Erratum: Efficient gene delivery in differentiated human embryonic stem cells. Exp Mol Med 2005;37:36-44

The authors would like to amend a reference (Lee et al., 2003) that was cited in "Cell culture" section of "Materials and Methods". Instead of "(Lee et al., 2003)", we would like to change the reference to "(Kim et al., 2003)". In "References", it also needs to include the following reference. Kim YY, Seol HW, Ahn HJ. Temporal expression of differentiation markers in embryoid bodies from various human embryonic stem cell line. International Society for Stem Cell Research 1st Annual Meeting, Washington, DC. U.S.A. June 8-11, 2003, Abstract No. 35. The authors apologize for any inconvenience.

mRNA Expression of the Regulatory Factors for the Early Folliculogenesis in vitro / 대한불임학회지

OBJECTIVE: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Mullerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. MATERIALS AND METHODS: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in alpha-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. RESULTS: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. CONCLUSION: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

Identification of New Proteins in Follicular Fluid from Mature Human Follicles by Direct Sample Rehydration Method of Two-Dimensional Polyacrylamide Gel Electrophoresis

Human follicular fluid (HFF) includes various biologically active proteins which can affect follicle growth and oocyte fertilization. Thus far, these proteins from mature follicles in human follicular fluid have been poorly characterized. Here, two-dimensional polyacrylamide gel electrophoresis (2-DE) with matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to identify new proteins in HFF. Mature follicular fluids were obtained from five females after oocyte collection during in vitro fertilization (IVF). We directly rehydrated HFF samples, obtained high-resolution 2-DE maps, and processed them for 2-DE and MALDI-MS. One hundred eighty spots were detected and 10 of these spots were identified. By the 2-DE database, six of them had been reported, as proteins already existing in HFF. Hormone sensitive lipase (HSL), Unnamed protein product 1 (UPP1), Unnamed protein product 2 (UPP2), and apolipoprotein A-IV precursor were newly detected. HSL and apolipoprotein A-IV participate in lipid metabolism. UPP1 has a homology with selenocysteine lyase. We found by RT-PCR that these genes are expressed from human primary granulosa cells. The proteins identified here may emerge as potential candidates for specific functions during folliculogenesis, hormone secretion regulation, or oocyte maturation. Further functional analysis of these proteins is necessitated to determine their biological implications.

Identification of a putative transactivation domain in human Nanog

Nanog is a newly identified divergent homeodomain protein that directs the infinite propagation and sustains the pluripotency of embryonic stem cells. It has been reported that murine Nanog has two potent transactivation domains in N-terminal and C-terminal regions. Human Nanog (hNanog) polypeptide shares about 58% and 87% identity to the open reading frame and homeodomain of murine Nanog, respectively. However, the functional domains and molecular mechanisms of hNanog are poorly understood. In this study, for the first time, we presented that only C-terminus of hNanog contains a potent transactivation domain. Based on the amino acid sequences of homeobox domain, we roughly divided hNanog open reading frame into the three regions such as N-terminal, homeodomain and C-terminal regions and constructed either the fusion proteins between hNanog individual and Gal4 DNA binding domain or the context of native hNanog protein. Reporter assays by using reporter plamid containing Gal4 or Nanog binding site revealed that the only C-terminal region exhibited the significant fold induction of transactivation. However, interestingly, there was no significant activation through N-terminal region unlike murine Nanog, suggesting that C-terminal region may have more critical roles in the transcriptional activation of target genes. Taken together, the finding of a putative transactivation domain in hNanog may contribute to the further understanding of molecular mechanism on the regulation of downstream genes involved in self-renewal and pluripotency of human stem cells.

