The clinical application of whole chromosome painting probes in preimplantation genetic diagnosis for translocation carriers / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To make preimplantation genetic diagnosis (PGD) for female translocation carriers by analyzing first polar bodies (1PBs) with whole chromosome painting probe (WCP).</p><p><b>METHODS</b>WCP 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSION</b>WCP can differentiate normal, balanced and unbalanced oocytes accurately and can be used as an efficient PGD method for female carriers of translocation.</p>

Analysis of chromosome mosaicism in preimplantation embryos by using 2 sequential rounds of fluorescence in situ hybridization / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the mechanism and factors affecting mosaicism in human preimplantation embryos by using 2 sequential rounds of fluorescence in situ hybridization(FISH).</p><p><b>METHODS</b>Totally 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.</p><p><b>RESULTS</b>Among 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 <or= 4 cell stage to 40.0% in 5-8 cell stage embryos. The aneuploidy rate for the patients over 35 years of age was significantly higher than that of the patients under 35 years (57.1% vs 23.3%).</p><p><b>CONCLUSION</b>Mosaicism 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.</p>

Characteristics of IGF-II gene imprinting in twin placentas / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>One 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.</p><p><b>RESULTS</b>Although 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%).</p><p><b>CONCLUSION</b>The risk of IGF-II gene imprinting loss is higher in female twins and has no relationship with assisted reproductive technology and zygosity.</p>

Sperm sex chromosome analysis and preimplantation genetic diagnosis of patients with sex chromosome anomalies / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the constitution of abnormal spermatozoa from patients with sex chromosome anomalies.</p><p><b>METHODS</b>Triple 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.</p><p><b>RESULTS</b>An 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.</p><p><b>CONCLUSION</b>Using 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.</p>

Growth differentiation factor-9 gene expression in in vitro cultured oocytes in mice / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the relation between oocyte maturation and growth differentiation factor-9 (GDF-9) gene expression.</p><p><b>METHODS</b>Ovariectomy 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.</p><p><b>RESULTS</b>Follicle 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).</p><p><b>CONCLUSION</b>MII 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.</p>

Closely linked polymorphic marker: successful application in preimplantation genetic diagnosis for beta-thalassemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Single 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSION</b>This 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.</p>

Establishment of human embryonic stem cell line from gamete donors / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Human 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.</p><p><b>METHODS</b>Three 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.</p><p><b>RESULTS</b>Four 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.</p><p><b>CONCLUSIONS</b>HES cell lines can be established from gamete donors at a relatively highly efficient rate. The establishment will exert a widespread impact on biomedical research.</p>

Cryopreservation of human embryonic stem cells by vitrification / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The 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.</p><p><b>METHODS</b>Human 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.</p><p><b>RESULTS</b>Eighty-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.</p><p><b>CONCLUSIONS</b>Vitrification is effective in cryopreserving human ES cells. During a prolonged culture, human ES cells retain their pluripotency after cryopreservation.</p>

Birth of healthy children after preimplantation diagnosis of beta-thalassemia / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Clinical 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>Of 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.</p><p><b>CONCLUSIONS</b>We 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.</p>

Sex determination of human preimplantation embryo using nested polymerase chain reaction / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis.</p><p><b>METHODS</b>One (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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>The KOH method for DNA preparation is superior to the other methods evaluated. Dual blastomere biopsy and independent blastomere analysis may improve preimplantation diagnostic reliability.</p>

Outcome of repeated epididymal sperm aspiration or testicular sperm extraction in azoospermic patients / 中华男科学杂志

<p><b>OBJECTIVE</b>To review the outcome of repeated percutaneous sperm aspiration (PESA) and testicular sperm extraction (TESE) for intracytoplasmic sperm injection (ICSI).</p><p><b>METHODS</b>Forty-three cycles of 31 cases of azoospermic patients which underwent at least two PESA or TESE for ICSI from January 2001 to December 2002 were collected. The sperm retrieval, fertilization, implantation and clinical pregnancy were analyzed.</p><p><b>RESULTS</b>Twenty-four cases underwent PESA and 7 cases underwent TESE. There were not any complications in these patients. Compared with the first cycle of 154 cases, the fertilization rate, implantation rate and clinical pregnancy rate were 78.39% vs 73.64%, 19.68% vs 18.38% and 34.88% vs 37.91%, respectively(P > 0.05).</p><p><b>CONCLUSIONS</b>Repeated PESA or TESE is safe and well tolerated in azoospermic patients. Compared with the first cycle, the differences of repeated PESA or TESE cycles in fertilization rate, implantation rate and clinical pregnancy rate were not statistically significant.</p>

