocol and 136 cycles of the control group were performed by using GnRH long protocol. We compared patients characteristics, controlled ovarian hyperstimulation outcomes and IVF-ET outcomes between two groups. RESULTS: Patients characteristics and baseline hormone levels were not different between the two groups. The duration of stimulation was significantly shorter in study group comparing with control group (12.8+/-1.5 days vs 13.7+/-1.7 days, p<0.05). There were no differences between the two groups in the number of follicles, endometrial thickness and serum E2 level on hCG day. The pregnancy rate seemed to be lower in the study group (32.4% vs 35.4%), but the difference was not statistically significant. There were also no differences in number of oocytes retrieved, matured oocytes, fertilized oocytes and transferred embryos between two groups. CONCLUSION: GnRH antagonist multi dose protocol in COH might be a simple and effective method compared with GnRH agonist long protocol.

The utility of an erythroblast scoring system and gender-independent short tandem repeat (STR) analysis for the detection of aneuploid fetal cells in maternal blood / 대한산부인과학회지

OBJECTIVE: The aim of this study was to determine whether fetal nucleated red blood cells (NRBCs) could be distinguished from maternal cells in peripheral blood using an erythroblast scoring system. Presumptive fetal NRBCs were further analyzed through the use of fluorescent PCR amplification with polymorphic STR markers to prove fetal origin. METHODS: NRBCs were isolated by density gradient separation, CD15/45 depletion, and gamma hemoglobin positive selection from peripheral blood of seven women who had undergone termination of pregnancy because of fetal trisomy 21 (n=4), 18 (n=1), and 13 (n=2). Candidate fetal NRBCs, based on four discrete morphological and hemoglobin staining criteria, were then subjected to fluorescent PCR amplification of chromosome 21 short tandem repeat (STR) markers (D21S1411, D21S11) and chromosome 18 STR markers (D18S535). RESULTS: In all cases candidate fetal NRBCs were accurately identified based on erythroblast scoring system and confirmed to be fetal in origin based on the presence of shared and non-shared polymorphic DNA alleles when compared to DNA isolated from maternal cells. Also in five cases aneuploid fetal cells in maternal blood were identified through the use of fluorescent PCR amplification with polymorphic STR markers. CONCLUSION: We were able to distinguish fetal NRBCs from maternal cells and prove fetal origin independent of gender. These results suggest that this novel combined approach to fetal cell isolation through using an erythroblast scoring system and genetic analysis by STR analysis is a promising method for noninvasive prenatal diagnostic applications.

Microchimerism of Fetal Origin in Ovarian Tissues from Women with Pelvic Endometriosis / 대한산부인과학회지

OBJECTIVE: To investigate whether fetal microchimeric cells were detected in ovarian tissues with pelvic endometriosis. METHODS: Ovarian tissues with endometriosis were obtained from five women who had at least one live-born son and who underwent enucleation of endometriotic cyst or oophorectomy after a diagnosis of endometriotic cyst. Control tissues were obtained from five women with endometriosis who had no pregnant history. Tissue sections were analyzed with fluorescence in situ hybridization for the presence of fetal cells, defined by X and Y chromosome. RESULTS: Fluorescence in situ hybridization using paraffin-embedded ovarian specimens was performed successfully. Male cells were found in ovarian tissues from all five patients. No male cells were found in ovarian tissues from all five controls. CONCLUSION: Fetal microchimeric cells, possibly from feto-maternal cell trafficking were detected in ovarian tissues with endometriosis were obtained from women who had prior male pregnancies. Further study is necessary to understand the role of persistent fetal microchimeric cells in the progression of endometriosis.

Comparison of anti-gamma hemoglobin antibody and CD71 antibodies in isolation of fetal nucleated erythrocytes from maternal blood / 대한산부인과학회지

OBJECTIVE: In an attempt to further maximize the potential of genetic analysis from fetal cells isolation, fetal nucleated red blood cell (FNRBC) recovery with direct anti-gamma hemoglobin staining after density gradient and depletion was compared with three different whole blood magnetic separations (1-step and 2-step ferrofluid, 2-step Dynal beads). METHODS: In model systems such as quantitatively defined spikes of fetal into adult blood, as well as blood samples after surgical termination procedures, fetal cell yield and purity through the results of fluorescence in situ hybridization (FISH), quantitative real time polymerase chain reaction (PCR), and fluorescence-activated cell sorting (FACS) were calculated. RESULTS: The yield of total number of cells with a XY signal after FISH was the highest on direct anti-gamma hemoglobin staining. After normalizing the results of each experiment to the corresponding result from anti-gamma hemoglobin staining (1), ratio is 0.42 in 1-step ferrofluid, 0.33 in 2-step ferrofluid, and 0.76 in 2-step dynal beads. The fetal cell purity is clearly better in direct anti-gamma hemoglobin staining than those of the magnetic separations from whole blood. The median ratio is 56.3% in anti-gamma hemoglobin staining, 7.7% in 1-step ferrofluid, 6.5% in 2-step ferrofluid, and 31.4% in 2-step dynal beads. CONCLUSION: This study shows that the direct anti-gamma staining is the best fetal cell recovery system and it is very useful to isolate fetal nucleated red blood cells as a non-invasive genetic source.