Overexpression of SOX9 in mouse embryonic stem cells directs the immediate chondrogenic commitment

Mouse embryonic stem (mES) cells are capable of undergoing chondrogenesis in vitro. To enhance this process, the human SOX9 (hSOX9) cDNA was delivered into mES cells and the clones overexpressing hSOX9 (denoted as mES-hSOX9 cells) were verified by Western blot analysis. The transcripts of collagen IIA (a juvenile form), aggrecan and Pax1 were expressed in mES-hSOX9 cells grown on feeder layers, suggesting the immediate effect of exogenous SOX9 on chondrogenesis. However, SOX9 overexpression did not affect the cell cycle distribution in undifferentiated mES cells. Upon differentiation, collagen IIB (an adult form) was detected in day 3 immature embryoid bodies. In addition, the overexpression of exogenous SOX9 significantly induced transcriptional activity driven by SOX9 binding site. Taken together, we for the first time demonstrated that constitutive overexpression of exogenous SOX9 in undifferentiated mES cells might have dual potentials to induce both chondrogenic commitment and growth capacity in the undifferentiated status.

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

No abstract available.

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.

Clinical Outcomes of Frozen-thawed Embryo Transfer after Microsurgical Removal of Damaged Blastomere / 대한불임학회지

OBJECTIVE: Human infertility clinics have been faced the demand for improving clinical results. The purpose of this study was to evaluate the effect of microsurgical removal of damaged blastomeres (DB) in frozen-thawed embryos on the clinical outcomes. METHODS: From January 2003 to May 2004, out of 258 thawing ET cycles were divided into three groups: Group-1 (n=46): Intact cleavaged embryos after thawing. Remained cycles with embryos containing DB were randomly divided into two groups. Group-2 (n=102): Drilling zona pellucida (ZP) of frozen-thawed embryos by acidified Tyrode's solution. Group-3 (n=110): Drilling ZP and removal of DB. Embryos after microsurgical manipulation were transferred into the uterus of patients. RESULTS: Clinical profiles and the mean number of transferred embryos among three groups were not different. Pregnancy and implantation rates were similar in three groups. It were 30.4% and 9.3% in Group-1, 29.4% and 7.8% in Group-2, and 26.4% and 7.6% in group-3, respectively. Miscarriage rate in Group-3 (37.9%) was slightly higher than those in Group-1 and Group-2 (14.3% and 23.3%), but it was not statistically significant. CONCLUSION: Intact cleaving embryos after DB removal showed higher potent of pregnancy and implantation. We could not find any improvement of clinical outcome by removal of DB in frozen-thawed embryos.

Efficient gene delivery in differentiated human embryonic stem cells

Human embryonic stem (hES) cells are capable of differentiating into pluralistic cell types, however, spontaneous differentiation generally gives rise to a limited number of specific differentiated cell types and a large degree of cell heterogeneity. In an effort to increase the efficiency of specified hES cell differentiation, we performed a series of transient transfection of hES cells with EGFP expression vectors driven by different promoter systems, including human cellular polypeptide chain elongation factor 1 alpha (hEF1alpha), human cytomegalo-virus, and chicken beta-actin. All these promoters were found to lead reporter gene expression in undifferentiated hES cells, but very few drug-selectable transfectants were obtained and failed to maintain stable expression of the transgene with either chemical or electroporation methods. In an attempt to increase transfection efficiency and obtain stable transgene expression, differentiated hES cells expressing both mesodermal and ectodermal markers were derived using a defined medium. Differentiated hES cells were electroporated with a hEF1alpha promoter-driven EGFP or human noggin expression vector. Using RT-PCR, immunocytochemistry and fluorescence microscopy, the differentiated hES cells transfected with foreign genes were confirmed to retain stable gene and protein expression during prolonged culture. These results may provide a new tool for introducing exogenous genes readily into hES cells, thereby facilitating more directed differentiation into specific and homogenous cell populations.

Cryopreservation of Human Embryos / 대한산부인과학회잡지

Technology for the long-term preservation of gamete and embryo has improved greatly over the past 20 years and currently is used for supporting various assisted reproductive technologies (ART). Recent progress in cryobiology and its related sciences have made it possible to preserve human embryos effectively, and several cryopreservation methods also have been developed. Successful freezing of supernumerary embryos has allowed patients undergoing ART the opportunity to achieve pregnancies from more than one embryo transfer without being subjected to controlled ovarian hyperstimulation and oocyte retrieval each time. It also allows a delay in embryo transfer where certain adverse conditions exist for fresh transfer, e.g. when the patient is at risk for ovarian hyperstimulation syndrome or when there is poor endometrial development during the retrieval cycle. Cryopreservation of all available embryos from retrieval is utilized when an oocyte recipient is not properly synchronized with oocyte donor's cycle. In this paper is to review the current status and perspectives of embryo cryopreservation in ART program. Also, briefly discuss the oocyte cryopreservation for the establishment of ovum bank.

