Birth of a healthy baby after preimplantation genetic diagnosis in a carrier of mucopolysaccharidosis type II: The first case in Korea / 대한생식의학회지

Mucopolysaccharidosis type II (MPS II) is a rare X-linked recessive lysosomal storage disease caused by mutation of the iduronate-2-sulfatase gene. The mutation results in iduronate-2-sulfatase deficiency, which causes the progressive accumulation of heparan sulfate and dermatan sulfate in cellular lysosomes. The phenotype, age of onset, and symptoms of MPS II vary; accordingly, the disease can be classified into either the early-onset type or the late-onset type, depending on the age of onset and the severity of the symptoms. In patients with severe MPS II, symptoms typically first appear between 2 and 5 years of age. Patients with severe MPS II usually die in the second decade of life although some patients with less severe disease have survived into their fifth or sixth decade. Here, we report the establishment of a preimplantation genetic diagnosis (PGD) strategy using multiplex nested polymerase chain reaction, direct sequencing, and linkage analysis. Unaffected embryos were selected via the diagnosis of a single blastomere, and a healthy boy was delivered by a female carrier of MPS II. This is the first successful application of PGD in a patient with MPS II in Korea

Pregnancy and fertilization potential of immature oocytes retrieved in intracytoplasmic sperm injection cycles / 대한생식의학회지

OBJECTIVE: The goal of this study was to evaluate the pregnancy potential of immature (metaphase I or germinal vesicle stage) oocytes retrieved in intracytoplasmic sperm injection (ICSI) cycles. METHODS: A total of 1,871 couples with infertility underwent 2,984 ICSI cycles. Cycles in which three or fewer oocytes were retrieved were included in this study in order to evaluate the pregnancy potential of immature oocytes. Cycles were divided into five groups (group I-V), according to the maturation status of the oocytes at the time of cumulus cell removal and ICSI. The fertilization and pregnancy rates after ICSI were analyzed and compared among the study groups based on the maturation status of the retrieved oocytes. RESULTS: The retrieval of only immature oocytes was associated with a significant decrease in the fertilization rate (76.1%+/-37.3% vs. 49.0%+/-49.1%, 66.7%+/-48.7%; group I vs. group II, group III, respectively) and the average number of transferred embryos (1.5+/-0.7 vs. 1.1+/-0.4, 1.1+/-0.6). The cycle cancellation rate was significantly higher when only immature oocytes were retrieved. The clinical pregnancy rate decreased significantly when the transferred embryos had originated from immature oocytes (16.9% vs. 10.3%, 1.2%). CONCLUSION: In ICSI cycles, the fertilization potential and pregnancy potential of the immature oocytes retrieved in ICSI cycles were inferior to those of mature oocytes. Therefore, increasing the number of injectable oocytes and transferrable embryos by using immature oocytes after their spontaneous in vitro maturation does not necessarily improve pregnancy outcomes.

Artificial oocyte activation in intracytoplasmic sperm injection cycles using testicular sperm in human in vitro fertilization / 대한생식의학회지

OBJECTIVE: Artificial oocyte activation (AOA) is an effective method to avoid total fertilization failure in human in vitro fertilization-embryo transfer (IVF-ET) cycles. AOA performed using a calcium ionophore can induce calcium oscillation in oocytes and initiate the fertilization process. We evaluated the usefulness of AOA with a calcium ionophore in cases of total fertilization failure in previous cycles and in cases of severe male factor infertility patients with non-motile spermatozoa after pentoxifylline (PF) treatment. METHODS: The present study describes 29 intracytoplasmic sperm injection (ICSI)-AOA cycles involving male factor infertility at Cheil General Hospital from January 2006 to June 2013. Patients were divided into two groups (control, n=480; AOA, n=29) depending on whether or not AOA using a calcium ionophore (A23187) was performed after testicular sperm extraction-ICSI (TESE-ICSI). The AOA group was further split into subgroups according to sperm motility after PF treatment: i.e., motile sperm-injected (n=12) and non-motile sperm-injected (n=17) groups (total n=29 cycles). RESULTS: The good embryo rate (52.3% vs. 66.9%), pregnancy rate (20.7% vs. 52.1%), and delivery rate (10.3% vs. 40.8%) were lower in the PF/AOA group than in the control group. When evaluating the effects of restoration of sperm motility after PF treatment on clinical outcomes there was no difference in fertilization rate (66.6% vs. 64.7% in non-motile and motile sperm, respectively), pregnancy rate (17.6% vs. 33.3%), or delivery rate (5.9% vs. 16.7%) between the two groups. CONCLUSION: We suggest that oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles regardless of restoration of sperm motility after PF treatment. AOA may be useful in selected patients who have a low fertilization rate or total fertilization failure.

