What was the fate of human embryos following long-term cryopreservation (≥12 years) and frozen embryo transfer?

STUDY QUESTION: Do human embryos survive long-term cryopreservation (CP) (≥12 years) and implant after frozen embryo transfer (ET)? SUMMARY ANSWER: Human embryos remain usable after long-term CP. WHAT IS KNOWN ALREADY: Several cohort studies have reported the live birth rate or neonatal outcomes of human embryos after CP for up to 5 years. Only a few case reports have described successful live births from human embryos after long-term CP up to 12 years. STUDY DESIGN, SIZE, DURATION: This retrospective observational study in China included 20 patients (128 embryos) from March 2016 to April 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twenty patients who had at least one live birth during their previous IVF/ICSI treatments and had surplus embryos cryopreserved were observed. Data concerning frozen embryo recovery, pregnancy and obstetric outcomes following frozen ET were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 128 embryos of 20 patients were observed. The embryo storage duration was 12.0-17.1 years, with a mean of 13.9 ± 1.73 years. In all, 115 embryos were thawed to transfer, with a survival rate of 74%. Sixty embryos were further cultured, which resulted in 20 blastocysts with a blastocyst formation rate of 33%. There were 21 cleavage-stage embryos and 13 blastocysts transferred in a total of 12 and 11 cycles, respectively, which resulted in one biochemical pregnancy, one first trimester miscarriage, two ectopic pregnancies, three singletons and one case of twins, with a clinical pregnancy rate of 25% (D3 ET) and 36% (blastocyst transfer) and a live birth rate of 17% (D3 ET) and 27% (blastocyst transfer). Two of the four patients who had live birth developed gestational diabetes mellitus. One of the five live births was a preterm delivery. LIMITATIONS, REASONS FOR CAUTION: The sample size was small due to the unique study population, and all the embryos underwent slow freezing. The fate of long-term cryopreserved embryos after vitrification is still unclear. WIDER IMPLICATIONS OF THE FINDINGS: The results provide evidence to support the use of embryos after extended CP to preserve patients' fertility. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by grants from the National Key Research and Development Programme of China (2016YC1000205) and the Guangzhou Scientific Programme (201508020006). None of the authors has any conflicts of interest to declare.

Pregnancy derived from human zygote pronuclear transfer in a patient who had arrested embryos after IVF.

Nuclear transfer of an oocyte into the cytoplasm of another enucleated oocyte has shown that embryogenesis and implantation are influenced by cytoplasmic factors. We report a case of a 30-year-old nulligravida woman who had two failed IVF cycles characterized by all her embryos arresting at the two-cell stage and ultimately had pronuclear transfer using donor oocytes. After her third IVF cycle, eight out of 12 patient oocytes and 12 out of 15 donor oocytes were fertilized. The patient's pronuclei were transferred subzonally into an enucleated donor cytoplasm resulting in seven reconstructed zygotes. Five viable reconstructed embryos were transferred into the patient's uterus resulting in a triplet pregnancy with fetal heartbeats, normal karyotypes and nuclear genetic fingerprinting matching the mother's genetic fingerprinting. Fetal mitochondrial DNA profiles were identical to those from donor cytoplasm with no detection of patient's mitochondrial DNA. This report suggests that a potentially viable pregnancy with normal karyotype can be achieved through pronuclear transfer. Ongoing work to establish the efficacy and safety of pronuclear transfer will result in its use as an aid for human reproduction.

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.

Preimplantation genetic diagnosis for α-and ß-double thalassemia.

PURPOSE: To evaluate the use of multiple displacement amplification (MDA) for preimplantation genetic diagnosis (PGD) of α- and ß-double thalassemia. METHOD: Whole genome of a single cell was directly amplified using MDA and its products were used as templates in fluorescent gap polymerase chain reaction (PCR) analysis of α-thalassemia and in PCR-reverse dot blot analysis, singleplex fluorescent PCR of ß-28 and CD17 mutation and HumTH01 for ß-thalassemia. RESULTS: 1) MDA from single cell could produce enough DNA templates for the detection of both α and ß-thalassemia; 2) The established MDA-PGD protocol for α- and ß-double thalassemia was successfully applied in PGD of six embryos, among which, three were transferred, but no pregnancy ensued. CONCLUSIONS: The use of MDA as a universal step allows for the simultaneous diagnosis of two or more hereditary defects.

[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.

Supraphysiological estrogen levels adversely impact proliferation and histone modification in human embryonic stem cells: possible implications for controlled ovarian hyperstimulation assisted pregnancy.

OBJECTIVE: Controlled ovarian hyperstimulation (COH) results in supraphysiologic levels of maternal serum estradiol (E(2)) during the luteal phase, thus promoting oocyte production at unknown risk to the subsequently developing embryo. Human embryonic stem cells (hESCs) have been identified as a model system to assess the impact of COH on early embryonic development, specifically 17ß-estradiol mediated effects on proliferation, gene expression, and histone modification. STUDY DESIGN: Cell proliferation and associated factors, such as HDAC1, as well as histone modification patterns were evaluated in ERα and ß expressing hESCs after exposure to 17ß-estradiol (1×10(-10) M to 1×10(-7) M), as well as in an untreated control. RESULTS: Resultant data revealed that while physiologically relevant E(2) levels (1×10(-9)M E(2)) induced cell cycle progression from G1 to the proliferation phase, supraphysiologic levels akin to those observed after COH (1×10(-7) M E(2)) adversely affected hESCs proliferation via down regulation of HDAC1. Modification of H3K9me2, PhH3S10, H4K5ac, and H2A.Z histone patterns were also dependent on 17ß-estradiol concentration. CONCLUSION: While physiologic levels of 17ß-estradiol induced cell proliferation, possibly via HDAC1 involvement in histone modification, cell proliferation in hESCs was suppressed at supraphysiologic levels.

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.

[Evaluation of the fidelity of multiple displacement amplification from small number of cells].

OBJECTIVE: To evaluate the fidelity of multiple displacement amplification (MDA) from small number of cells (1-10 cells) by 10K 2.0 SNP mapping array. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

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.

Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array.

The scarce amount of DNA contained in a single cell is a limiting factor for clinical application of preimplantation genetic diagnosis mainly due to the risk of misdiagnosis caused by allele dropout and the difficulty in obtaining copy number variations in all 23 pairs of chromosomes. Multiple displacement amplification (MDA) has been reported to generate large quantity of products from small amount of templates. Here, we evaluated the fidelity of whole-genome amplification MDA from single or a few cells and determined the accuracy of chromosome copy number assessment on these MDA products using an Affymetrix 10K 2.0 SNP Mapping Array. An average coverage rate (86.2%) from single cells was obtained and the rates increased significantly when five or more cells were used as templates. Higher concordance for chromosome copy number from single cells could be achieved when the MDA amplified product was used as reference (93.1%) than when gDNA used as reference (82.8%). The present study indicates that satisfactory genome coverage can be obtained from single-cell MDA which may be used for studies where only a minute amount of genetic materials is available. Clinically, MDA coupled with SNP mapping array may provide a reliable and accurate method for chromosome copy number analysis and most likely for the detection of single-gene disorders as well.

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.