Clinical analysis of 100 preimplantation genetic diagnosis cycles / 中华妇产科杂志

Objective To investigate influence of chromosomal translocations on early embryo development and to evaluate the efficacy and feasibility of preimplantation genetic diagnosis (PGD)techniques through clinical analysis on PGD cycles. Methods Embryo development, efficacy of PGD and clinical outcome of 100 cycles were studied retrospectively, including 23 cycles with Robertsonian translocations, 19 cycles with reciprocal translocations, and 58 cycles for α-Thalassaemia. Results Among 354 embryos biopsied by PGD for translocations, 321 (90. 7% ) presented fluorescence in situ hybridization (FISH) results. The rate of normal/balanced embryos in the Robertsonian translocation was 38. 3% (64/167),which was significantly higher than 20. 8% (32/154) in the reciprocal translocation group. Amplification was achieved in 443 blastomeres from 537 embryos in Thalassaemia group, which given to an amplification efficiency rate of 82. 5% ( 443/537 ). Totally, 140 normal homozygous, 112 heterozygotes and 155 affected homozygous embryos were identified, while 36 embryos had uncertain result. The successful diagnostic rate was 75.8% (407/537). After 3 days in the translocation groups, the rate of normal and/or balanced translocations in biopsed embryos with ≥7 cells was 34. 4% (77/224), which was significantly higher than 19. 6% ( 19/97 ) of biopsed embryos with ＜ 7 cells. After 4 days, the compaction rate in normal/balanced embryos was 59.4% ( 57/96 ), which was significantly higher than 34. 2% ( 77/225 ) in imbalanced embryos significantly. Seventy-five embryos transferred in 37 cycles with translocations group led to clinical pregnancy rate of 27.0% (10/37), and 170 embryos transferred in 58 cycles with Thalassaemia got a clinical pregnancy rate of 43. 1% ( 25/58 ) . Conclusions PGD can provide management efficiently for both chromosome translocations and Thalassaemia. Translocations might have slightly negative impact on embryo development before implantation.

Evaluation of the fidelity of multiple displacement amplification from small number of cells / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To evaluate the fidelity of multiple displacement amplification (MDA) from small number of cells (1-10 cells) by 10K 2.0 SNP mapping array.</p><p><b>METHODS</b>A fibroblast cell line (Tri-18; GM02732, 47, XY, +18) was used as the template, and 6 groups were set up in the study. Groups A and B were positive and negative control, respectively; groups C-F were experimental groups involving the MDA products from 1, 2, 5 and 10 cells respectively. In combination of single nucleotide polymorphism (SNP) array, the product of each group was assessed based on the genome coverage, loss of heterozygosity (LOH) rate and allele dropout (ADO) rate.</p><p><b>RESULTS</b>The nonspecific product of negative control presented an average call rate of 3.2%. The genome coverage of the MDA product increased from 86.4% to 96.4% with the increasing number of template from 1 to 10 cells, while the LOH rate and ADO rate decreased significantly (P<0.05).</p><p><b>CONCLUSION</b>MDA is a highly efficient and reliable method for whole genome amplification. The fidelity of MDA will be improved significantly with the increasing number of template cells. 10K 2.0 SNP mapping array is a quick, accurate and comprehensive method to evaluate the fidelity of amplified DNA products, but the ADO SNPs should be distinguished from those of preferential amplification from the LOH loci to avoid errors.</p>

Nude Mouse Models for Green fluorescence Human Endometriosis / 中山大学学报(医学科学版)

