Recently, through the development of the primer extension preamplification(PEP) method which amplifies the whole genome, simultaneous multiple DNA analysis has become possible. Whole genome from each single cell can be amplified using 15 base oligonucleotide random primer. The greatest advantage of PEP-PCR is the ability to investigate several loci simultaneously and confirm results by analysing multiple aliquots for each locus. This technique led to the development of preimplantation genetic disease diagnosis using blastomere from early embryo, sperm, polar body and oocyte. In this study, we applied PEP-PCR in 20 cases of single amniocyte and 20 cases of single chorionic villus cell for the clinical application of the prenatal and preimplantational genetic diagnosis. We analysed 7 gene loci simultaneously which are 46, 47 exons related to dystrophin gene, two VNTR (variable number tandem repeat) markers using 5'toysIII, 3'CA related to dystrophin gene and DYZ1, DYZ3, DYS14 regions on chromosome Y. In all the tests, 97.5% of PEP-PCR amplifications with single cells were successful. We obtained 38/40 (95%) accuracy in gender determination through chromosome analysis comparison. Therefore, these results have significant implications for a sperm or oocyte analysis and prenatal or preimplantational genetic diagnosis.
Blastomeres ; Chorionic Villi ; Diagnosis ; DNA ; Dystrophin* ; Embryonic Structures ; Exons ; Genome ; Oocytes ; Polar Bodies ; Spermatozoa

OBJECTIVE: This study was performed to evaluate the efficiency of preimplantation genetic diagnosis (PGD) using fluorescence in-situ hybridization (FISH) in Robertsonian or balanced reciprocal translocation carriers in human IVF-ET programm. METHOD: FISH was carried out in 25 cycles of 15 couples. Two-color FISH analysis was performed on 54 polar bodies in 3 cycles and 234 blastomeres in 22 cycles. After FISH analysis, the embryos with normal FISH signals were transferred into mother's uterus. RESULTS: In FISH analysis of polar bodies, 18 nuclei of polar bodies were normal and 12 embryos were transferred in 3 cycles. FISH efficiency per oocyte was 95.0% in cases using polar bodies. In FISH analysis of blastomeres, 49 embryos were normal and transferred in 21 cycles. FISH efficiency per embryo was 92.7% using blastomeres. At present, three pregnancies were achieved. A girl and a boy were delivered. Both of them were translocation carriers. The other conceptus showed normal karyotype. CONCLUSIONS: According to this study, PGD using FISH can be successfully applied for the patients with translocations of chromosomes.
Blastomeres ; Embryonic Structures ; Family Characteristics ; Female ; Fluorescence* ; Humans* ; Karyotype ; Male ; Oocytes ; Polar Bodies ; Pregnancy ; Preimplantation Diagnosis* ; Prostaglandins D ; Uterus

OBJECTIVE: To verify the expression of leptin receptor (OB-R) in oocytes and preimplantation embryos, the involvement of mitogen activated protein kinase (MAPK or Erk1/2) in the leptin signaling, and effect of leptin on the oocyte maturation in mice. METHOD: RT-PCR analysis of OB-R was conducted in germinal vesicle (GV)-intact and MII stage oocytes, and 1, 2, 8-cell embryos and blastocysts. Germinal vesicle breakdown (GVB), polar body extrusion, monitored in the presence or absence of leptin (1 microM). Following the leptin treatment, temporal changes in MAPK activity were verified by immunoprecipitation and in vitro kinase assay in MII oocytes. RESULTS: The expression of OB-R mRNA was found in GV and MII oocyte but not in the embryos. MAPK activity of the MII oocytes was significantly increased by brief incubation in the HTF supplemented with leptin (1 microM). Priming of PD098059, a MEK inhibitor to leptin treatment attenuated the activation of MAPK by leptin in MII oocytes. Following 24 hrs of culture of the GV oocytes, leptin significant increased the GVB and 1st polar body extrusion. CONCLUSION: This result suggested that functional interaction between leptin and OB-R resulted in potentiation of MAPK (Erk1/2) activity in MII oocytes through MEK activation and that leptin might be a local regulator of meiotic maturation of the mouse oocytes.
Animals ; Blastocyst ; Embryonic Structures ; Immunoprecipitation ; Leptin* ; Mice* ; Oocytes* ; Phosphotransferases ; Polar Bodies ; Protein Kinases* ; Receptors, Leptin ; RNA, Messenger