Observation of sperm-head vacuoles and sperm morphology under light microscope / 대한생식의학회지

OBJECTIVE: The presence of sperm-head vacuoles has been suspected to be deleterious to the outcomes of assisted reproductive technology (ART). It is difficult to accurately distinguish morphologically abnormal sperm with vacuoles under a light microscope. This study was performed to analyze the result of the observation of sperm-head vacuoles using Papanicolaou staining under a light microscope and whether the male partner's age affects these vacuoles. METHODS: Sperm morphology with vacuoles was evaluated using Papanicolaou staining and observed under a light microscope (400x) in 980 men. The normal morphology was divided into three categories (group A, 14% of normal morphology). The criteria for the sperm-head vacuoles were those given in the World Health Organization manual. For the analysis of the age factor, the participants were divided into the following groups: 26-30 years, 31-35 years, 36-40 years, 41-45 years, and 46-50 years. RESULTS: The percentage of sperm-head vacuoles increased with normal sperm morphology (group A vs. groups B, C) (p<0.05). In the case of the age factor, a statistically significant difference was not observed across any of the age groups. CONCLUSION: A majority of the sperm-head vacuoles showed a statistically significant difference among normal morphology groups. Therefore, we should consider the probability of the percentage of sperm-head vacuoles not increasing with age but with abnormal sperm morphology. A further study is required to clarify the effect of the sperm-head vacuoles on ART outcomes.

Preimplantation genetic diagnosis for Charcot-Marie-Tooth disease / 대한생식의학회지

OBJECTIVE: Preimplantation genetic diagnosis (PGD) is an assisted reproductive technique for couples carrying genetic risks. Charcot-Marie-Tooth (CMT) disease is the most common hereditary neuropathy, with a prevalence rate of 1/2,500. In this study, we report on our experience with PGD cycles performed for CMT types 1A and 2F. METHODS: Before clinical PGD, we assessed the amplification rate and allele drop-out (ADO) rate of multiplex fluorescent polymerase chain reaction (PCR) followed by fragment analysis or sequencing using single lymphocytes. We performed six cycles of PGD for CMT1A and one cycle for CMT2F. RESULTS: Two duplex and two triplex protocols were developed according to the available markers for each CMT1A couple. Depending on the PCR protocols, the amplification rates and ADO rates ranged from 90.0% to 98.3% and 0.0% to 11.1%, respectively. For CMT2F, the amplification rates and ADO rates were 93.3% and 4.8%, respectively. In case of CMT1A, 60 out of 63 embryos (95.2%) were diagnosed and 13 out of 21 unaffected embryos were transferred in five cycles. Two pregnancies were achieved and three babies were delivered without any complications. In the case of CMT2F, a total of eight embryos were analyzed and diagnosed. Seven embryos were diagnosed as unaffected and four embryos were transferred, resulting in a twin pregnancy. Two healthy babies were delivered. CONCLUSION: This is the first report of successful pregnancy and delivery after specific PGD for CMT disease in Korea. Our PGD procedure could provide healthy babies to couples with a high risk of transmitting genetic diseases.

Complete androgen insensitivity syndrome due to androgen receptor gene mutation in the amino acid position 607 which bilateral testes situated in the normal ovary sites / 대한산부인과학회지

Androgen insensitivity syndrome (AIS) is a disorder of male sexual differentiation caused by mutations within the androgen receptor gene, represents a variety of phenotypes from females with 46,XY karyotype over individuals with ambiguous genitalia to infertile males. Single base mutations resulting in amino acid substitution represent the most common mutations of the androgen receptor (AR) gene and are associated with complete AIS. The location of the gonads can be variable including, the intra-abdominal cavity, the labioscrotal folds, and the inguinal regions. Testicular descent is a two-stage process comprising transabdominal and transinguinal phases. The first phase is not controlled by androgen and may be regulated by mullerian inhibiting substance, by contrast the second phase is androgen dependent. Recently we have identified a point mutation CGA to TGA at position 607 of exon 3 in complete AIS patient, so we report it with brief review of literatures.