[Objective]To establish a novel noninvasive fluorescent animal model for endometriosis in vitro and in vivo.[Methods]Adenovirus encoding enhancing green fluorescent protein(Ad-eGFP)was used to transfect endometrial glandular cells and stromal cells(cells transfection and injection,Method No.1),and fragments(tissues transfection and injection,Method No.2).Transfection efficiencies were compared between the two methods in vitro.Then GFP transfected glandular cells and stromal cells suspension were injected into nude mice subcutaneously(Method No.1),taking Method No.2 as a comparison.In vivo observation last for 25 days,and positive rates and duration times of fluorescent lesions were calculated.Histological examination was used to confirmed lesion formation.[Results]On the fifth day after injection,lesion positive rate of Method No.1 was 88.9%,which was statistically significantly higher than that of Method No.2(22.2%),P=0.015＜0.05.The fluorescent positive duration of Method No.1 and No.2 were 12 ± 8 days and 7±4 days.The structures of lesions were all identified as human original endometrium by histological examination,including HE staining and immunofluoresceney.[Conclusion]Noninvasive animal model of endometriosis can be built up by subcutaneously injection of Ad-EGFP transfected endometrial glandular cells and stromal cells suspension with higher positive rate and longer observation time

Detection of Aneuploidy from Single Cells by Array-Comparative Genetic Hybridization / 中山大学学报(医学科学版)

[Objective] To set up an optimized protocol for aneuploidy detection from single cells through Array- Comparative Genetic Hybridization (CGH).[Method] Two cell lines,trisomy 18 (Tri-18;GM02732,47,XY,+18) and chromosome 4 segment deletion [sDel-4;GM00343,46,XY,4(del) (qter ＞ p14)],were used in the study.In combination of 10 k 2.0 SNP mapping array platform and multiple displacement amplification (MDA),the diagnostic accurate rates of MDA product from single cells of the two cell lines using gDNA and single-cell MDA product as reference were compared.[Result] An extremely lower call rate (3.2 ± 1.2)% in the negative control group was observed compared to the experiment groups.When the single-cell MDA product was used as reference,the standard deviations of Log2 (signal intensity ratio) were significantly decreased in both groups,compared with when the gDNA as reference (P ＝ 0.004).Through CNAT analytic software,some specific chromosomes (16,17,19,and 22) presented obvious preferential amplification (PA) when the gDNA was used as reference,but this PA could be eliminated when single-cell MDA product was used as reference.[Conclusion] 10 k 2.0 SNP mapping array in combination with MDA could be a quick,highly efficient and accurate method to detect aneuploidy in single cells.

Comparing Results with Immature Germinal Vesicle Oocytes From Different In-vitro Fertilization Cycles / 中山大学学报(医学科学版)

[Objective] This study compared outcomes of in vitro maturation (IVM) and in vitro fertilization (IVF) intracytoplasmic sperm injection (ICSI) cycles after IVM of immature germinal vesicle (GV) oocytes.[Methods] ICSI was performed on metaphase II (MII) oocytes retrieved in 163 IVF-ICSI cycles (group I;n ＝ 987) or matured from GV stage oocytes in IVF-ICSI ( group II;n ＝ 132) and 37 IVM cycles ( group III;n ＝ 235).Fertilization and cleavage rates and embryo quality were compared among the three groups.[Results] The fertilization rate,cleavage rate and top quality embryos rate were higher in group I than group II and group III (84.9%,98.1%,and 61.6%;72.0%,90.5% and 22.1%l;75.3%,94.4%,and 25.1%,respectively).Blastomere numbers and morphology scores were highest in group I (P ＜ 0.05),but no significant differences existed between group II and group III.[Conclusion] The morphology of embryos developed from in vivo MII oocytes was superior to those from in vitro matured MII oocytes.No significant difference was observed in embryo morphology from immature GV oocytes in IVF and IVM cycles.

Efficiency comparison between two preimplantation genetic diagnostic methods for chromosomal translocation carriers / 中华妇产科杂志

