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In vitro culture and in vitro maturation of mouse preantral follicles with recombinant gonadotropins

OBJECTIVE To develop an effective method for in vitro maturation of preantral follicles isolated from mice ovarian tissue. DESIGN Isolated preantral follicles were randomly allocated to designed experimental groups for study. SETTING University-based research lab. PATIENT(S) Healthy, normal mice. INTERVENTION(S) Superovulation with pregnant mare serum gonadotropin and hCG. MAIN OUTCOME MEASURE(S) Morphological changes and E(2) production were assessed. RESULT(S) To obtain competent oocytes, preantral follicles must be cultured with medium containing insulin and recombinant gonadotropins (i.e., recombinant FSH and recombinant LH), with a change of medium daily. A high initial recombinant LH or recombinant FSH facilitates E(2) secretion, enhances granulosa cell outgrowth, and has earlier antral formation. However, prolonged culture in high-recombinant LH or recombinant FSH triggers early differentiation and luteinization of granulosa cells, which results in low metaphase II oocyte and blastocyst formation. CONCLUSION(S) We have developed a culture system that allows the successful maturation of preantral follicles in vitro. The matured follicles are a physiologically functional unit that not only secrete E(2) but also generate competent oocytes. In a special condition, 90% of the cultured follicles survived, 53.5% of them produced MII oocytes, and 50% of the derived MII oocytes were fertilized and reached the blastocyst stage after culture in vitro.

Expression of apoptosis-related genes in human oocytes and embryos.

AbstractPurpose: The objective was to study whether apoptosis occurs in human embryogenesis. Methods: Human viable, arrested, and nonviable embryos and immature, and nonfertilized oocytes donated by our patients were used to detect apoptosis by Tunel labeling, annexin staining, and single-cell reverse transcriptase–polymerase chain reaction (RT-PCR). Results: DNA fragmentation and phosphotidylserine translocation, the two markers for apoptosis, were detected frequently in fragmented human embryos derived from in vitro fertilization–embryo transfer (IVF-ET). Using RT-PCR, apoptotic genes also were detected in these embryos. The frequencies of gene expression in viable embryos, arrested embryos, nonviable embryos, immature oocytes, and nonfertilized oocytes were: 7/8, 5/5, 5/6, 0/6, 0/3, for Bax; 8/8, 5/5, 7/7, 0/4, 0/5 for Fas; 2/8, 0/2, 0/3, 0/5, 0/3 for BCL-2; 0/8, 1/3, 0/2, 0/3, 0/2 for Fas-ligand; and 8/8, 17/17, 21/21, 24/24, 15/15 for actin, respectively. Conclusions: Our preliminary data did not show a significant difference in the expression frequency of all studied genes between viable embryos and nonviable or arrested embryos. However, the expression of Bax and Fas was noticeably higher in nonviable embryos than in viable embryos as judged by the intensities of amplicons visualized after ethidium bromide staining. In addition, BCL-2 was only detected in viable embryos. Whether embryos quality is related to the regulation of BCL-2, Bax, and Fas expressions requires further study.

Simultaneous detection of multiple gene expression in mouse and human individual preimplantation embryos

OBJECTIVE To detect simultaneously multiple gene expression in mouse and human individual embryos by reverse transcriptase-polymerase chain reaction. DESIGN Transcripts involved in the insulin-like growth factor (IGF) system were detected in mouse and human preimplantation embryos. SETTING An academic teaching hospital. MAIN OUTCOME MEASURE(S) Transcripts of the IGF family genes. RESULT(S) In the mouse, genes are expressed differentially and messenger RNA transcripts of maternal origin in nonfertilized ova decline gradually until the initiation of the embryonic genome transcription. Insulin-like growth factor-binding protein-2 (IGFBP)-2, -3, -4, and beta-actin transcripts appear to be initiated at the two- to four-cell stage, whereas IGFBP-1, -5, and -6 transcripts are initiated at later stages. Transcription, once initiated, appears to continue through to the blastocyst stage. In humans, almost all genes of the IGF system were expressed in preimplantation embryos. This is the first report of the assessment of IGF family transcripts in individual embryos, and introduces a novel method for research and clinical diagnosis of preimplantation embryos.

Expression of IGFs and their receptors is a potential marker for embryo quality.

PROBLEM: Insulin‐like growth factors (IGFs) and insulin have been demonstrated to stimulate oocyte maturation and embryo development. Therefore, the expression of IGFs and their receptors may be an important intrinsic factor for embryo growth and may be a potential marker for embryo quality.

Dicer is a key player in oocyte maturation.

ObjectiveApply Dicer siRNA to study functions of Dicer and miRNA during oogenesis.Materials and MethodsMouse oocytes were injected with Dicer siRNA and negative control siRNA and then matured in vitro. After IVM, oocytes were examined for maturation rates, spindle and chromosomal organization, and various gene expressions.ResultsDicer siRNA significantly reduced maturation rates, increased abnormal spindle and chromosomal organization, and reduced the transcripts of Dicer miRNAs, spindle formation proteins (plk1 and AURKA) and spindle check points (Bub1, Bublb). Depletion of bulb16 markedly prohibited the first polar body extrusion and increased the incidence of misaligned chromosomes and abnormal meiotic spindle assembly.ConclusionDicer siRNA triggered a cascade reduction for gene expressions starting from Dicer to miRNAs than to spindle assembly proteins and checkpoints which led to abnormal spindle and chromosomal organization. Thus, Dicer and miRNA appeared to play an important role during oogenesis and were essential for meiotic completion.

