[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to determine the efficacy of in vitro fertilization and embryo transfer (IVF-ET) in patients with hyper-response to ovulation induction for intrauterine insemination (IUI).
Patients with polycystic ovary syndrome (PCOS) who were initially treated with IUI in our centre between Jan 2007 and Oct 2010 were retrospectively analyzed. The ovarian hyper-stimulation syndrome (OHSS) found in 50 patients was then treated with IVF-ET following informed consent.
The fresh transfer had 42 cycles and a total of 87 embryos were transferred. Urine pregnancy tests were positive in 15 patients and fetal heart beat was detected in 12 patients by transvaginal ultrasound, from which 3 patients had two fetuses, 2 patients had three fetuses, and 7 patients had a single fetus. The overall clinical pregnancy rate was 28.5% (12/42) for the fresh embryo transfer. A total of 21 cycles of frozen embryo transfer with up to 55 embryos were conducted for patients who were not pregnant at the end of fresh embryo transfer cycles or who did not receive fresh embryo transfer in the first place. Urine pregnancy tests were positive in 10 patients and fetal heart beat was detected in 8 patients. The clinical pregnancy rate was 38.1% (8/21) for frozen embryo transfer.
We conclude that IVF-ET is an effective method for patients with hyper-response to ovulation induction in IUI.
Biomedical papers of the Medical Faculty of the University Palacky, Olomouc, Czechoslovakia 06/2012; 156(2):159-63. DOI:10.5507/bp.2012.044 · 1.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-viral gene delivery systems based on cationic polymers have faced limitations related to their relative low gene transfer efficiency, cytotoxicity and system instability in vivo. In this paper, a flexible and pompon-like dendrimer composed of poly (amidoamine) (PAMAM) G4.0 as the inner core and poly (L-glutamic acid) grafted low-molecular-weight polyethylenimine (PLGE) as the surrounding multiple arms was synthesized (MGI dendrimer). The novel MGI dendrimer was designed to combine the merits of size-controlled PAMAM G4.0 and the low toxicity and flexible chains of PLGE. In phosphate-buffered saline dispersions the well-defined DNA/MGI complex above a N/P ratio of 30 showed good stability with particle sizes of approximately 200 nm and a comparatively low polydispersity index. However, the particle size of the DNA/25 kDa polyethylenimine (DNA/PEI 25K) complex was larger than 700 nm under the same salt conditions. The shielding of the compact amino groups at the periphery of flexible PAMAM and biocompatible PLGE chains in MGI resulted in a dramatic decrease of the cytotoxicity compared to native PAMAM G4.0 dendrimer. The in vitro transfection efficiency of DNA/MGI dendrimer complex was higher than that of PAMAM G4.0 dendrimer. Importantly, in serum-containing medium, DNA/MGI complexes at their optimal N/P ratio maintained the same high levels of transfection efficiency as in serum-free medium, while the transfection efficiency of native PAMAM G4.0, PEI 25K and Lipofectamine 2000 were sharply decreased. In vivo gene delivery of pVEGF165/MGI complex into balloon-injured rabbit carotid arteries resulted in significant inhibition of restenosis by increasing VEGF165 expression in local vessels. Therefore, the pompon-like MGI dendrimer may be a promising vector candidate for efficient gene delivery in vivo.
[Show abstract][Hide abstract] ABSTRACT: In fertile women the patterns of proteome during the prereceptive (day 2 after LH surge) and receptive (day 7 after LH surge) phases were analyzed by two-dimensional fluorescence difference gel electrophoresis and mass spectrometry. A total of 31 differentially expressed proteins (17 up-regulated and 14 down-regulated) were identified between the two phases, which is helpful in understanding the biological mechanisms underlying human endometrial receptivity.
Fertility and sterility 10/2010; 95(3):1161-3. DOI:10.1016/j.fertnstert.2010.09.033 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study examined the expression of human leukocyte antigen (HLA)-G and HLA-I (which includes HLA-A, -B, -C, -E and -F, but is without HLA-G) in the cleavage embryo and its supernatant, and related the results to embryo development including growth rate and grade. In total, 136 day-3 cleavage embryos were used for detection of HLA-G and 24 embryos for HLA-I without HLA-G by immunohistochemistry. The expression of HLA-I was examined by western blot in the lysates of a further 63 day-3 cleavage embryos; soluble HLA-I in the culture supernatant of embryos with detectable HLA-I was also examined by western blot. It was found that 90 of 136 (66.2%) cleavage embryos expressed HLA-G, whereas 23 of 24 (95.8%) embryos expressed HLA-I without HLA-G. HLA-G expression typically showed an even and symmetrical pattern of distribution in each blastomere. HLA-I without HLA-G in cleavage-stage embryos is typically scattered around the blastomere surface. The expression of HLA-G but without HLA-I in cleavage-stage embryos was significantly associated with embryo grade (P < 0.001) and cell number (P = 0.03). In conclusion, HLA-I is expressed on day-3 cleavage embryos, and HLA-G expression on preimplantation embryos is related to embryo development, including embryo growth rate and embryo grade.
