Preimplantation genetic diagnosis is the integration of both assisted reproductive technologiesand molecular genetic technologies.Since the birth of the first healthy females after PGD in 1990,re-markable advances have been achieved in this field.Most research in PGD is focused on new methods toimprove the sensitivity and accuracy of single cell analysis.The principal problems in single cell PCR in-clude amplification failure,ADO and contamination.Fluorescent PCR with multiplex amplifications ofhighly polymorphic markers is a highly effective strategy to avoid contamination and detect ADO.The ad-vantages and disadvantages of fluorescence in situ hybridization to detect age-related aneuploidy are stillunder debate.We summarize the most recent developments in this review,and also introduce our own ex-periences in PGD.

BACKGROUNDThe efficiency of traditional cryopreservation of human embryonic stem (ES) cells is low, and there have been few attempts to prove new cryopreservation methods effective. This study was designed to evaluate the efficiency of cryopreservation of human ES cells using vitrification method.

METHODSHuman ES cells clumped from an identical cell line were randomly allocated to be cryopreserved by vitrification or by slow freezing. The recovery rates, the growth and differentiation potential of thawed human ES cells were compared between these two groups. The pluripotency of human ES cells after thawing was identified.

RESULTSEighty-one point nine percent (59/72) of human ES cell clumps were recovered after vitrification, while only 22.8% (16/70) were recovered after slow freezing (P < 0.01). The colonies after vitrification manifested have not only faster growth but also a lower level of differentiation when compared to colonies subjected to the slow freezing protocol. However, the rates of growth and differentiation in undifferentiated colonies from both groups were identical to the rates in those of non-cryopreserved stem cells after a prolonged culture period. Passage 6 of vitrified human ES cells retained the properties of pluripotent cells, a normal karyotype and expressed the transcription factor OCT-4, stage specific expressed antigen-4 (SSEA-4) and SSEA-3. Teratoma growth of these cells demonstrated the ability to develop into all three germ layers.

CONCLUSIONSVitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.

Cell Differentiation ; Cell Survival ; Cryopreservation ; methods ; Embryo, Mammalian ; cytology ; Humans ; Osmotic Pressure ; Stem Cells ; cytology

BACKGROUNDHuman embryonic stem (HES) cell derived from human blastocyst can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent. It has exciting potential in human developmental biology, drug discovery, and transplantation medicine. But there are insufficient HES cell lines for further study.

METHODSThree oocyte donors were studied, and 3 in vitro fertilization (IVF) cycles were carried out to get blastocysts for the establishment of HES cell line. Isolated from blastocysts immunosurgically, inner cell mass (ICM) was cultured and propagated on mouse embryonic fibroblasts (MEFs). Once established, morphology, cell surface markers, karyotype and differentiating ability of the cell line were thoroughly analyzed.

RESULTSFour ICMs from 7 blastocysts were cultured on MEFs. After culture, one cell line (cHES-1) was established and met the criteria for defining human pluripotent stem cells including a series of markers used to identify pluripotent stem cells, morphological similarity to primate embryonic stem cells and HES reported else where. Normal and stable karyotype maintained over 60 passages, and demonstrated ability to differentiate into a wide variety of cell types.

CONCLUSIONSHES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.

Blastocyst ; cytology ; Cell Differentiation ; Cell Line ; DNA-Binding Proteins ; analysis ; Female ; Fertilization in Vitro ; Humans ; Karyotyping ; Male ; Octamer Transcription Factor-3 ; Stem Cells ; cytology ; Tissue Donors ; Transcription Factors ; analysis

OBJECTIVETo explore the relation between oocyte maturation and growth differentiation factor-9 (GDF-9) gene expression.

METHODSOvariectomy was performed in 50 Kunming female mice of 10 days old, and the preantral follicles were isolated from the ovaries and cultured in medium drops for 12 days. Oocytes and somatic cells were mechanically isolated. The oocytes cultured in vitro for 2, 4, 6, 8, 10, and 12 days constituted the in vitro cultured group and the oocytes obtained from female mice of 12, 14, 16, 18, 20, and 22 days old served as the in vivo group. Semi-quantitative RT-PCR and agar gel electrophoresis were performed to quantify GDF-9 gene expression in each oocyte.