OBJECTIVE: Melatonin, which is secreted by pineal gland play an important role in the regulation of ovarian function via seasonal rhythm and sleep in most mammals. It also has a role in the protection of cells by removing toxic oxygen free radicals brought about by metabolism. In the present study, effects of melatonin on the mouse oocyte maturation were examined using two different culture conditions provided with 5% or 21% oxygen concentration. MATERIAL AND METHOD: Immature mouse oocytes were obtained from the ovarian follicles of 3~4 weeks old ICR strain mice intraperitoneally injected with 5 I.U. PMSG 44 hour before. Under stereomicroscope, morphologically healthy oocytes with distinct germinal vesicle (GV) were liberated from the graafian follicles and collected using mouth-controlled micropipette. They were then cultured for 17 hour at 37degrees C, 5% CO2 and 21% O2 (95% air) or 5% CO2, 5% O2 and 90% N2. New modified Hank's balanced salt solution (New MHBS) was used as a culture medium throughout the experiments. Effects of melatonin were examined at a concentration of 0.0001 micrometer, 0.01 micrometer or 1.0 micrometer. For the prevention of spontaneous maturation of immature oocytes during culture, dibutyryl cyclic AMP (dbcAMP) and/or hypoxanthine were included in the medium. RESULTS: Under 21% oxygen condition, oocytes cultured in the presence of 0.01 micrometer melatonin showed a significantly higher maturation rates, in terms of germinal vesicle breakdown (95.0% vs 89.0%) and polar body formation (88.1% vs 75.4%), compared to those cultured with 0.0001 micrometer or 1.0 micrometer melatonin. However, no difference was observed in oocytes cultured under 5% oxygen whether they were treated with melatonin or not. In the presence of 0.01 micrometer melatonin, oocytes either cultured under 21% or 5% oxygen exhibited no difference in the polar body formation (85.6% vs 86.7%). However, in the absence of melatonin, oocytes cultured under 21% oxygen exhibited lower polar body formation (74.7%). When oocytes were cultured in the presence of dbcAMP alone or with varying concentrations of melatonin, those treated with both compounds always showed better maturation, i.e., germinal vesicle breakdown and polar body formation, compared to those cultured with dbcAMP alone. At the same concentration of melatonin, however, oocytes exposed to 21% oxygen showed poor maturation than those to 5% oxygen. Similar results were obtained from the experiments using hypoxanthine instead of dbcAMP. CONCLUSION: Based upon these results, it is suggested that melatonin could enhance the meiotic maturation of mouse oocytes under 21% oxygen concentration, and release oocytes from the meiotic arrest by dbcAMP or hypoxanthine regardless of the concentration of oxygen, probably via the removal of oxygen free radicals.
Animals ; Bucladesine ; Female ; Free Radicals ; Hypoxanthine ; Mammals ; Melatonin* ; Metabolism ; Mice* ; Oocytes* ; Ovarian Follicle ; Oxygen ; Pineal Gland ; Polar Bodies ; Seasons

