[Effect of domestic highly purified urinary follicle stimulating hormone on outcomes of in vitro fertilization-embryo transfer in controlled ovarian stimulation].

OBJECTIVE: To investigate the effect of domestic urine-derived high-purity follicle- stimulating hormone (HP-FSH, Lishenbao) on the outcome of in vitro fertilization(IVF) embryo transfer (ET) in controlled ovarian stimulation (COS). METHODS: From 1 September 2010 to 31 March 2011, total of 3178 infertility patients from 14 Reproductive Center with IVF or intracytoplasmic sperm injection (ICSI) indications who accepted first IVF or ICSI cycle were studied retrospectively. Their causes of infertility include all infertility factors except ovulatory dysfunction infertility and uterine factor infertility. The only long luteal phase gonadotropin-releasing hormone agonist (GnRH-a) protocol was included. Patients were divided into 2 groups according to the type of follicle-stimulating hormone (FSH) agents used: 1932 cases in HP-FSH group and 1246 cases in recombinant FSH (rFSH)group. Patients in both groups were combined with human menopausal gonadotropin (hMG) at doses of 150 U when follicle with diameter reached to 14-16 mm. When 3 dominate follicle with diameter reached 18 mm, hCG at dose of 5000 to 10 000 U or recombinant hCG at dose of 250 µg was administered by intramuscular injection. After 34 to 36 hours, oocytes were obtained guided by ultrasound, then IVF-ET were underwent in their Reproductive Center. The primary endpoint was comparison of live birth rate between the two groups. The secondary endpoints were comparisons of clinical pregnancy rate, miscarriage rate, and implantation rate, as well as COS and IVF outcome between the two groups. RESULTS: (1) There were significantly differences in baseline characteristics of the patients between two groups. The mean age was elder(32 ± 4 versus 30 ± 4, P < 0.01) , the infertility duration was longer (5 ± 4 versus 5 ± 3, P < 0.01) , and antral follicle count (AFC) was less (11 ± 5 versus 13 ± 7, P < 0.01) in patients of HP-FSH group compared with those in patients of rFSH group. (2) As compared with rFSH, the total doses of gonadotropin needed was (2348 ± 1011) U in HP-FSH group versus (2022 ± 659) U in rFSH group, the number of oocytes 13 ± 6 in HP-FSH group and 14 ± 7 in rFSH group, the rate of embryo frozen cycle of 66.30% (1281/1932) in HP-FSH group and 74.88% (933/1246) in rFSH group, which all reached statistical difference (P < 0.01). However, there were no significant different implantation rate [30.49% (1111/3644) versus 32.45% (737/2271)] between two groups. The other clinical parameters did not show significant difference, including clinical pregnancy rate per started cycle [41.61% (804/1932) versus 41.97% (523/1246) ] , clinical pregnancy rate per ET cycle[46.58% (804/1726) versus 48.47% (523/1079)], live birth rate per started cycle[34.21% (661/1932) versus 34.19% (426/1246)], live birth rate per ET cycle [38.30% (661/1726) versus 39.48% (426/1079)], miscarriage rate[13.6% (109/804) versus 16.4% (86/523)], and moderate/severe ovarian hyperstimulation syndrome (OHSS) rate [5.80% (112/1932) versus 7.78% (97/1246)](P > 0.05).(3) Treatment cost: the cost of gonadotropins needed for the patients in HP-FSH group was lower than that in rFSH group (4005 ± 1650 versus 6482 ± 2095, P < 0.01). CONCLUSION: In IVF/ICSI treatment cycles, domestic HP-FSH has similar live birth rate and lower financial burden when compared with rFSH.

[Clinical analysis of 100 preimplantation genetic diagnosis cycles].

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.

Improved nude mouse models for green fluorescence human endometriosis.