RESULTSFollicle survival, antrum formation and maturation rate was 89.5%, 51.8% and 56.6% in the in vitro cultured follicles, respectively. GDF-9 gene expression on days 2, 4, 6, 8, 10, and 12 in in vitro cultured oocytes was 0.83-/+0.08, 0.52-/+0.09, 0.45-/+0.13, 0.49-/+0.09, 0.49-/+0.09, and 0.68-/+0.08, respectively; GDF-9 gene expression in in vivo grown oocytes of 12, 14, 16, 18, 20, and 22 days were 0.64-/+0.35, 0.48-/+0.10, 0.52-/+0.10, 0.66-/+0.08, 0.72-/+0.09, and 0.91-/+0.11, respectively. Between days 8 and 12, GDF-9 gene expression in in vitro cultured oocyte was significantly lower than that in in vivo grown oocytes (P<0.05).

CONCLUSIONMII oocytes can be obtained from in vitro culture of the preantral follicles. GDF-9 gene expression in the oocytes varies with their growth stages. Between days 8 and 12 of in vitro culture, GDF-9 gene expression in the cultured oocytes is different from that in in vivo grown oocytes.

Animals ; Animals, Newborn ; Bone Morphogenetic Protein 15 ; Cell Survival ; Cells, Cultured ; Electrophoresis, Agar Gel ; Female ; Gene Expression ; Growth Differentiation Factor 9 ; Intercellular Signaling Peptides and Proteins ; genetics ; Mice ; Oocytes ; cytology ; growth & development ; metabolism ; Ovarian Follicle ; cytology ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

The formation of antisperm antibodies (AsAb) results from the disruption of the blood-testis barrier by a variety of mechanisms, which leads to exposure of immunogenic sperm antigens to the immune system and initiates an immune response. AsAb can impair the fusion of sperm and egg and even the embryo development, resulting in infertility. The etiology of AsAb, effect of AsAb on assisted reproduction and treatment of AsAb in the literature are reviewed in this article.
Antibodies ; immunology ; Antibody Formation ; Humans ; Infertility, Male ; etiology ; Male ; Reproductive Techniques ; Spermatozoa ; immunology

OBJECTIVETo investigate the mechanism and factors affecting mosaicism in human preimplantation embryos by using 2 sequential rounds of fluorescence in situ hybridization(FISH).

METHODSTotally 51 normal fertilized embryos, which were not suitable for embryo transfer and cryopreservation, were analyzed on day 3 after fertilization by using two sequential rounds of FISH. Chromosomes 13, 16, 18, 21, 22, X and Y were analyzed.

RESULTSAmong 51 embryos, 16 (31.4%) were mosaic, 12 (23.5%) were chaotic, and the remaining were either normal (27.5%) or non-mosaic abnormal (17.6%). The incidence of mosaic embryos was related to embryo developmental stage, for the incidence of mosaicism increased from 12.5% in embryos

CONCLUSIONMosaicism is common in human preimplantaion embryos, which may be one of the important factors affecting the success rates in IVF-ET. Most of the chromosomal abnormalities can be identified by two sequential rounds of FISH.

Aneuploidy ; Blastocyst ; Chromosomes, Human ; Embryo Transfer ; Female ; Humans ; In Situ Hybridization, Fluorescence ; methods ; Mosaicism ; chemically induced ; embryology ; Preimplantation Diagnosis

0.05.Conclusions The UF effectively removed BUN in sheep with low flow VV-ECMO.The application of UF didn't cause blood shunt in ECMO.The low flow VV-ECMO effectively eliminated carbon dioxide and rerformed oxygenation.

OBJECTIVETo make preimplantation genetic diagnosis (PGD) for female translocation carriers by analyzing first polar bodies (1PBs) with whole chromosome painting probe (WCP).