Ultrasonographic findings of early abortion: suggested predictors

To investigate predictable ultrasonographic findings of early abortion. To investigate objective rules for the screening of abortion Ultrasonographic examination of 111 early pregnancies between the sixth and ninth week in women who had regular 28 day menstrual cycles was performed. Ultrasonographic measurements of the gestational sac. Crown rump length and fetal heart rate were performed using a linear array real time transducer with doppler ultrasonogram. All measurements of 17 early abortions were compared to those of 94 normal pregnancies. Most of early aborted pregnanices were classified correctly by disciminant analysis with G-SAC and CRL (GSAC=0.5 CRL+15. Sensitivity 76.5%, specificity 96.8%). With the addition of FHR, 94.1 of early abortions could be predicted. In conclusion, ultrasonographic findings of early intrauterine growth retardation. Small gestational sac and bradycardia can be predictable signs suggestive of poor prognosis of early pregnancies.

Analysis of the Gene Expression by Laser Captured Microdissection (I): Minimum Conditions Required for the RNA Extraction from Oocytes and Amplification for RT-PCR / 대한불임학회지

OBJECTIVE: Recently, microdissection of tissue sections has been used increasingly for the isolation of morphologically identified homogeneous cell populations, thus overcoming the obstacle of tissue complexity for the analysis cell-specific expression of macromolecules. The aim of the present study was to establish the minimal conditions required for the RNA extraction and amplification from the cells captured by the laser captured microdissection. METHODS: Mouse ovaries were fixed and cut into serial sections (7 micrometer thickness). Oocytes were captured by laser captured microdissection (LCM) method by using PixCell IITM system. The frozen sections were fixed in 70% ethanol and stained with hematoxylin and eosin, while the paraffin sections were stained with Multiple stain. Sections were dehydrated in graded alcohols followed by xylene and air-dried for 20 min prior to LCM. All reactions were performed in ribonuclease free solutions to prevent RNA degradation. After LCM, total RNA extraction from the captured oocytes was performed using the guanidinium isothiocyanate (GITC) solution, and subsequently evaluated by reverse transcriptase -polymerase chain reaction (RT-PCR) for glyceraldehyde-3-phosphate-dehydrogenase (GAPDH). RESULTS: With the frozen sections, detection of the GAPDH mRNA expression in the number of captured 25 oocytes were not repeatable, but the expression was always detectable from 50 oocytes. With 25 oocytes, at least 27 PCR cycles were required, whereas with 50 oocytes, 21 cycles were enough to detect GAPDH expression. Amount of the primary cDNA required for RT-PCR was reduced down to at least 0.25 microl with 50 oocytes, thus the resting 19.75 microl cDNA can be used for the testing other interested gene expression. Tissue-to-slide, tissue-to-tissue forces were very high in the paraffin sections, thus the greater number of cell procurement was required than the frozen sections. CONCLUSION: We have described a method for analyzing gene expression at the RNA level with the homogeneously microdissected cells from the small amount of tissues with complexity. We found that LCM coupled with RT-PCR could detect housekeeping gene expression in 50 oocytes captured. This technique can be easily applied for the study of gene expression with the small amount of tissues.