Objective To compare the diagnostic efficiency between blastomere preimplantation genetic diagnosis (PGD) and polar body PGD for chromosomal translocation carriers. Methods Group A had 8 cycles using whole painting probes for the first polar body diagnosis, while group B had 29 cycles using two subtelomeric probes and one centromeric probe for the blastomere diagnosis. Results The fertilization rate of group A was significantly lower than group B [66. 1% (72/109) vs 85.2% (304/357) , P < 0.05]. There was no significant difference in the successful biopsy rate between two groups. However, group A had a significantly higher loss rate during fixation and higher no signal rate after fluorescence in situ hybridization [ FISH, 9. 6% (12/104) vs 1.6% (4/252), 11.2% (10/89) vs 3.0% (7/233) ]. Totally, the diagnostic efficiency in group A (72. 5% ,79/109 ) was significantly lower than that in group B( 89. 8%, 230/256, P < 0. 05 ). Although both the clinical pregnancy rate( 3/7 ) and implantation rate( 22. 2% ,4/18 ) of group A were higher, the differences were not statistically significant ( P > 0.05 ). Conclusion Both methods can be used efficiently in the PGD for chromosomal translocation carriers. Blastomere PGD has a higher diagnostic rate.

Variance of serum prolactin in controlled ovarian stimulafion / 中华妇产科杂志

Objective To investigate the variance of peripheral blood prolactin(PRL)in controlled ovarian stimulation.Methods Seventy-two patients,with totally 106 cycles receiving a long protocol of gonadotropin-releasing hormone agonist combined with gonadotropin(Gn)were randomly enrolled in this retrospective study.During controlled ovarian stimulation,peripheral blood hormones were measured by chemiluminescent microparticle immunoassay.Results Prolactin was positively correlated with estradiol (r=0.5897.P＜0.01)while there was no significant correlation between luteinizing hormone and PRLProgesterone had a positive relation with prolactin(r=0.1412,P＜0.01).Conclusions During controlled ovarian stimulation,prolactin secretion is not affected by Gn but may be stimulated by estradiol.Progesterone has a positive relation with prolactin.

Preimplantation gender diagnosis by fluorescence in situ hybridization / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To describe the clinical application of fluorescence in situ hybridization (FISH ) in preimplantation gender diagnosis.</p><p><b>METHODS</b>Preimplantation gender diagnosis was performed in 2 female hemophilia A carriers, 1 male patient with glucose-6-phosphate dehydrogenase (G-6-PD) deficiency and 2 male patients with Y chromosome abnormality. Embryo sex was identified by FISH in total of 6 treatment cycles.</p><p><b>RESULTS</b>A total of 123 cumulus-oocytes were retrieved in 6 treatment cycles. Sixty-one embryos were available for embryo biopsy. The success rate of biopsy was 86.9% (53/61), with a further cleavage rate of 62.3% (33/53). In the FISH procedure, one cell was lost during fixation, leading to a 98.1% (52/53) fixation rate. Totally, 16 female embryos and 3 male embryos were transferred to 5 patients in 6 cycles. Three healthy babies were born. The diagnosis was confirmed by subsequent analysis of amniocytes and embryonic buds after embryo reduction.</p><p><b>CONCLUSIONS</b>FISH is an efficient and reliable technique for determining the sex of human preimplantation embryos. Selective abortion and births of affected children can be avoided by preimplantation gender diagnosis.</p>

STUDIES ON THE CLONAL GROWTH AND PASSAGE OF HUMAN EMBRYONIC STEM CELLS / 解剖学报

Objective Try to determine the relationship between blastocyst quality,the clonal growth of inner cell mass(ICM)and the establishment of human embryonic cell line. Methods Coculture D 3 discarded embryos with mouse embryonic fibroblast cells(MEFs).Then remove trophoectoderm by immunosurgery after getting different quality blastocysts.Culture ICM and passage these cells on MEFs. Results Human embryonic stem cells derived from good quality blastocysts could be passaged further than that from poor quality blastocysts,and ICMs growing fast could be passaged more quickly and efficiently.Conclusion The establishment of human embryonic stem cells is closely related with blastocyst quality and the original growth of ICM.

Preimplantation genetic diagnosis / 中华医学遗传学杂志

Preimplantation genetic diagnosis is a very early form of prenatal diagnosis aimed at eliminating embryos carrying serious genetic diseases before implantation. The basic techniques currently used involve embryo biopsy, the polymerase chain reaction and fluorescence in situ hybridization. In the current review, a number of problems arising from the use of these technologies as well as the possible solutions and new developments are discussed.