[Show abstract][Hide abstract] ABSTRACT: To explore the association of altered expression of annexin IV in human endometrium during the implantation window and endometrial receptivity.
A comparative proteomic strategy, in a combination of two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS), was adopted to search for proteome alternations of pre-receptive (day LH + 2) versus receptive (LH + 7) endometria. The location and abundance of the identified differentially expressed protein- annexin IV were analyzed by immunostaining and western blot.
By comparing protein profiles of LH + 2 and LH + 7 samples, we found a protein up-regulated 2.12 times in LH + 7 samples, with a relative molecular weight of 36,000 and an isoelectric point near pH 5.8. It was characterized using mass spectrometry and was identified as annexin IV. Immunohistochemical analysis revealed an altered localization of annexin IV--in the epithelia on day LH + 2, and both in the epithelia and stroma cells on day LH + 7. Protein levels of annexin IV were up-regulated on day LH + 7 compared with that on day LH + 2 by Western blot. Integrated optical density of the object (OPTDI) was 46.249 +/- 32.376 and 249.507 +/- 31.959, respectively (P = 0.004).
Our study indicates endometrial samples obtained by microbiopsy are available for proteomics studies. It seems possible that the increased expression of annexin IV during the implantation window plays an important role in the morphological differentiation of the uterus to the receptive state.
[Show abstract][Hide abstract] ABSTRACT: In southern China, the average carrier rates of alpha-thalassemia and beta-thalassemia in the population are as high as 10.3% and 2.8%, respectively. Because of the high rates, they are known as ‘social diseases’ in some regions. In this study, the fluorescent gap PCR, which can detect the alpha-thalassemia Southeast Asia deletion (SEA deletion), was applied in four clinical applications of preimplantation genetic diagnosis (PGD) on four couples, among whom both partners were alpha-thalassemia carriers. Two patients became pregnant and two healthy babies were born, which confirmed the PGD results. The single cell multiplex nested PCR followed by reverse dot blot (RDB), which can simultaneously detect the 16 beta-thalassemia mutations in the Chinese population, was applied in four clinical PGD cycles on four couples among whom both partners were beta-thalassemia carriers. One pregnancy was achieved and it resulted in a live healthy birth, which confirmed the results of PGD. The amplification efficiencies of the two protocols described above were 89.5% and 93.9%, respectively. The allele drop-out (ADO) rates of these two protocols were 5.9% and 10.9%, respectively.
These studies represent the successful applications of PGD protocols that can detect more than 95% of alpha- and beta -thalassemia mutations in the Chinese population. Copyright
[Show abstract][Hide abstract] ABSTRACT: To develop single-cell fluorescent gap polymerase chain reaction (PCR) assay for preimplantation genetic diagnosis (PGD) in couples at risk of having child with alpha-thalassemia.
Single cell fluorescent gap PCR which can detect the alpha-thalassemia Southeast Asia deletion (-SEA deletion), was applied to single lymphocytes and blastomeres which coming from four clinical PGD cycles.
The Single cell fluorescent gap PCR can detect the alpha-thalassemia -SEA deletion, which account for 94% of hydrop fetalis in Chinese population with an amplification efficiency of 90.0% (72/80) and allele drop-out (ADO) rate of 8.3% (6/72) in lymphocytes. In four clinical PGD cycles, a total 38 embryos were detected and 38 blastomeres were obtained. Thirty-four blastomeres were amplified with the amplification efficiency of 89.5% (34/38) and ADO rate of 5.9% (2/34). Eleven embryos were shown to be normal homozygous, eight embryos were shown to be heterozygous and 15 embryos were shown to be affected homozygous. Eleven embryos were transferred back to the uterus of the patients. Two pregnancy achieved, resulting in two live healthy births, which confirmed the results of PGD.
This first reported unaffected pregnancy resulting from PGD using fluorescent gap PCR for alpha-thalassemia demonstrates that this technique, as an alternative to prenatal diagnosis, is a reliable and effective way to help those carrier couples to get a healthy baby.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 09/2005; 22(4):391-5.
[Show abstract][Hide abstract] ABSTRACT: To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia.
Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed.
200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30).
PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby.
[Show abstract][Hide abstract] ABSTRACT: To find out the expression of soluble human leukocyte antigen G (sHLA-G) and its relationship to the cleavage embryo development.