Clinical Application of 'Scrotogram' / 대한비뇨기과학회지

A study was undertaken during the past 2 years of patients who visited the infertility clinic of this hospital. The patients included those with male infertility, scrotal mass, scrotal pain or impotence. These patients were studied by means of reviewing the clinical applications of 'Scrotogram' and the following results were obtained. A total of 122 cases underwent a 'Scrotogram' study and among them 80 cases were diagnosed as varicocele, which included 14 cases( 18%) of subclinical varicocele which are difficult to be diagnosed by initial physical examination. There was statistical significance in the varicocele index between the control group and the Grade I varicocele group(p<0.01) and between the control group and subclinical varicocele group(p<0.01), and between the Grade I and Grade II group.(p<0.01), and between the Grade II and Grade II group(p<0.05). Among 66 clinical varicocele patients, 63 cases(80% ) were diagnosed by varicocele index, and 54 cases(82% ) were diagnosed by static image. A combination of the above two methods allowed 61 cases(92%) to be diagnosed. Among 48 patients who underwent high ligation of internal spermatic vein, 25 patients were subject to postoperative follow-up 'Scrotogram', which revealed 12 cases with excellent results, 8 cases with good results, and 5 cases with poor results. The average varicocele index before and after surgery was 1.78 and 1.24 and there were statistical significance(p<0.01). There was statistical significance, in sperm count and motility between the control group and clinical varicocele group(p <0.01) and between the control group and subclinical varicocele group(p <0.01). However, there appeared no statistical significance in semen character between the clinical varicocele and subclinical varicocele group. Among the 13 patients who were subject to postoperative follow-up semen analysis 11 patient showed improved results, and there was marked improvement in sperm count(p<0.05). There was slight improvement in sperm motility and morphology but showed no statistical significance. At present, 2 patients were impreged. Among 110 patients with infertility and olieoasthenoteratozoospermia, 22 were initially diagnose as varicocele by physical examination only, and 7 patients were diagnosed as subclinical varicocele after a 'Scrotogram' was taken. In conclusion, the 'Scrotogram' is able to identify varicocele objectively, and also identify subclinical varicocele which is an important contributing factor to male infertility. Also, this method of study is able to assess the postoperative result of varicocele accurately.

Predictable ultrasonographic findings of early abortion

Early fetal growth delay and early oligohydramnios have been suspected as signs of embryonal jeopardy. However, little information is available for the prediction of early abortion. Sonographic examination of 111 early pregnancies between the sixth and ninth gestational week with regular, 28 day menstrual cycles was performed to investigate predictable sonographic findings of early abortion. Sonographic measurements of the gestational sac (G-SAC), crown-rump length (CRL) and fetal heart rate (FHR) were performed using a linear array real time transducer with Doppler. All measurements of 17 early abortions were compared to those of 94 normal pregnancies to investigate the objective rules for the screening of early abortion. Most of the early aborted pregnancies were classified correctly by discriminant analysis with G-SAC and CRL (G-SAC = 0.5222 CRL + 14.6673 = 0.5 CRL + 15, sensitivity 76.5% specificity 96.8%). With the addition of FHR, 94.1% of early abortions could be predicted. In conclusion, sonographic findings of early intrauterine growth retardation, early oligohydromnios and bradycardia can be predictable signs for the poor prognosis of early pregnancies.

A Case of Diamnionic Monochorionic Monozygotic Twin Which was Conceived by In Vitro Fertilization / 대한산부인과학회잡지

Monozygotic twinning is a relatively rare event in vivo conception, being estimated to occur in 0.42% of all birth. The underlying mechanism for monozygotic twin formation is the division of the embryo early in its development. Separation of cells may theoretically occur before or after inner cell mass formation. The incidence of monozygotic twinning following assisted reproduction techniques is higher than the commonly accepted incidence after in vivo conception. Patients at particular risk of monozygotic twins are those aged > 35 years and those who had manipulation techniques for assisted fertilization. Hence, it seems prudent to counsel these patients about the potential obstetric complications of monozygotic multiple gestations prior to the initiation of their treatment.

A Case of Transverse Vaginal Septum with Microperforation Which got Pregnant by Intrauterine Insemination / 대한산부인과학회잡지

The mullerian ducts join the sinovaginal bulb at a point known as the mullerian tubercle. Canalization of the mullerian tubercle and sinovaginal bulb is necessary to give a normal vaginal lumen. If the area of junction between these structures is not completely canalized, a transverse vaginal septum will occur. This may be partial or complete and generally lies at the junction of the upper third and lower two thirds of the vagina. It occurs in about 1 per 75,000 females. Partial transverse vaginal septa have been reported in diethylstilbestrol (DES)-exposed females. In the prepubertal state, diagnosis is generally not made unless there is the development of a mucocolpos or mucometrium behind the septum. At puberty, however, if the septum is complete, hematocolpos and hematometrium may occur in a fashion similar to that seen in the imperforate hymen, except that there is no bulging at the introitus. The patient with an incomplete transverse septum may bleed somewhat but will still develop hematocolpos and hematometrium over time and may also complain of foul smelling vaginal discharge.