Experimental of Human Cord Blood Hematopoietic Stem Cells in Ut ero Transplantation through Abdominal Cavity of Fetal Rats / 中山大学学报(医学科学版)

【Objective】 To explore the complication and engra ftment of human cord blood hematopoietic stem cells in utero transplantation thr o ugh abdominal cavity of fetal rats , and to establish an animal model for clini cal application. 【Methods】　Human cord blood (MNC) cells were transplanted into th e abdominal cavity of fetal rats, the complications and the outcome of pregn ancy were observed. The condition of engraftment was detected by flow cytometr y and immunohistochemistry methods after the fetus were born. 【Results】 Huma n CD3 cells were detected in rats and the engraftment rate was 64%. At 1 and 2 months of age, the mean value of human CD3 cells were 0.28%±0.05% and 0.41 %± 0.05% respectively （P＜0.05）.Human CD3 、CD20及 CD+34 ce lls were also detected in liver、spleen and thymus of rats at 2 months of age. The i ncidence of complication was significantly different between transplanted grou p and non-transplanted group. 【Conclusion】　Human cord blood cells transfused into the abdominal cavity of fetal rats were engrafted . There were some complication s occurred during operations which affected the outcome of pregnancy.

Preimplantation Genetic Diagnosis for Robertsonian Translocation Carrier / 中山大学学报(医学科学版)

【Objective】To perform preimplanation genetic diagnosis by using dual color fluorescent in-situ hybridization (FISH).【Methods】The chemical and mechanical division methods were used to perform embryo biopsy in 3 cases of Robertsonian translocation t (13q14q).Vysis LSI 13q14 and Tel Vysion 14q probes were used to detect the blastomeres biopsied from the IVF embryos of the patients.FISH analysis was performed to select normal or balanced karyotype embryos ,which then were transfered into the uterus.【Results】Total of 23 oocytes were retrieved in 3 treatment cycles.Fertilization rate was 79%.14 embryos were available for embryo biopsy.Among them,9 embryos were biopsied by chemical division method,with further cleavage rate of 67%;5 embryos were biopsied by mechanical division method,with further cleavage rate of 40%.Single embryo was diagnosed as normal karyotype or balanced respectively in 2 treatment cycles.Both of them were transfered into the uterus.One clinical normal on-going pregnancy was achieved,the diagnosis was confirmed by amniocyte karyotype analysis.【Conclusion】Preimplanation genetic diagnosis can be used to resolve the problem of fertility for Robertsonian Translocation Carriers.

Treatment Options of Clomiphene resistant Polycystic Ovary Syndrome Related Infertility / 中华妇产科杂志

Objective To investigate the treatment options of clomiphene(CC) resistant polycystic ovary syndrome (PCOS) related infertility. Methods Figty nine PCOS patients with CC resistant anovulation were accepted and treated by the following three protcols :(1)follicle stimulating hormone (FSH) group, 49 cycles; (2)FSH +pulsatile gonadotropin releasing hormone(GnRH) group, 13 cycles; (3)conventional in vitro fertilization (IVF)_embryo transfer(ET) group, 19 cycles. Suppressive treatment on serum luteinising hormone (LH)and testosterone (T) levels was given in the first and second groups in advance. Serum estradiol level; pregnancy rate; miscarriage rate, ovarian hyperstimulation syndrome (OHSS) and multiple pregnancy rate were compared among the three groups. Results The pregnancy rate in FSH group, FSH+GnRH group and IVF ET group werte 37%,54% and 22% respectively. The highest rate and multiple pregnancy rate was found in IVF ET group. Conclusion The rationale treatment options for CC resistant PCOS related infertility was the addition pre suppressive treatment, low dose FSH stimulated regimen subsequent pulsatile GnRH infusion. IVF were only accepted after failure with gonadotropin therapy.