The role of angiotensin II to regulation of steroidogenic pathway in human ovarian follicle / 대한산부인과학회지

OBJECTIVE: The exact mechanism of angiotensin II to steroidogenesis is still speculative in spite of many researches especially in human and these were performed indirectly with serum or follicular fluid. Under the hypothesis that ovarian RAS increases androgen, decreases progesterone synthesis in normal human ovary, we investigated the exact action of angiotnesin II on human ovary. METHODS: After appliance of angiotensin II and saralasin to the normal human ovarian follicles, we measured sex steroids like progesterone, testosterone, DHEA and enzymes like HSD3beta2, CYP 17 to see the action of angiotensin II and its antagonist, saralasin. The results were analyzed by ANOVA test. RESULTS: Angiotensin II increased androgen synthesis but did not affect progesterone synthesis. There were no difference of HSD 3beta2 mRNA expression in angiotensin II and saralasin group compared with control group. The expression of CYP17 mRNA was increased by angiotensin II but did not reach statistically significant level. CONCLUSION: Angiotensin II could increase androgen production probably via overexpression of CYP17, but had no efffect on progesterone production.

The Regulators of VEGF Expression in Mouse Ovaries

The objectives of this study were to explore whether ovarian vascular endothelial growth factor (VEGF) expression in mice can be regulated by IL-6 (interleukin-6), angiotensin II, FSH, and hCG; and to test whether the mouse ovarian VEGF expression can result in angiogenesis. The ICR mice were sacrificed, and their ovaries were recovered. Recovered ovaries were treated with IL-6, angiotensin II, FSH, and hCG separately and incubated for 24 hours in alpha-MEM. Expression of mRNA and protein of VEGF were assessed by RT-PCR and immunohistochemistry. The resulting angiogenesis was evaluated through immunohistochemical analysis for CD34. Treatment of mice ovaries with IL-6, FSH, and hCG resulted in a significant increase of VEGF mRNA, and IL-6 was the most potent inducer of VEGF. IL-6 and FSH resulted in increased neovascularization in the follicular phase of mouse ovaries. In contrast, angiotensin II could not increase VEGF expression or neovascularization. We documented an in vitro increase in VEGF expression by IL-6, FSH, and hCG; and reaffirmed that the proliferative response of murine ovarian endothelial cells paralleled an increase of VEGF expression.

Toxic Effect of Cryoprotectants on Embryo Development in a Murine Model / 대한불임학회지

OBJECTIVES: The aim of this study was to assess toxicities of cryoprotectants. METHODS: Toxicities of two cryoprotectants, dimethyl sulfoxide (DMSO) and 1,2-propanediol (PROH), were investigated using a murine embryo model. Female F-1 mice were stimulated with gonadotropin, induced ovulation with hCG and mated. Two cell embryos were collected and cultured after exposure to either DMSO or PROH. Embryo development was evaluated up to the blastocyst stage. Blastocysts were stained with bis-benzimide to evaluate the cell count and with terminal deoxynucleotidyl transferase mediated dUTP nick labeling (TUNEL) to assess apoptosis. RESULTS: The total cell count of blastocysts that were treated with DMSO at the 2-cell stage was significantly lower than that were treated with PROH (75.9+/-27.0) or the control (99.0+/-18.3) (p<0.001). On comparison of two cryoprotectant treated groups, the DMSO treated group showed a decreased cell count compared with the PROH treated group (p<0.05). Both DMSO (14.2+/-1.5) and PROH (11.2+/-1.4) treated groups showed higher apoptosis rates of cells in the blastocyst compared with the control (6.2+/-0.9, p<0.0001). In addition, the DMSO treated group showed more apoptotic cells than the PROH treated group (p<0.001). CONCLUSIONS: The potential toxicity of cryoprotectants was uncovered by prolonged exposure of murine embryos to either DMSO or PROH at room temperature. When comparing two cryoprotective agents, PROH appeared to be less toxic than DMSO at least in a murine embryo model.

The Modulation of IL-12 by Phosphodiesterase IV Inhibitor and Subsequent Changes of Th-1, Th-2 Cytokines in Human Endometrial Tissue / 대한산부인과학회잡지

OBJECTIVE: To assess the capability of phosphodiesterase type IV inhibitor (rolipram) to suppress IL-12 in human decidua and the subsequent changes of Th-2 cytokine (IL-10) and Th-1 cytokine (TNF-alpha). METHODS: Decidual tissues of 10 first-trimester pregnant women and 10 first-trimester pregnant women diagnosed as missed abortion were collected by dilatation and currettage. The decidual tissues were treated with rolipram for 6 hours. Protein and mRNA expression in the tissues were analysed by western blotting, immunohistochemistry and reverse transcription-polymerase chain reaction. RESULTS: Rolipram, in the concentration above 1 microgram/ml, could decrease the expression of IL-12p35 (control: 46.37+/-7.38, rolipram: 24.34+/-8.46) and IL-12p40 mRNA (control: 31.7+/-5.8, rolipram: 14.9+/-4.6) and protein (control: 52.4+/-8.9, rolipram: 40.9+/-12.1). However, the expression of IL-10 and TNF-alpha mRNA and protein did not changed by rolipram. There was no difference in the cytokine expression pattern between the decidual tissues of normal pregnancy and missed abortion. CONCLUSION: Rolipram, the phosphodiesterase type IV inhibitor, could induce the decrease of IL-12 in human decidua. In human decidual tissue, unlike other human tissues, the decrease of IL-12 by rolipram did not modulate other Th-1/Th-2 cytokines. Inability of IL-12 to modulate other Th-1/Th-2 cytokines might be related with unique cytokine network in human decidua rather than its small extent of decrease.