A Case of the Carrier of Reciprocal Translocation Which was Inherited from Patient with Recurrent Spontaneous Abortion / 대한산부인과학회잡지

A balanced translocation in a parent may produce unbalanced gametes leading to abortions or defective liveborn children, or interval infertility. It also may give rise to a balanced gamete resulting in a balanced carrier, or it may produce a cytogenetically normal gamete. The incidence of balanced chromosomal translocations in couples with multiple abortions was reported as 0% to 31%. This wide variation is related to the heterogeneous criteria used for patient selection. Because parents with balanced chromosomal rearrangements and history of only repeated abortions have a significant chance with each pregnancy of having a child with normal or balanced karyotype, the usual criteria for investigation include at least two abortions or reproductive losses. There is no evidence from several reported series that increasing the number of losses to three or more leads to any change in the yield of chromosomal rearrangements detected.

A Case of Pregnancy Using Recombinant Follicle Stimulating Hormone and Gonadotropin Releasing Hormone Antagonist / 대한불임학회지

OBJECTIVE: To report the pregnancy which was made by in vitro fertilization using recombinant follicle stimulating hormone and gonadotropin releasing hormone antagonist. MATERIAL AND METHOD: Case report. RESULTS: Six oocytes were retrieved and all were fertilized by intracytoplasmic sperm injection. Six embryos were transferred and the pregnancy was confirmed. CONCLUSION: It is envisaged that the availability of recombinant gonadotropins and gonadotropin releasing hormone antagonists will ultimately lead to shorter, cheaper and safer treatments, using reduced dosages.

A Study of Lupus Anticoagulants and Anticardiolipin Antibodies in Patients with Infertility and Recurrent Spontaneous Abortion / 대한불임학회지

OBJECTIVE: To report the prevalence of lupus anticoagulants and anticardiolipin antibodies in patients with recurrent spontaneous abortion and infertility. MATERIAL AND METHOD: Lupus anticoagulants and anticardiolipin antibodies were analyzed by Diluted Russell's Viper Venom Test (DRVVT) and solid phase enzyme immunoassay, respectively. RESULTS: In 200 patients with infertility, there were 6 cases (3%) with positive lupus anticoagulants or anticardiolipin antibodies. Of these, 3 patients (1.5%) showed positive lupus anticoagulants and anticardiolipin antibodies, respectively. In 120 patients with recurrent spontaneous abortion, there were 13 cases (10.8%) of positive lupus anticoagulants or anticardiolipin antibodies. Of these, one patient (1%) showed lupus anticoagulants and 12 patients (10%) showed anticardiolipin antibodies. But in two groups, there was no cases with positive lupus anticoagulants and anticardiolipin antibodies. CONCLUSION: Lupus anticoagulants and anticardiolipin antibodies are definite cause of recurrent spontaneous abortion. There has been a speculation that they might be associated with infertility and repeated IVF failures. But it was found that the role of lupus anticoagulants and anticardiolipin antibodies in these cases are not clear.

The Study of Antithrombin III Deficiency in Patients with Recurrent Spontaneous Abortion / 대한불임학회지

OBJETIVE: To analyze the antithrombin III deficiency in patients with recurrent spontaneous abortion. MATERIALS AND METHOD: The blood samples were tested by chromogenic assay to evaluate the activity of antithrombin III. RESULTS: There was only one case of antithrombin III deficiency. This patient experienced one neonatal death after delivery and one FDIU (fetal death in utero). And also this patient showed a lupus anticoagulant and the prolongation of PTT. CONCLUSIONS: Women with recurrent miscarriage who have no obvious identified cause should consider hematologic screening. Antithrombin III deficiency could be a cause of recurrent spontaneous abortion. But the incidence is very rare in Korean patients.