OBJECTIVE: During stimulated IVF cycles, up to 15% of oocytes are recovered as immature. The purpose of this study was to investigate the trend of oocyte maturity in repeated ovarian stimulation for IVF. METHODS: One hundred forty-eight patients were selected who underwent two consecutive IVF cycles using same stimulation protocol during 2008 to 2010. Ovarian stimulation was performed with FSH and human menopausal gonadotropin and flexible GnRH antagonist protocol in both cycles. Oocyte maturity was assessed according to presence of germinal vesicle (GV) and the first polar body. Immature oocyte was defined as GV stage or metaphase I oocyte (GV breakdown with no visible polar body) and cultured up to 48 hours. If matured, they were fertilized with ICSI. RESULTS: Percentages of immature oocytes were 30.8% and 32.9% (p=0.466) and IVM rates of immature oocytes were 36.2% and 25.7% (p=0.077), respectively. A significant correlation was noted between percentage of immature oocytes in the two cycles (R=0.178, p=0.03). Women with >40% immaturity in both cycles (n=21) showed lower fertilization rate of in vivo matured oocytes (56.4% vs. 72.0%, p=0.005) and lower pregnancy rate (19.0% vs. 27.1%, p=0.454) after the second cycle when compared with women with <40% immaturity (n=70). In both groups, female age, number of total retrieved oocyte and embryos transferred were similar. CONCLUSION: In repeated ovarian stimulation cycles for IVF, the immature oocyte tended to be retrieved repetitively in consecutive IVF cycles.
Embryonic Structures ; Female ; Fertilization ; Gonadotropin-Releasing Hormone ; Gonadotropins ; Humans ; Metaphase ; Oocytes ; Ovulation Induction ; Polar Bodies ; Pregnancy Rate

OBJECTIVE: It is widely accepted that aging decreases women’s fertility capacity. The aim of this study was to assess correlations between maternal age and the morphokinetic parameters and cleavage pattern of embryos. METHODS: The morphokinetics of embryos derived from women <30, 30–35, 36–40, and >40 years of age were compared retrospectively in terms of time of second polar body extrusion, time of pronuclei appearance, time of pronuclei fading, and time of two to eight discrete cells (t2–t8). Furthermore, abnormal cleavage patterns such as uneven blastomeres at the two-cell stage, cell fusion (Fu), and trichotomous mitoses (TM) were assessed. RESULTS: Only t5 occurred later in women aged 36–40 and >40 years when compared with those aged <30 and 30–35 years (p<0.001). Other morphokinetic timing parameters, as well the presence of uneven blastomeres, were comparable between the groups (p>0.05). However, Fu and TM were more common in women aged >40 years than in younger women (p<0.001). CONCLUSION: Maternal age was correlated with the cleavage pattern of embryos. Therefore, evaluating embryo morphokinetics may contribute to optimal embryo selection, thereby increasing fertility in patients with advanced maternal age.
Aging ; Blastomeres ; Cell Fusion ; Embryonic Structures ; Female ; Fertility ; Humans ; Maternal Age ; Mitosis ; Polar Bodies ; Retrospective Studies ; Sperm Injections, Intracytoplasmic

OBJECTIVE: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. METHODS: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. RESULTS: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. CONCLUSION: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.
Embryo Transfer ; Embryonic Development ; Embryonic Structures ; Female ; Fertilization ; Humans ; Oocytes ; Ovulation Induction ; Polar Bodies ; Pregnancy ; Pregnancy Outcome ; Retrospective Studies ; Sperm Injections, Intracytoplasmic ; Spermatozoa