Effect of GM-CSF on the Embryonic Development and the Expression of Implantation Related Genes of Mouse Embryos / 대한불임학회지

OBJECTIVE: The purpose of the current series of experiments were to assess the effect of GM-CSF, as a medium supplement, on the development of mouse embryos and the expression of LIF and IL-1beta mRNA. MATERIALS AND METHODS: Mouse 2-cell embryos were collected from the oviducts of 6 weeks old ICR mice at 48 hours after hCG injection. Embryos were cultured in P-1 medium supplemented with mouse GM-CSF(0,1,5,10 ng/ml). The embryo development to blastocysts and hatching blastocysts was assessed and the cell number in blastocyst was also examined. Using RT-PCR, the expressions of LIF and IL-1beta mRNA in blastocyst were evaluated in the GM-CSF supplemented group and control group. RESULTS: In mouse, the addition of GM-CSF increased the percentage of blastocysts(65.5%, 68.6%, 73.0% and 76.1% for control and 1, 5 and 10 ng/ml, respectively), and increased the proportion of hatching blastocysts(35.2%, 36.4%, 43.2% and 53.0% for control and 1, 5 and 10 ng/ml, respectively). The mean cell numbers in blastocyst were significantly increased in GM-CSF supplemented groups compared to control group. LIF and IL-1beta expression in blastocysts were significantly higher in GM-CSF supplemented group than in control group. CONCLUSION: The results of experiment by mouse embryos showed beneficial effects of GM-CSF as a medium supplement. Furthermore, the addition of GM-CSF significantly increased the expression of LIF and IL-1beta in mouse embryos. These results suggest that GM-CSF might be a important molecule in embryo implantation.

Effects of Xenoestrogens on Gene Expression of Cytochrome P450 Genes in in vitro Cultured Mice Spermatogenic Cells / 대한불임학회지

No abstract available.

Effect of Partial Laser Assisted Hatching on Mouse Embryos / 대한불임학회지

OBJECTIVE: The present study was performed to investigate the efficiency of partial laser assisted hatching (p-LAH; lased 1/2 ZP width from ZP edge) on hatching of mouse blastocysts. METHODS: We used non-contact 1.48 micrometer diode laser (MTM, Switzland) to create a precise hole on zona pellucida. 2-cell embryos were collected from the mouse (ICR) oviduct at 48 hours after hCG administration. Collected 2-cell embryos were cultured in the P-1 medium supplemented with 0.4% BSA. For experiments, embryos at 8-cell stage were used after 20~22 hours in culture. After conventional (c-LAH) or partial laser assisted hatching, the embryos were further cultured in P-1 medium supplemented with 0.4% BSA for 3 days. To compare efficiency of complete and partial laser assisted hatching, hatching rate, hatching time and blastocyst diameter and zona pellucida thickness at hatching time were investigated. Embryos were examined every 12 hours. Blastocyst diameter and zona pellucida thickness at hatching time were measured with an ocular micrometer. RESULTS: Hatching rates of p-LAH group (84.2%) was significantly higher than that of control group (39.3%), but there was no difference between the p-LAH (84.2%) and c-LAH (91.2%). p-LAH group was hatched 12 hours earlier than control group, but hatched 12 hours later than c-LAH group. The diameter of blastocyst at hatching time of p-LAH group (113.1+/-6.4 micrometer) was smaller than that of control group (122.2+/-5.0 micrometer), but larger than that of c-LAH group (102.2+/-2.7 micrometer). Zona pellucida thickness at hatching time of p-LAH group (6.4+/-0.9 micrometer) was thicker than that of control group (4.5+/-1.5 micrometer), but thinner than that of c-LAH group (10.0+/-0.8 micrometer). CONCLUSION: These results suggest that p-LAH may maintains the cell arrangement of early embryos to ensure successful development and prevent precocious hatching of blastocyst when compare to c-LAH and conventional (acidic tyrode) AH. Thus, p-LAH may provide a valuable and effective AH technique for human ART program.