Diagnosis, management and pregnancy outcomes of twin-twin transfusion syndrome / 中华围产医学杂志

Objective To investigate the diagnosis, treatment and pregnancy outcomes of twin-twin transfusion syndrome(TTTS). Methods Eighteen cases of TTTS(TTTS group) were studied retrospectively from Jan 1991 to Oct 2005 in our hospital, and 620 twin pregnancies unaffected by TTTS (control group) were compared. Results (1)The overall incidence of TTTS was 2.8% in all twin pregnancies, and 8.1% in monochorionic twin pregnancies. (2) Fourteen cases of TTTS were staged, and 10 were terminated. Seven cases opted to be managed (1 case at stage Ⅱ, 5 at stage Ⅲ and 1 at stage Ⅳ). Transabdominal amnioreduction was performed in 3 cases, 2 of them progressed to stage Ⅴ and was terminated. One case was treated by fetoscopic laser coagulation and 2 neonates survived.(3)The ratio of conception by assisted reproductive techniques in TTTS group was lower(11.1% vs 40.0%,P

Isolation and culture of human primordial germ cells in vitro / 中国病理生理杂志

AIM: To investigate the optimal materials and culture system of human primordial germ cells (PGCs) in order to establish human embryonic germ (EG) cell lines. METHODS: Human embryos of different gestational age were collected to isolate human PGCs. The isolated human PGCs were cultured in different medium and on different feeder layers, then their growth, proliferation and differentiation in different culture systems were observed. RESTILTS: The formation rate of primary colonies was higher when human PGCs were obtained with enzyme-mechanical method from 8-and 9-weeks gestational age human embryos than that from 7-weeks. Human PGCs grew better and maintained undifferentiating when mouse embryonic fibroblast or STO cells served as feeder layers and in conditional medium with hLIF, hbFGF, hSCF. CONCLUSION: 8-and 9-week gestational age human embryo are optimal material for isolating human PGCs. Enzyme-mechanical method is simple and available to isolate human PGCs. Feeder layer and growth factors are necessary for human PGCs culture in vitro.

Rescue activation of calcium ionophore A23187 on unfertilized human oocytes after conventional in-vitro fertilization and intracytoplasmic sperm injection / 中国病理生理杂志

AIM: To explore the activation effect of calcium ionophore A23187 on unfertilized human mature oocytes after conventional in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHODS: Thirty-seven unfertilized mature oocytes from IVF and 41 after ICSI were included in our experiment. They were incubated in 5 ?mol/L calcium ionophore A23187 for 5 minutes. Second polar body extrusion and pronuclear formation were recorded 12-16 hours later. The activated oocytes were cultured for another 2 days in vitro. RESULTS: Activation rate of unfertilized oocytes from conventional IVF and ICSI were 64.9%(24/37)and 73.2%(30/41), respectively. Among 41 unfertilized oocytes after ICSI treated with A23187, 30 were activated and 24 had 2 polar body (PB) and 2 pronuclear (PN). But for the unfertilized oocytes from conventional IVF only 20% activated oocytes had 2 PN and 2 PB. The percentage difference of oocytes containing 2 PB and 2 PN between the two groups was significant ( P

Premature response to luteinizing hormone of granulosa cells from women with polycystic ovary syndrome / 中国病理生理杂志

AIM:To demonstrate the relationship between hormones in follicular fluid and the expression of LH receptor in granulosa cells(GC) in anovulatory women with polycystic ovary syndrome(PCOS).METHODS: Follicles were obtained from 12 women with PCOS and 15 women with normal menstrual period through surgery at time between day 7 and day 10 of menstrual cycle.The accumulations of estrogen(E2),progesterone(P),luteinizing hormone(LH),follicle stimulating hormone(FSH) and insulin in follicular fluid were determined by a automatism chemiluminescent microparticle immunoassay(CMIA) for the quantitative determination.The accumulation of androstenediol(A) was determined by ELISA.The amounts of the mRNA expressions of LH receptors from GC and theca cells(TC) respectively were measured by RT-PCR using ?-actin as intra-control simultaneously.RESULTS: The levels of LH [(3.8?2.1 vs 1.7?0.8)IU/L,P