METHODSWCP was used in fluorescence in situ hybridization (FISH) analysis of 1PBs for four female Robertsonian carriers presented for PGD with 45 XX, der(13;14)(q10;q10) karyotype. All the patients underwent ovarian stimulation and during 6 h after oocyte retrieval 1PBs were biopsied and WCP were used in FISH. On day 3 after fertilization embryos diagnosed as normal or balanced were transferred.

RESULTSA total of 61 oocytes were collected in 4 PGD cycles. Of the 54 matured oocytes, 50 were biopsied and 45 were fixed successfully. Results were obtained in 40 1PBs. Overall, 74.1% (40/54) oocytes were diagnosed. The fertilization rate and good embryo rate were 64.8% (35/54) and 65.7% (23/35) respectively. Two clinical pregnancies were obtained. One patient delivered a normal female baby with karyotype 46, XX in June 2006. For another patient, the fetus spontaneously aborted at 9th week of pregnancy with karyotype of 45, X confirmed by amniotic villus diagnosis.

CONCLUSIONWCP can differentiate normal, balanced and unbalanced oocytes accurately and can be used as an efficient PGD method for female carriers of translocation.

Adult ; Chromosome Painting ; methods ; Female ; Heterozygote ; Humans ; In Situ Hybridization, Fluorescence ; Oocytes ; metabolism ; Pregnancy ; Preimplantation Diagnosis ; methods ; Translocation, Genetic ; genetics

OBJECTIVETo compare insulin-like growth factor II (IGF-II) gene imprinting in twin placentas with singleton ones and to determine whether imprinting was influenced by assisted reproductive technology, zygosity and fetal sex.

METHODSOne hundred and sixty cases of twin placentas and 42 cases of singleton ones were recruited. Allele-specific IGF-II expression was determined by reverse transcription-PCR combined with analysis of an Apa I-sensitive restriction fragment length polymorphism.

RESULTSAlthough the incidence of IGF-II imprinting loss was higher in normal twin placentas than in singleton ones (20.6% vs 8.7%), there was no statistical significance. There were no significant differences between twins conceived by assisted reproductive technology and those conceived spontaneously (17.9% vs 24.4%), and between dizygotic and monozygotic twins (22.4% vs 16.7%). The incidence of IGF-II imprinting loss in placenta of female twins was statistically higher than that of male ones (26.4% vs 9.8%).

CONCLUSIONThe risk of IGF-II gene imprinting loss is higher in female twins and has no relationship with assisted reproductive technology and zygosity.

Adult ; Female ; Genomic Imprinting ; genetics ; Humans ; Infant, Newborn ; Insulin-Like Growth Factor II ; genetics ; Male ; Maternal Age ; Placenta ; metabolism ; Polymorphism, Genetic ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Twins ; genetics ; Young Adult

OBJECTIVETo investigate the constitution of abnormal spermatozoa from patients with sex chromosome anomalies.

METHODSTriple color fluorescence in situ hybridization (FISH) was used to determine the sex chromosome constitution of spermatozoa from three patients with sex chromosome anomalies (case 1:46,XY/47,XXY, case 2:45,XO/46,X,Yqh-, case 3:47,XXY). The preimplantation genetic diagnosis (PGD) was performed to case 2.

RESULTSAn increased ratio (2.05 vs 1) of X-bearing to Y-bearing spermatozoa was only observed in case 2, who also had an increased incidence of total abnormal spermatozoa (29.71%). An increased incidence of total abnormal spermatozoa (4.91%) was also observed in case 3. Among the constitution of abnormal spermatozoa, case 2 had the increased proportions of XY18 disomy, O18 monosomy and XO monosomy, while case 3 had an increase proportion of XY18 disomy (1.87%). PGD was performed to case 2 and one embryo with XX1818 was selected for implanting.

CONCLUSIONUsing FISH to detect the sperm sex chromosomes in patients with sex chromosome anomalies can provide the useful information to evaluate the risk of sex chromosome anomalies in preimplantation embryos.

Adult ; Chromosomes, Human, X ; genetics ; Chromosomes, Human, Y ; genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Preimplantation Diagnosis ; methods ; Sex Chromosome Aberrations ; Spermatozoa ; metabolism