Simultaneous bilateral tubal pregnancy following in-vitro fertilization and embryo transfer / 대한산부인과학회지

A case of simultaneous bilateral tubal pregnancy following in-vitro fertilization and embryo transfer is presented. On the 22 days after ET, the patient complained of low abdominal pain and vaginal spotting for one day and was suspected of left tubal pregnancy by transvaginal ultrasonography. However, laparoscopy revealed the bilateral tubal pregnancy and laparoscopic bilateral salpingectomy was performed. This unusual type of ectopic pregnancy must be kept in mind when evaluating a patient suspected of a possible early abnormal gestation after assisted reproductive technologies. It is critical to perform a close inspection of the abdomen, pelvis, and contralateral tube during surgery.

A Study of Clinical efficacy of GnRH Antagonist (Cetrorelix(R)) and GnRH Agonist for Controlled Ovarian Hyperstimulation / 대한산부인과학회지

OBJECTIVE: To compare the clinical outcomes of GnRH antagonist (cetrorelix(R)) with those of conventional GnRH agonist for down-regulation in assisted reproductive cycle. Materials and Method: Ninety-nine women undergoing IVF or ICSI were treated with either GnRH antagonist (cetrorelix(R)) or GnRH agonist (Lucrin(R)) for pituitary down regulation. The patient characteristics, basal hormone profile and IVF outcome were compared. RESULTS: There were no significant differences in age and duration of infertility between two groups. E2 (pg/mL)/LH (mIU/mL)/FSH (mIU/ mL) on the 3 day of menstrual period as a baseline were also not significantly different between two groups. The number of hMG amples administered (30.5+/-11.2 versus 47.6+/-16.4 ample/cycle) and the duration of stimulation (11.0+/-1.7 versus 14.1+/-2.2 days) were significantly lower in the cetrorelix(R) group. There were no significant differences in the fertilization and pregnancy rates, the number of embryo transferred, the number of mature oocyte and the number of embryo obtained between two groups. CONCLUSION: The cycles using an antagonist protocol shows a shorter duration of stimulation with comparable outcomes with few injections than those with an agonist protocol. GnRH antagonist can be effectively used as GnRH agonist for pituitary down regulation in IVF-ET cycles.

Gene Expression Analysis of Cultured Amniotic Fluid Cell with Down Syndrome by DNA Microarray

Complete or partial triplication of human chromosome 21 results in Down syndrome (DS). To analyze differential gene expressions in amniotic fluid (AF) cells of DS, we used a DNA microarray system to analyze 102 genes, which included 24 genes on chromosome 21, 28 genes related to the function of brain and muscle, 36 genes related to apoptosis, 4 genes related to extracellular matrix, 8 genes related to other molecular function and 2 house-keeping genes. AF cells were collected from 12 pregnancies at 16-18 weeks of gestation in DS (n=6) and normal (n=6) subjects. Our DNA microarray experiments showed that the expressions of 11 genes were altered by at least 2-folds in DS, as follows. Ten genes, COL6A1, CASP5, AKT2, JUN, PYGM, BNIP1, OSF-2, PRSS7, COL3A1, and MBLL were down-regulated and GSTT1 was only up-regulated. The differential expressions of GSTT1 and COL3A1 were further confirmed by semi-quantitative RT-PCR for each sample. The gene dosage hypothesis on chromosome 21 may explain the neurological and other symptoms of DS. However, our results showed that only two genes (COL6A1 and PRSS7), among 24 genes on chromosome 21, were down-regulated in the AF cells of DS. Our data may provide the basis for a more systematic identification of biological markers of fetal DS, thus leading to an improved understanding of pathogenesis for fetal DS.

HIDE, a Testis Specific Deubiquitinating Enzyme, Interacts with HSP90 / 대한불임학회지

No abstract available.