AIM: To establish an improved noninvasive fluorescent animal model for endometriosis. MATERIAL AND METHODS: Adenovirus encoding enhanced green fluorescent protein (Ad-eGFP) was used to transfect primary culture endometrial glandular cells and stromal cells (purified cell transfection and mixed injection, Group 1) as well as endometrial fragments (tissues transfection and injection, Group 2). Transfection results were compared between the cells and tissues in vitro. The GFP-transfected cells suspension of Group 1 or endometrial fragments of Group 2, with similar weight, were injected into nude mice subcutaneously and noninvasively observed every 5 days until day 15 (Subgroup 1, N = 5), day 20 (Subgroup 2, N = 5) or day 25 (Subgroup 3, N =11). The positive rates and duration times of the fluorescent lesions were calculated. RESULTS: After 18 h of incubation, glandular cells and stromal cells all had higher GFP-positive rates. In vivo imaging showed that the GFP positive rates of Group 1 were significantly higher than those of Group 2. The fluorescent-positive durations of Groups 1 and 2 were 23.636 ± 4.523 days and 5.909 ± 5.394 days, respectively (P < 0.001). In vivo analysis demonstrated that on days 15, 20, and 25, there were more typical lesions and fluorescent-positive lesions formed in Group 1 and that the lesion weight in Group 1 was greater. The structures of the lesions were all identified as human origin. CONCLUSION: A noninvasive animal model for endometriosis created by subcutaneous injection of an Ad-eGFP-transfected endometrial glandular and stromal cells suspension had higher a positive rate, longer duration time of fluorescent imaging and greater lesion weight.

Expression of certain HLA-I types in cleavage-stage embryos.

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.

Preimplantation genetic diagnosis for alpha-thalassaemia in China.

PURPOSE: To report the usage of PGD for alpha-thalassaemia with the - -(SEA) genotype. METHOD: A PGD protocol using fluorescent gap PCR was performed for 51 cycles on 43 couples with the - -(SEA) genotype. Allele drop-out and amplification failure rates were retrospectively analyzed. RESULTS: A total of 472 embryos were biopsied. Amplification was achieved in 390 blastomeres, accounting for an amplification rate of 82.6%. In total, 120 wild-type, 94 heterozygotes and 140 homozygous mutant embryos were diagnosed. The successful diagnosis rate was 75.0%. The ADO rate in 49 blastomeres from six donated embryos was 16.4%. One hundred and fifty four embryos were transferred, resulting in 25 clinical pregnancies with an implantation rate of 24.0%. CONCLUSIONS: Single-round fluorescent gap PCR is a feasible and effective strategy in the PGD for alpha-thalassaemia with the - -(SEA) genotype.

Effects of cilostamide and forskolin on the meiotic resumption and embryonic development of immature human oocytes.

BACKGROUND: In an attempt to allow for acquisition of oocyte cytoplasmic maturation, PDE3 specific inhibitor, cilostamide and adenylate cyclase activator, forskolin were used to extend pre-maturation culture of immature human oocytes. METHODS: Cumulus-oocyte complexes retrieved from unstimulated ovaries were continuously cultured under 20 microM cilostamide or 50 microM forskolin, alone or in combination for 6, 12, 24 or 48 h, respectively. Levels of intercellular gap junction communication (GJC) and maturational status were examined at these designated time points. Metaphase II oocytes obtained following 54 h biphasic culture (with meiotic inhibitors from 0 to 24 h, no meiotic inhibitors from 24 to 54 h) were subject to intracytoplasmic sperm injection and embryos were cultured for five more days. RESULTS: Both cilostamide and forskolin delayed spontaneous meiotic progression after continuous culture with immature human oocytes. Combined treatment of cilostamide and forskolin significantly lowered the rates of germinal vesicle breakdown (GVBD) at 6, 12, 24 or 48 h after meiotic inhibitory culture, when compared with the control (all P < 0.05). A delay of 6 h for the loss of GJC was also observed under the combined treatment of cilostamide and forskolin. The fertilization rate was significantly higher under the combined treatment of cilostamide and forskolin than that of the control. Although the rates of oocyte maturation and embryo cleavage were similar among groups, there was a slight but non-significant increase in blastocyst formation rate with the treatment of cilostamide and forskolin. CONCLUSIONS: Combined treatment of cilostamide and forskolin positively influences oocyte developmental competence by exhibiting a synergistic effect on the prevention of GJC loss and resumption of meiosis.