One hundred and seventy-seven day 3 cleavage embryos were detected for sHLA-G by immunohistochemistry.
sHLA-G was detected in 57.1% cleavage embryos. The positive rate of sHLA-G in cleavage embryos developed from dipronucleate fertilized eggs was 66.2% (90/136), that developed from tripronucleate fertilized eggs was 26.8% (11/41). There was significant difference between these two groups (P < 0.01). The positive rate of sHLA-G in the grade 1 cleavage embryos developed from dipronucleate fertilized eggs was 64.3% (18/28), that in the grade 2 cleavage embryos developed from dipronucleate fertilized eggs was 91.7% (66/72), that in the grade 3 cleavage embryos developed from dipronucleate fertilized eggs was 16.7% (6/36), there were significant differences between these different embryo grades (P < 0.01), and the intensity of sHLA-G had negative relationship with the embryo grades (r = -0.503). The positive rate of sHLA-G in the first class cleavage embryos developed from tripronucleate fertilized eggs was 88.9% (32/36), that from dipronucleate fertilized eggs was 64.3% (18/28). There was significant difference in these two groups (P < 0.01). The intensity of sHLA-G in cleavage embryos developed from tripronucleate fertilized eggs was higher than that from dipronucleate fertilized eggs. The positive rate of sHLA-G in the cleavage embryos developed from dipronucleate fertilized eggs whose cell number was less than 4 was 56.7% (34/60), that from dipronucleate fertilized eggs whose cell number ranged from 5 to 6 was 67.9% (36/53), and that from dipronucleate fertilized eggs whose cell number ranged from 7 to 8 was 87.0% (20/23). There were no significant differences in these three groups. The intensity of sHLA-G had no significant difference between embryos with different cell number (P > 0.05), but it had relationship with the cell number (r = 0.267).
Cleavage embryos express sHLA-G which is related with the embryo development.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
Chinese medical journal 05/2004; 117(4):483-7. · 1.05 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
These studies represent the successful application of PGD for beta-thalassemia in China.
Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 11/2003; 20(5):447-8.
[Show abstract][Hide abstract] ABSTRACT: Preimplantation genetic diagnosis (PGD) offers couples at risk for transmitting an inherited disorder the possibility to avoid the need to terminate affected pregnancies. PGD for monogenic diseases is most commonly accomplished by blastomere biopsy from cleavage-stage embryos, followed by PCR-based DNA analysis. However, the molecular heterogeneity of many monogenic diseases requires a diagnostic strategy capable of detecting a range of mutations and compound genotypes. With the above considerations, we developed an accurate and reliable strategy for analysis of beta-globin gene mutations, applicable for PGD for the wide spectrum of beta-thalassemia major mutations in the Chinese population. The strategy involves primer-extension preamplification (PEP), followed by nested PCR and reverse dot blot (RDB) for mutation detection since it facilitates simultaneous analysis of more than one mutation in a single cell. This report describes the application of the strategy in two clinical IVF/PGD cycles at risk for transmitting beta-thalassemia major, which resulted in the first thalassemia-free children born after PGD in China.
[Show abstract][Hide abstract] ABSTRACT: To investigate the effect of in vitro fertilization and embryo transfer (IVF-ET) and preimplantation genetic diagnosis (PGD) for the couples at risk of having children with beta-thalassemia.
Four couples carrying different thalassemia mutations received standard IVF treatment. Embryo biopsy was conducted. Single blastomeres were genotyped by a protocol involving primer extension preamplification, nested polymerase chain reaction and reverse dot-blot analysis. Only the unaffected embryos were transferred to the uterus.
A total of 97 oocytes were retrieved from the four female carriers. Among them, 83% showed two pronuclei. Embryo biopsy was performed on 47 of these embryos. The amplification efficiency was 84.8%. The average ADO rate was 14.9%. Ten unaffected embryos were transferred. A twin pregnancy with one blighted ovum was confirmed at 7 weeks' gestation by ultrasonography and one normal baby and one carrier of thalassemia mutation were born finally.
This unaffected pregnancy resulting from PGD for beta-thalassemia demonstrates that PGD technique can be a powerful diagnostic tool for couples carrying beta-thalassemia mutations who desire a healthy child and wish to avoid abortion of an affected fetus.
[Show abstract][Hide abstract] ABSTRACT: To study the expression of soluble human leukocyte antigen G (sHLA-G) in trophoblast cells of spontaneous abortion after in vitro fertilization and embryo transfer (IVF-ET) and choriocarcinoma, and explore the relationship between sHLA-G expression in trophoblast cells and the early embryo development.
14 cases of trophoblast cells of spontaneous abortion after IVF-ET and 6 cases of choriocarcinoma tissue has been detected, and which were compared with the 10 cases of trophoblast cells of normal villus.
(1) One case (1/14) has positive sHLA-G expression in IVF-ET group, and other cases in this group are negative or faint sHLA-G expression; ALL cases in choriocarcinoma group are strong positive sHLA-G expression; All cases in normal villous group are positive sHLA-G expression. (2) sHLA-G is expressed on the cytotrophoblast cells, especially on the extravillus cytotrophablast. The expression level of HLA-G is obviously decreased in the spontaneous abortion group compared with normal group. For single cells, the staining on the choriocarinoma cells are stronger than that of the normal villus cells.
High expression of sHLA-G in choriocarcinoma trophoblast cells and in trophoblast cells of normal villus and low expression in spontaneous abortion trophoblast cells indicating sHLA-G in trophoblast cells has some relationship with the early embryo development.