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.

[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.

High follicle-stimulating hormone increases aneuploidy in human oocytes matured in vitro.

OBJECTIVE: To study the effect of FSH on the aneuploidy risk of human oocytes matured in vitro. DESIGN: Prospective study. SETTING: Hospital-based IVF center. PATIENT(S): Patients with male factor infertility undergoing intracytoplasmic sperm injection (ICSI) cycles. INTERVENTION(S): Immature oocytes were put into five groups according to the FSH concentration (0, 5.5, 22, 100, and 2,000 ng/mL) in in vitro maturation (IVM) medium. Spindles were observed under a polarized microscope before polar body biopsy. Fixed polar bodies and corresponding oocytes were examined on chromosomes 13, 16, 18, 21, and 22 by fluorescence in situ hybridization. Oocytes matured in 5.5 and 2,000 ng/mL FSH were immunostained for tubulin and chromatin. MAIN OUTCOME MEASURE(S): Aneuploidy rate, spindle visualization rate, and spindle morphology. RESULT(S): The frequency rates of aneuploidy were 26.7%, 23.3%, 36.75%, 46.67%, and 63.3% in the five FSH groups, respectively. There was a significantly higher aneuploidy rate in oocytes matured in the 2,000 ng/mL FSH group. The spindle visualization rates assessed under PolScope were not significantly different between aneuploid and normal oocytes. There was no difference in spindle morphology between the 2,000 and 5.5 ng/mL FSH groups. CONCLUSION(S): High-concentration FSH in IVM medium significantly increased the first meiotic division error, resulting in more aneuploid oocytes during IVM.

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.

Imprinting and Promoter Usage of Insulin-Like Growth Factor II in Twin Discordant Placenta.

Case reports from infant twins suggest that abnormal genomic imprinting may be one of the important causes of twin discordance, but it is unknown whether abnormal genomic imprinting occurs in the placenta. Therefore, we sought to determine the relationship between the imprinting of insulin-like growth factor II (IGF-II) in placenta and twin discordance. We analyzed the imprinting and promoter usage of IGF-II in placenta of normal twins (T0 group), weight discordance (T1 group), and phenotype discordance (T2 group). We found the incidence of loss of imprinting (LOI) for IGF-II was higher in the T2 group than that in the T0 and T1 groups, while there was no difference between T0 and T1 groups. The transcripts of promoter 3 were lower in the T2 group than in the T0 and T1 groups, and lower in the twin placenta with LOI than in those with normal imprinting. Our findings indicate that the promoter 3 specific LOI of the IGF-II gene may be closely related with phenotype discordance, not weight discordance.

Mechanically expanding the zona pellucida of human frozen thawed embryos: a new method of assisted hatching.

OBJECTIVE: To determine whether a new assisted hatching (AH) method increases the implantation and clinical pregnancy rates of frozen-thawed day-3 (D3) embryos. DESIGN: Prospective study. SETTING: A university hospital in vitro fertilization (IVF) program. PATIENT(S): Patients who had their first IVF/intracytoplasmic sperm injection (ICSI) cycles between June 1, 2006, and December 31, 2008, with fresh IVF-embryo transfer failures or without fresh embryo transfer. INTERVENTION(S): The couples were randomized into thawed embryo transfer after AH versus no AH. In the AH group, the zona pellucida (ZP) of D3 frozen-thawed embryos was expanded by injected hydrostatic pressure after thawing. In the control group, embryos were pierced by ICSI needles without expanding the ZP. MAIN OUTCOME MEASURE(S): Clinical pregnancy and implantation rates. RESULT(S): The morphologic features of the blastomeres were carefully monitored and recorded. In the AH group, 244 embryos were thawed, and 178 (73.0%) survived; in the control group, 259 embryos were thawed, and 190 (73.4%) survived. Despite the transfer of a similar number of embryos, the AH group resulted in statistically significantly higher implantation and clinical pregnancy rates compared with the no AH group. CONCLUSION(S): Mechanically expanding the ZP of frozen-thawed D3 embryos with injected hydrostatic pressure after thawing increases the implantation rate compared with control embryos.

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.

RNA interference targeting of sphingomyelin phosphodiesterase 1 protects human granulosa cells from apoptosis.

AIM: Sphingomyelin phosphodiesterase 1 (SMPD1) plays an essential role in initiating the female germ cell death signal. To evaluate whether RNA interference has potential as a new approach in germ cell protection, we tested the effect of SMPD1 knockdown on human granulosa cells in vitro. METHODS: We designed and synthesized three small interference RNA (siRNA) sequences targeted on SMPD1 and transfected them into human luteinizing granulosa cells (hGC) in vitro. Forty-eight hours after transfecting with siRNAs, hGC were treated with mitomycin C (MMC) to induce apoptosis. mRNA was detected with quantitative RT-PCR and protein was detected with Western blot. Methyl thiazolyl tetrazolium (MTT) assay was used to measure cell survival rate and detection of apoptotic rate of cells with Annexin V-PI staining by flow cytometer (FCM). Study groups were compared with liposome (lipofectamine 2000), MMC control and negative control siRNA. RESULTS: After treatment with siRNA targeted to SMPD1, significant SMPD1 suppression occurred. After knockdown expression of SMPD1, the survival rate of hGC increased from 32.3% to 40.3%, and the apoptosis rate decreased from 68.3% to 44%. CONCLUSION: siRNA targeted on SMPD1 can protect hGC cells from apoptosis. These results reveal SMPD1 as a significant and effective target site for RNAi in the protection of human germ cells, which may have a direct bearing on future therapeutic research.