[Variance of serum prolactin in controlled ovarian stimulation].

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 PRL Progesterone 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.

[Efficiency comparison between two preimplantation genetic diagnostic methods for chromosomal translocation carriers].

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.

[Analysis of chromosome mosaicism in preimplantation embryos by using 2 sequential rounds of fluorescence in situ hybridization].

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

[The clinical application of whole chromosome painting probes in preimplantation genetic diagnosis for translocation carriers].

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

[Meiotic spindle location and embryo development of in vivo and in vitro matured human oocytes].

OBJECTIVE: To analyze the relationship between meiotic spindle location and embryo developmental potential of in vivo and in vitro matured human oocytes. METHODS: One hundred and thirty-four in vivo matured oocytes and 45 in vitro matured oocytes were observed with polscope at the time of intracytoplasm sperm injection (ICSI). RESULTS: Meiotic spindle was detected in 83.6% (112/134) and 82.2% (37/45) in in vivo and in vitro matured oocytes respectively. In vivo matured oocytes which showed a minimal angle (0-5 degrees ) between the meiotic spindle and the first polar body had a higher fertilization rate (93.3%) than the others. The frequency of the oocytes which had a 0-5 degrees spindle angle in in vivo and in vitro matured oocytes was 22.4% and 17.8%, respectively, and that of oocytes which had a 6 degrees - 45 degrees, 46 degrees-90 degrees and > 90 degrees spindle angle was 55.2% vs 51.1%, 3.0% vs 8.9%, and 3.0% vs 4.4%. No significant difference was found between them. No relationship was found between the position of meiotic spindle and embryo quality. CONCLUSIONS: There is some relationship between the angle of the meiotic spindle with the first polar body and fertilization rate. No significant difference is found in the position of the meiotic spindle between in vivo and in vitro matured human oocytes.

[Characteristics of IGF-II gene imprinting in twin placentas].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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%). CONCLUSION: The risk of IGF-II gene imprinting loss is higher in female twins and has no relationship with assisted reproductive technology and zygosity.

[Sperm sex chromosome analysis and preimplantation genetic diagnosis of patients with sex chromosome anomalies].

OBJECTIVE: To investigate the constitution of abnormal spermatozoa from patients with sex chromosome anomalies. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Influence of the depth of embryo transfer on pregnancy outcome in in vitro fertilization-embryo transfer].

OBJECTIVE: To detect whether the depth of embryo transfer has influence on pregnancy outcome in in vitro fertilization-embryo transfer (IVF-ET). METHODS: The distance between the high echogenic transfer dot and the fundal endometrium was measured under guidance of transabdominal ultrasound. The average distance 0.75 cm was used to divide patients into two groups. Group 1 included 44 patients with a distance > 0.75 cm, while group 2 had 48 patients with a distance

[Study of altered expression of annexin IV and human endometrial receptivity].

OBJECTIVE: To explore the association of altered expression of annexin IV in human endometrium during the implantation window and endometrial receptivity. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

[Features of morphometry of ultrasound and endometrial histology in the anovulatory polycystic ovary syndrome women].

OBJECTIVE: To investigate the relationship between ultrasonographic features of endometrium and the relation of histological staging of the endometrium and sexual hormone levels in anovulatory polycystic ovary syndrome (PCOS) women. METHODS: Seventy-six anovulatory PCOS patients and 32 women with normal ovulation were enrolled in this study. Ultrasonographic examination, and transmission electron microscope were used to observe endometrium. The expressions of nulear antigen associated with cell proliferation Ki-67 and calcitonin were analyzed by immunohistochemistry. The sexual hormone levels were measured by chemiluminescent microparticle immunoassay. RESULTS: In 11 patients the endometrium showed secretory change out of 76 anovulatory PCOS patients. The frequency of secretory change of the endometrium was not increased with the increase of menses-biopsy interval (P > 0.05). The frequency of abnormal stroma was significantly lower in tripleline endometria than those in non-tripleline endometria (9% vs 43%, P < 0.05). Compared with the control group, the anovulatory PCOS group showed a significant higher expression of Ki-67 in the glandular cell of the secretory phase endometrium (P < 0.05). In the proliferative endometrium, anovulatory PCOS group had more cell organelles than those of the control group. The endometrium showed insufficient secretory changes in the anovulatory PCOS patients. CONCLUSIONS: Proliferative and secretory stage of the endometrium in the anovulatory PCOS group show abnormal features. The abnormal stroma may contribute to the hyperechonic images of the endometrium in the anovulatory PCOS patients.