Application of DNA chip techniques for Yq microdeletion analysis in infertile males

Our aim was to apply DNA chip technology as a diagnostic tool in infertility research and clinics. Six loci, including a sex-determining region on the Y chromosome and five sequence-tagged sites in azoospermia-factor regions were investigated in infertile male patients. Our method produced a sensitive signal, which showed the presence or absence of the STS regions on the Y chromosome. The results from 93 patients with non- obstructive azoospermia, oligoathenoteratozoospermia, or oligozoospermia were identical when analyzed with either the DNA chip technique or conventional PCR-gel electrophoresis. We have demonstrated its application in the molecular diagnosis of male infertility. This system provides an economic and high-throughput method for detecting the deletion of genomic DNA sequences of large groups of infertile patients, and a completely new approach to male infertility screening. The application of DNA chip technology to identify Yq deletions can also facilitate our understanding of male infertility.

G769A Variation of Inhibin alpha -gene in Korean Women with Premature Ovarian Failure

Premature ovarian failure (POF) is menopause before the age of 40 years. The frequency of POF is about 1% of all women. Recently inhibin alpha gene (INHalpha) has been indicated as candidate in POF pathogenesis. Inhibin, a glycoprotein, is a gonadal hormone, which can inhibit the synthesis and secretion of pituitary follicle-stimulating hormone (FSH), which has an important role in the recruitment and development of ovarian follicles during the folliculogenesis. G769A variation of INH alpha, alanine, is highly conserved across species, and has an important role of its receptor binding. We screened a G769A transition in the INHalpha from the total population of the patients of 84 women with POF and 100 normal fertile women. We found no variation between the normal subjects and the POF patients. G769A variation of INHalpha is rare in Korea women with POF.

The Quantitative Analysis of Mitochondrial DNA Copy Number in Premature Ovarian Failure Patients Using the Real-time Polymerase Chain Reaction / 대한산부인과학회잡지

OBJECTIVE: To design a screening method which identifies women with a potential of progression to the premature ovarian failure (POF) in near future, particularly among young women who have high FSH level but no symptoms and signs of POF. METHODS: Peripheral venous blood samples were collected from 30 patients with POF (age range, 19- 39 years) and from 30 controls with normal serum FSH level who had delivered two or more naturally conceived babies (age range, 32-39 years). mtDNA/28S rRNA ratio was determined by real-time quantitative PCR. Comparative threshold cycle (CT) method was adopted for the relative quantitation between two groups and the effectiveness of the method was evaluated. RESULTS: A significant decrease in mtDNA/28S rRNA ratio was found in the POF group (0.58 0.38) when compared with the control group (1.15 0.67) (P<0.01). In both control and POF groups, there was positive correlation between mtDNA and mtDNA/28S rRNA ratio (r=0.774, P<0.001; r=0.556, P=0.001, respectively) and negative correlation between 28S rRNA and mtDNA/28S rRNA ratio (r=-0.677, P<0.001; r=-0.627, P=0.001, respectively), which indicated the suitability of the method. CONCLUSION: This study showed a significant decrease in mtDNA copy number in the peripheral venous blood of POF patients. The comparative CT method was found to be an effective and efficient alternative for the screening purposes. With this basis, further studies on the early diagnostic and/or screening method for the POF might be enriched.

The Study of X Chromosome Inactivation Mechanism in Klinefelter's Syndrome by cDNA Microarray Experiment

To investigate the XIST gene expression and its effect in a Klinefelter''s patient, we used Klinefelter''s syndrome (XXY) patient with azoospermia and also used a normal male (XY) and a normal female (XX) as the control, We were performed cytogenetic analysis, Y chromosomal microdeletion assay (Yq), semi-quantitative RT-PCR, and the Northern blot for Klinefelter''s syndrome (KS) patient, a female and a male control, We extracted total RNA from the KS patient, and from the normal cells of the female and male control subjects using the RNA prep kit (Qiagen), cDNA microarray contained 218 human X chromosome-specific genes was fabricated. Each total RNA was reverse transcribed to the first strand cDNA and was labeled with Cy-3 and Cy-5 fluorescein, The microarray was scanned by ScanArray 4000XL system. XIST transcripts were detected from the Klinefelters patient and the female by RT-PCR and Northern blot analysis, but not from the normal male, In the cDNA microarray experiment, we found 24 genes and 14 genes are highly expressed in KS more than the normal male and females, respectively. We concluded that highly expressed genes in KS may be a resulted of the abnormal X inactivation mechanism.