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.

In vitro-matured rat oocytes have low mitochondrial deoxyribonucleic acid and adenosine triphosphate contents and have abnormal mitochondrial redistribution.

OBJECTIVE: To study the development and function of mitochondria in in vitro-matured rat oocytes derived from follicles of different sizes. DESIGN: Experimental animal study. SETTING: Department of Anatomy at the University of Hong Kong. ANIMAL(S): Immature female Sprague-Dawley rats that were 25 days of age. INTERVENTION(S): Immature oocytes were collected from rat ovarian follicles of different sizes and were induced to mature in vitro. MAIN OUTCOME MEASURE(S): The number of copies of mitochondrial DNA, mitochondrial activity, adenosine triphosphate content of matured oocytes, and rates of fertilization and blastulation were determined. RESULT(S): The mitochondrial DNA copy number of oocytes increased linearly with the diameter of antral follicles. The mitochondrial DNA copy number, adenosine triphosphate content, and proportion of oocytes with peripheral distribution of mitochondria in in vitro-matured oocytes from small antral follicles were significantly lower than those from preovulatory follicles and in vivo-matured oocytes. Compared with in vitro-matured oocytes from small antral follicles, those from preovulatory follicles and in vivo-matured oocytes also had significantly better fertilization potential and higher blastulation rate. CONCLUSION(S): The inferior developmental potential of in vitro-matured oocytes may be attributed partly to a reduced number of mitochondria, resulting in insufficient production of adenosine triphosphate for required developmental events.

Preimplantation genetic diagnosis for Duchenne muscular dystrophy by multiple displacement amplification.

OBJECTIVE: To evaluate the use of multiple displacement amplification (MDA) in preimplantation genetic diagnosis (PGD) for female carriers with Duchenne muscular dystrophy (DMD). DESIGN: MDA was used to amplify a whole genome of single cells. Following the setup on single cells, the test was applied in two clinical cases of PGD. One mutant exon, six short tandem repeats (STR) markers within the dystrophin gene, and amelogenin were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products of single blastomeres. SETTING: Center for reproductive medicine in First Affiliated Hospital, Sun Yat-sen University, China. PATIENT(S): Two female carriers with a duplication of exons 3-11 and a deletion of exons 47-50, respectively. INTERVENTION(S): The MDA of single cells and fluorescent PCR assays for PGD. MAIN OUTCOME MEASURE(S): The ability to analyze single blastomeres for DMD using MDA. RESULT(S): The protocol setup previously allowed for the accurate diagnosis of each embryo. Two clinical cases resulted in a healthy girl, which was the first successful clinical application of MDA in PGD for DMD. CONCLUSION(S): We suggest that this protocol is reliable to increase the accuracy of the PGD for DMD.

[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.

[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.

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.

[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

Visualization of meiotic spindle and subsequent embryonic development in in vitro and in vivo matured human oocytes.

PURPOSE: The aim of this study is to investigate the relationship between spindle location and embryonic development of in vivo and in vitro matured human oocytes. METHODS: The spindles of 134 in vivo matured, 105 in vitro matured oocytes were examined by Polscope at the time of ICSI. RESULTS: The spindles were visualized in 83.6 and 77.1% of in vivo and in vitro matured oocytes respectively. The rate of fertilization of in vivo matured oocytes with spindles beneath or adjacent to the first polar body (angle of 0-5 degrees) was significantly higher (93.3%) than all other groups. The proportions of various spindle positions did not differ statistically in in vivo and in vitro matured oocytes. CONCLUSIONS: Meiotic spindle location with regard to the first polar body appears to influence fertilization rate.

[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.

[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.

Low mitochondrial DNA and ATP contents contribute to the absence of birefringent spindle imaged with PolScope in in vitro matured human oocytes.

BACKGROUND: Birefrigent meiotic spindle in live human oocytes can be visualized by the PolScope. This study investigated the relationship between birefrigent meiotic spindle and cytoplasmic mitochondrial DNA (mtDNA) and ATP contents in in vitro matured human oocytes. METHODS: Oocytes at germinal vesicle stage were collected and cultured for 24-48 h with or without the metabolic inhibitor, carbonyl cyanide p-(tri-fluromethoxy) phenyl-hydrazone (FCCP). All in vitro matured oocytes were examined by PolScope for the presence of meiotic spindle, then the oocytes were used for either intracytoplasmic sperm injection or the measurement of mitochondrial quantity and ATP content. RESULTS: Meiotic spindles were observed in 51.3% (60/117) of the in vitro matured oocytes. Oocytes with detectable meiotic spindle contained significantly higher mtDNA copies (637 250 +/- 237 606 versus 491 454 +/- 153 406, P = 0.027) and ATP content (1.97 +/- 0.38 versus 1.65 +/- 0.32 pmol, P = 0.028) when compared with those without detectable meiotic spindle. However, in vitro matured oocytes showed a significantly reduced rate of positive meiotic spindle and a lower ATP content when cultured with FCCP. A lower incidence of normal fertilization and good quality embryos were observed if meiotic spindles were not detected. CONCLUSIONS: Low mtDNA and ATP content might contribute to the absence of birefringent spindle imaged with the PolScope in human in vitro matured oocytes.