OBJECTIVE: To investigate fertilization and embryo quality of dysmorphic mature oocytes with specific morphological abnormalities obtained from intracytoplasmic sperm injection (ICSI). METHODS: The fertilization rate (FR) and embryo quality were compared among 58 dysmorphic and 42 normal form oocytes (control 1) obtained from 35 consecutive ICSI cycles, each of which yielded at least one dysmorphic mature oocyte, performed over a period of 5 years. The FR and embryo quality of 441 normal form oocytes from another 119 ICSI cycles that did not involve dysmorphic oocytes served as control 2. Dysmorphic oocytes were classified as having a dark cytoplasm, cytoplasmic granularity, cytoplasmic vacuoles, refractile bodies in the cytoplasm, smooth endoplasmic reticulum in the cytoplasm, an oval shape, an abnormal zona pellucida, a large perivitelline space, debris in the perivitelline space, or an abnormal polar body (PB). RESULTS: The overall FR was significantly lower in dysmorphic oocytes than in normal form oocytes in both the control 1 and control 2 groups. However, embryo quality in the dysmorphic oocyte group and the normal form oocyte groups at day 3 was similar. The FR and embryo quality were similar in the oocyte groups with a single abnormality and multiple abnormalities. Specific abnormalities related with a higher percentage of top-quality embryos were dark cytoplasm (66.7%), abnormal PB (50%), and cytoplasmic vacuoles (25%). CONCLUSION: The fertilization potential of dysmorphic oocytes in our study was lower, but their subsequent embryonic development and embryo quality was relatively good. We were able to define several specific abnormalities related with good or poor embryo quality.
Abnormalities, Multiple ; Cytoplasm ; Embryonic Development ; Embryonic Structures* ; Endoplasmic Reticulum, Smooth ; Female ; Fertilization* ; Oocytes* ; Polar Bodies ; Pregnancy ; Sperm Injections, Intracytoplasmic ; Vacuoles ; Zona Pellucida

OBJECTIVE: Nitric oxide (NO) produced in ovary may contribute to follicle maturation, ovulation, oocyte maturation and luteinization. In this study, the effect of nitric oxide on the spontaneous maturation of mouse oocyte was observed. Method: The index of oocyte maturation was checked by the germinal vesicle breakdown (GVBD) and appearance of polar body (PB) under microscope in the denuded oocytes and oocyte-cumulus complexes (OCCs) from mouse ovarian follicles after 24 hours pregnant-mare serum gonadotropin treatment. RESULTS: The GVBD appeared 50 %, 1 hour and 80 %, 2 hrs after changes of oocytes from dibutyryl cAMP (dbcAMP, 0.5 mM) contained media into dbcAMP-free media. dbcAMP (0.5 mM) completely blocked the GVBD until 24 hrs but dbcGMP (5 mM) delayed the GVBD by 1 hr. Sodium nitroprusside, the NO generator, inhibited the GVBD dose-dependently at 2 hr incubation in denuded and OCCs. The appearance of GVBD was not different between control and dbcGMP or SNP in denuded oocytes and OCCs at 24 hrs incubation. The guanylate cyclase activity in denuded oocyte cytosol was not detected whereas the guanylate cyclase activity in OCCs cytosol was 1.3 nmole/min/mg protein which was increased about 3 times by SNP (100 micrometer). CONCLUSION: These results suggest that the NO in ovary may delay the spontaneous oocyte maturation in early stage by acting on the maturation signaling protein as well as guanylate cyclase.
Animals ; Bucladesine ; Cytosol ; Female ; Gonadotropins ; Guanylate Cyclase ; Lutein ; Luteinization ; Mice* ; Nitric Oxide* ; Nitroprusside ; Oocytes* ; Ovarian Follicle ; Ovary ; Ovulation ; Polar Bodies ; Staphylococcal Protein A

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.
Abortion, Habitual ; Abortion, Spontaneous ; Alleles ; Aneuploidy ; Biopsy ; Blastocyst ; Chimera ; Chromosome Aberrations ; Comparative Genomic Hybridization ; Cryopreservation ; Embryonic Structures ; Endometrium ; Female ; Fluorescence ; Genetic Testing ; Genome ; Humans ; Live Birth ; Mass Screening ; Maternal Age ; Mitochondrial Diseases ; Mosaicism ; Ovarian Hyperstimulation Syndrome ; Patient Dropouts ; Polar Bodies ; Polymorphism, Single Nucleotide ; Pregnancy ; Preimplantation Diagnosis ; Prostaglandins D ; Translocation, Genetic ; Transportation ; Vitrification