Constitutive expression and changes of cytochrome P450 isozymes mRNAs by vehicles (petrolatum, DMSO, ethanol) in rat skin using semi-quantitative RT-PCR

Many drugs are primarily metabolized by the cytochrome P450s (CYPs). Drug metabolites would be important allergens for adverse drug reactions such as drug eruptions. Skin tests with a suspected drug have conducted to identify causative drugs of drug eruptions, with vehicles such as white petrolatum, DMSO, ethanol. This study will compare the expression of rat CYP isozyme mRNAs between the skin and the liver, with examining an effect of the vehicles on the cutaneous CYPs using semi-quantitative RT-PCR. Thirty-two Sprague-Dawley rats between the ages of six and eight weeks were divided as four groups. One group was used to compare the constitutive mRNA expression between skin and liver, while the others were to examine the effects of three vehicles. The ratios of expression of CYP1A2, CYP2B1/2, CYP2E1, CYP3A1, and CYP4A1 were significantly higher in the liver than the skin. However, CYP1A1 and CYP2C11 were higher in the skin than liver. The effects of vehicles were quite different; white petrolatum significantly induced CYP1A1 (p=0.012) and CYP2C11 mRNAs, while ethanol inhibited CYP1A1 and CYP2B1/2. DMSO did not make any changes. The results suggest that rat skin can participate in drug metabolism with their own CYP isozymes. The effects of vehicles on the cutaneous CYP expression should not be ignored and may be applied for determination of an appropriate vehicle for certain drug(s).

Expression of Testis Specific Genes of Male Germ Cells in Pre-pubertal Mouse using in vitro Culture System / 대한남성과학회지

PURPOSE: To establish cultures of premeiotic mouse spermatogenic cells that could allow study of the meiotic process in vitro and to investigate expression of sperm-specific genes in premeiotic and postmeiotic division. MATERIALS AND METHODS: Male ICR mice were used. To obtain the premeiotic spermatogenic cells, we examined the histologic appearance of the germ cells in hematoxylin-eosin-stained testis sections during prepubertal development. To confirm the identity of the premeiotic cells, we preformed reverse transcriptase-polymerase chain reaction (RT-PCR) for the genes for LDH-C4, acrosin, protamine-2 (P-2), and SP-10, which are testis-specific marker proteins. In order to establish the culture system, the premeiotic spermatogenic cells were cocultured with mouse Sertoli cells in DMEM containing 10% fetal bovine serum (FBS), amino acids, FSH, and testosterone at 32 degrees C for 6 days, After 2 days of culture, RT-PCR was done to detect the sperm-specific genes. RESULTS: The four genes was not expressed in 10-day-old mice, but expression was detectable in 17-day-old mice. Thus, mice of this age have initiated the meiotic process, and round spermatids were seen in the testis. Premeiotic germ cells isolated from 15-day-old mice expressed LDH-C4 and acrosin but not P-2 and SP-10, which are post-meiotic marker proteins. Beginning after 2 days of culture, expression of the P-2 and SP-10 genes was detected in cultured premeiotic germ cells as in 15-day-old mice. CONCLUSIONS: P-2 and SP-10 may be marker genes for the premeiotic stage of spermatogenesis. Premeiotic male germ cells are able to differentiate into postmeiotic forms during coculture with Sertoli cells.

Mutation at exon 10 of the fibroblast growth factor receptor 3 (FGFR3) in a fetus with thanatophoric dysplasia type I (TDI) / 대한산부인과학회잡지

Thanatophoric dysplasia (TD) is a sporadic lethal type of skeletal dysplasia featuring micromelia, decreased thoracic dimension and macrocephaly. To date, several kinds of mutation in fibroblast growth factor receptor 3 (FGFR3) has been identified in TD. We experienced a case of TD type I and underwent sequencing of the exon 7, 10 and the stop codon of FGFR3 to identify the type of mutation. TDI was diagnosed by the prenatal ultrasound at 25 weeks of gestation. The pregnancy was terminated and the diagnosis was confirmed by radiological and histologic examinations. The genomic DNA was extracted and the sequences of the exon 7, 10 and the stop codon of FGFR3 were amplified by PCR. The sequencing was performed for the each PCR products by dideoxyterminator method. The nucleotide transition from G to T was found in the nucleotide 1108, which is a part of the transmembrane domain, exon 10. To date, only one type of mutation (nucleotide 742) in the FGFR3 was identified in TD1 among Asian. This case firstly reveals the mutation of FGFR3 other than mutation at nucleotide 742 in TD1.