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 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

BACKGROUNDClinical programs for preventing beta-thalassemia are presently based on prospective carrier screening and prenatal diagnosis. This paper report an achievement of a pregnancy with unaffected embryos using in vitro fertilization and embryo transfer (IVF-ET), in combination with preimplantation genetic diagnosis (PGD), for a couple at risk of having children with beta-thalassemia.

METHODSA couple carrying different thalassemia mutations, both a codon 41 - 42 mutation and the IVS II 654 mutation, received standard IVF treatment, with intracytoplasmic sperm injection, embryo biopsiy, single cell polymerase chain reaction (PCR) and DNA analysis. Only unaffected or carrier embryos were transferred to the uterine cavity. After confirmation of pregnancy, a prenatal diagnosis was performed.

RESULTSOf a total of 13 embryos analyzed for beta-globin mutations, PGD indicated that 2 were normal, 3 were affected, and 6 were carriers. Diagnosis could not be made in the other 2 embryos. Three embryos were transferred to the uterus on the third day after oocyte retrieval. Ultrasonography revealed a twin pregnancy with one blighted ovum. The prenatal genetic diagnosis revealed that both fetuses were unaffected, and two healthy boys were born, confirming the results of PGD.

CONCLUSIONSWe developed a single-cell based primer extension preamplification (PEP)-PCR assay for the detection of beta-thalassemia mutations. The assays were efficient and accurate at all stages of the procedure, and resulted in the birth of PGD-confirmed beta-thalassemia free children in China. PEP was used here in PGD for beta-thalassemia.

Adult ; Embryo Transfer ; Female ; Fertilization in Vitro ; Humans ; Infant, Newborn ; Male ; Mutation ; Polymerase Chain Reaction ; Pregnancy ; Preimplantation Diagnosis ; beta-Thalassemia ; diagnosis ; genetics

OBJECTIVEUsing nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis.

METHODSOne (or two) lymphocyte and blastomere (n=50/group) were collected and prepared under the following conditions: (1) water only (H(2)O); (2) freeze-thaw liquid nitrogen, then boiling; (3) potassium hydroxide/dithiotheriol, heated to 65 degree centigrade, followed by acid neutralization (KOH). Cells were analyzed by PCR using nested primers amplification with amelogenin gene.

RESULTSThe amplification rate and allele dropout (ADO) rate for male lymphocytes by the three methods were 83%, 94%, 95% and 24%, 12%, 4%, respectively. Using two cells per reaction did not increase the amplification rate for the KOH method.

CONCLUSIONThe KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.

Amelogenin ; Blastocyst ; cytology ; metabolism ; Blastomeres ; cytology ; metabolism ; DNA ; genetics ; Dental Enamel Proteins ; genetics ; Female ; Genotype ; Humans ; Lymphocytes ; cytology ; metabolism ; Male ; Polymerase Chain Reaction ; methods ; Sex Determination Analysis ; methods

OBJECTIVETo develop a real-time fluorescent PCR protocol suitable for the routine screening of AZFc/DAZ microdeletions on the Y chromosome in azoospermic and oligozoospermic male infertility patients.

METHODSA set of real-time fluorescent PCR was established. Eighty-seven azoospermic and ligozoospermic patients undergoing ICSI in the IVF center and 30 azoospermic men undergoing testicular biopsy in the clinic of urology surgery were screened for AZFc/DAZ microdeletions of Y chromosome.

RESULTSEleven cases (9.4%) of AZFc/DAZ microdeletions were found in 117 cases of azoospermic and oligozoospermic patients by screening of realtime fluorescent PCR. Four cases (6.6%) were found in 61 oligozoospermic patients, and 7 cases (12.5%) were found in 56 azoospermic patients.

CONCLUSIONThe real-time fluorescent PCR protocol presented in this study is an easy and reliable method for detection of AZFc/DAZ microdeletions on the Y chromosome, which yields identical results to those of the multiplex PCR.

Chromosome Deletion ; Chromosomes, Human, Y ; Deleted in Azoospermia 1 Protein ; Fluorescence ; Humans ; Infertility, Male ; genetics ; Male ; Polymerase Chain Reaction ; methods ; RNA-Binding Proteins ; genetics

OBJECTIVETo evaluate the applicability of the polymorphic marker closely linked with beta-globin gene for the preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia.

METHODSSingle cell multiplex nested PCR which coamplifies the beta-globin gene and the closely linked polymorphic marker, HumTHO1 gene, was applied in six clinical PGD cycles for four couples with beta-thalassemia.

RESULTSIn six clinical PGD cycles, a total of 44 embryos were biopsied and 44 blastomeres were obtained. Forty-one blastomeres were amplified and thirty-five embryos were given definite diagnoses. Fourteen embryos were transferred back to the uterus of the patients and one pregnancy went on well and ended with one live healthy birth, which confirmed the results of PGD. The average amplification efficiency of single blastomere was 89.7% and the average allele drop-out(ADO) rate was 14.4%. The coamplification of HumTHO1 could help to detect the existence of ADO and contamination.

CONCLUSIONThis is the first report on unaffected pregnancy resulting from PGD using multiplex nested PCR in China. The simultaneous amplification of polymorphic marker closely linked to beta-globin gene(HumTHO1) could help to resist the risk of misdiagnosis in PGD caused by ADO and contamination.

Adult ; Female ; Humans ; Male ; Polymerase Chain Reaction ; Pregnancy ; Preimplantation Diagnosis ; methods ; beta-Globins ; genetics ; beta-Thalassemia ; diagnosis ; genetics

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 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

OBJECTIVETo achieve pregnancy with unaffected embryo using in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis(PGD) for the couples at risk of having children with beta-thalassemia.

METHODSA couple carrying different thalassemia mutations of codon 41/42 and codon IVS2 position 654 received standard IVF treatment and intracytoplasmic sperm injection, embryo biopsy, single cell polymerase chain reaction and DNA analyses, and only the unaffected or carrier embryos were transferred to uterus. Pregnancy confirmation, and prenatal diagnosis were done at 20 week's gestation.

RESULTSA total of 13 embryos were analyzed in the IVF cycle. PGD indicated that 2 were normal 18.1 , 3 were affected 27.3 , and 6 were carriers 54.5 ; diagnosis was not possible in 2. Three embryos were transferred to uterus on the third day after oocyte retrieval. Ultrasonography showed twin pregnancy with one blighted ovum. The prenatal diagnoses revealed that both fetuses were unaffected, one normal baby and one carrier were born.

CONCLUSIONThese studies represent the successful application of PGD for beta-thalassemia in China.

Adult ; Embryo Transfer ; Female ; Fertilization in Vitro ; Humans ; Male ; Mutation ; Pregnancy ; Pregnancy Outcome ; Preimplantation Diagnosis ; methods ; Prenatal Diagnosis ; methods ; beta-Thalassemia ; diagnosis ; genetics ; prevention & control