Real-time fluorescent PCR for screening AZFc/DAZ microdeletions on the Y chromosome in male infertility patients / 中华男科学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>Eleven 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.</p><p><b>CONCLUSION</b>The 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.</p>

Effect of antisperm antibodies on assisted reproduction / 中华男科学杂志

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.

Preimplantation genetic diagnosis for beta-thalassemia using whole genome amplification / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>A 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.</p><p><b>RESULTS</b>A 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.</p><p><b>CONCLUSION</b>These studies represent the successful application of PGD for beta-thalassemia in China.</p>

Establishment and preliminary application of screening methods for Y chromosome microdeletions in male infertility patients / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To develop a multiplex PCR protocol, which could be suitable for routine screening of microdeletions on the Y chromosome in azoospermic and oligozoospermic male infertility patients.</p><p><b>METHODS</b>Five multiplex sets were established. Eighty-seven azoospermic and oligozoospermic patients undergoing intracytoplasmic sperm injection (ICSI) in the in vitro fertilization (IVF) center and 30 azoospermic men undergoing testicular biopsy in the clinic of Urology Surgery were screened for microdeletions of Y chromosome.</p><p><b>RESULTS</b>A total of 19 (16.2%) cases of microdeletions were found in 117 azoospermic and oligozoospermic patients by screening of Y chromosome microdeletions. Of these, 11 cases (18.0%) were found in 61 oligozoospermic patients, and 8 cases (14.3%) were found in 56 azoospermic patients.</p><p><b>CONCLUSION</b>The multiplex PCR protocol presented in this study is an easy-to-do and reliable method for detecting microdeletions on the Y chromosome. Routine screening of microdeletions on the Y chromosome for azoospermic and oligozoospermic patients is essential.</p>

The latest development in preimplantation genetic diagnosis / 北京大学学报(医学版)

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.

Sperm retrieval methods and pregnancy outcome of 100 azoospermia patients / 中华男科学杂志

<p><b>OBJECTIVES</b>To review the retrospective treatment results of the azoospermia patients during January 2001 to January 2002 in the fertility center.</p><p><b>METHODS</b>One hundred males attempted intracytoplasmic sperm injection (ICSI) cycle for treatment of azoospermia. All patients were undergone sperm retrieval by percutaneous epididymal sperm aspiration (PESA) or testicular sperm extraction (TESE) while their wives received conventional ovarian hyperstimulation. The hormone levels, testicular histology, the rates of sperm retrieval, fertilization, implantation and pregnancy were analysed and evaluated.</p><p><b>RESULTS</b>Sperm were retrieved by PESA in 76 of 100 (76%) and by TESE in 23 of 100 (23%) men of azoospermia. The fertilization rate, implantation rate and clinical pregnancy rate were 71.3%, 20.35% and 42.11% respectively in PESA group, and 75.18%, 22.05% and 41.60% respectively in TESA group. Thirty-two clinical pregnancies were achieved with 15 ongoing pregnancies and subsequent live delivery for 15 cases in PESA group, and 2 cases of miscarriage, while 10 clinical pregnancies were achieved with 6 ongoing pregnancies, 2 cases of live delivery and 2 cases of miscarriage in TESA group. One case failed to retrieve sperm by TESE and canceled.</p><p><b>CONCLUSIONS</b>Hormonal levels and testicular histology are unable to predict which men with azoospermia will have sperm retrieved by PESA and TESE. PESA and TESE with ICSI are effective methods to treat azoospermia. There were no significant differences in fertilization, implantation and clinical pregnancy rate between two groups.</p>