Nonprofessional phagocytosis in trophectoderm cells of human preimplantation blastocysts.

UNLABELLED: Trophoblast phagocytosis has been considered important in pregnancy. However, whether human preimplantation blastocysts possess phagocytic activity is still unclear. In this study, we determined the phagocytosis potential in human trophectoderm cells of blastocysts prior to implantation. Fluorescent microspheres were used as markers for phagocytic analysis under transmission electron microscope (TEM) and fluorescence microscopy. Phagocytosis of 1 µm fluorescent microspheres was observed in most (9/11) day-6 and even some (2/9) day-5 blastocysts. More effective phagocytosis occurred in blastocysts at the morula-blastocyst stage of day-6. Furthermore, we observed an increased trend of phagocytic acitivities in polar trophectoderm. Our findings indicated phagocytic ability exists in human blastocysts prior to implantation and the differentiation between polar and mural trophectoderm may be associated with blastocyst implantation. ABBREVIATIONS: TEM: transmission electron microscope; ZP: zona pellucida; PGD: preimplantation genetic diagnosis; ICM: inner cell mass; FGF4: fibroblast growth factor 4.

Establishment of human embryonic stem cell line from gamete donors.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Closely linked polymorphic marker: successful application in preimplantation genetic diagnosis for beta-thalassemia.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

[Dynamic development of normal fetal circulating blood hematopoietic stem progenitor cell surface antigen in on-going pregnancy].

OBJECTIVE: To investigate the dynamic development of fetal circulating hematopoietic stem/progenitor cell (HSPC) surface antigen CD133, CD34 and CD38 in on-going normal pregnancy. METHODS: 0.2-2.0 ml fetal blood samples were obtained from 106 human fetuses between the gestational age of postmenstrual weeks 12 and 41, 96 being collected by ultrasound-guided percutaneous cordocentesis, 3 by ultrasound-guided selective feticide, and 7 being collected after birth. The nucleated cells (NCs) were separated, and the cell surface antigen CD34, CD38, and CD133 were labeled by bicolor immunofluorescence technique. Fluorescence activated cell sorting (FACS) was performed to analyze the concentration of CD34(+)/NC, CD38(-)/CD34(+), CD133(+)/NC, and CD133(+)/CD34(+) cell respectively. RESULTS: All operations were uneventful for the fetuses and mothers. The proportions of CD34(+)/NC, CD38(-)/CD34(+)cells, CD133+/NC, and CD133(+)/CD34(+) cells dramatically decreased when the gestational age advanced from week 12 to week 41. CD34+ cell/NC proportion decreased from 4.21% to 0.04% (r(2) = 0.90, x+/- s = 1.54% +/- 0.54%) with the peak at gestational week 12 and a the second peak at the week 20. (2) The percentage of CD38(-)/CD34(+) cells decreased from 58.5% to 10.7% (x+/- s = 28.66% +/- 9.88%) with advancing gestational age (r(2) = 0.82) with the peak at gestational week 12 and a second peak at week 22. (3) The percentage of CD133(+) cells among CD34(+) cells decreased with advancing gestational age, from 87.6% to 48.5% (x+/- s = 63.5% +/- 11.4%). (4) CD133(+) cells/NC concentration decreased with advancing gestational age, from 3.69% to 0.31% (x+/- s = 1.40% +/- 0.86%) with the peak time at gestational week 12., and showed a negative linear correlation with the gestational age (r(2) = 0.76). CONCLUSION: The immunophenotype of normal fetal circulating HSPC changes along with the gestational age in on-going pregnancy. The earlier the time, the more primitive the immunophenotype of the fetal circulating HSPCs. The more primitive fetal circulating HSPCs may be the ideal target for in utero gene therapy.