Cryopreservation of nuclear material as a potential method of fertility preservation

OBJECTIVE To establish a cryopreservation method for female nuclear materials with the aim of creating viable embryos or offspring by nuclear transfer. DESIGN A randomized controlled study. SETTING Clinical and academic research facility.B6D2F1 mice. INTERVENTION(S) Female pronuclei (FPNs) and second polar bodies (2PBs) isolated from superovulated mouse zygotes were cryopreserved, thawed, and transferred into donor zygotes. MAIN OUTCOME MEASURE(S) Survival rates after freeze-thaw and blastocyst formation rates were evaluated. RESULT(S) Female pronuclei and 2PBs preserved with three tested methods resulted in survival rates ranging from 11.5% to 85% for FPNs vs. 46.9% to 95.0% for 2PBs and blastocyst formation rates ranging from 0% to 35.5% for FPNs vs. 9.1% to 47.4% for 2PBs. Live birth offspring also resulted from both FPNs and 2PBs preserved with vitrification. CONCLUSION(S) We have established a new system to effectively revive frozen nuclei into viable embryos by a combination of nuclei preservation, zygote reconstruction, and coculturing of reconstructed zygotes with mouse embryonic fibroblast cells. Our data suggest that the preserved female genomic DNA material can be potentially used for future nuclear transfer to preserve female fertility.

Correlation of somatic cell steroid secretion and quality of generated oocytes after in-vitro stimulation of mouse follicles.

AbstractPurpose: To test the possibility of follicular somatic cell steroidogenesis as a marker for quality of their embraced oocytes. Methods: Mechanically isolated mouse preantral follicles were cultured and matured in-vitro (IVC/IVM) for study. Results: During IVC/IVM, oogenesis occurred concomitantly with folliculogenesis in a coordinated manner and simultaneously with progressive increments of somatic cell steroidogenesis. Follicular E2 production of matured oocytes were significantly higher than that of immature ones. The majority of MII oocytes (32/36) and all developed blastocysts(12/12) were associated with active E2 production prior to ovulation. In this study, 18 MII oocytes met both requirements for active and optimal E2 production. 13 of them were fertilized and 10 developed into blastocysts. Conclusion: Active somatic cell steroidogenesis prior to ovulation and an optimal steroid milieu at ovulation are prerequisites for generation of competent oocytes after follicular maturation in-vitro.

Recycling of a single human blastomere fixed on a microscopic slide for sexing and diagnosis of specific mutations by various types of polymerase chain reaction

OBJECTIVE To investigate the suitability of recycling single blastomeres to assess multiple genetic variables for preimplantation genetic diagnosis. DESIGN Prospective randomized study. SETTING An academic medical center. PATIENT(S) Patients undergoing IVF-ET. INTERVENTION(S) Blastomeres were disaggregated from donated embryos obtained from patients. MAIN OUTCOME MEASURE(S) Polymerase chain reaction (PCR) amplification products. RESULT(S) Fifty-eight blastomeres individually fixed on slides were separated into four groups. Sequential PCRs (group I, n = 30), primed in situ labeling (PRINS) before five sequential PCRs (group II, n = 10), staining with hematoxylin before performing five sequential PCRs (group III, n = 11) and preamplification of whole DNAs by degenerate oligonucleotide primer (DOP) before performing PCR were executed. The amplification efficiencies of five sequential PCRs were 100%, 100%, 96.6%, 83.3%, 56.7% for group I; 100% 100%, 100%, 80%, 40% for group II; 54.5%, 36.4%, 18.2%, 9.1% for group III; and 100%, 100%, 100%, 100%, 100% for group IV. CONCLUSION(S) Blastomeres fixed for PRINS can be recycled for PCR to obtain more genetic information. Hematoxylin staining appears to increase the incidence of failed amplification. Preamplification of whole genomic DNAs by DOP-PCR appears to facilitate diagnosis with high efficiency.

O-006 Recycling of a single human blastomere fixed on a microscopic slide for sexing and diagnosis of specific mutations by various types of PCR

Objective: To investigate the suitability of recycling single blastomeres biopsied from human embryos in order to obtain multiple genetic information for preimplantation genetic diagnosis. Design: Biopsied blastomeres were fixed on slides. Conventional polymerase chain reaction (PCR), primed in-situ labeling (PRINS) and degenerate oligonucleotide primer (DOP) were performed sequentially on the blastomeres. The rate of allele specific drop out (ADO) was monitored to evaluate the amplification efficiency. Materials and Methods: Sixty blastomeres were individually fixed on slides. Two were lost due to fixation. The remaining 58 blastomeres were separated into 4 groups to amplify the gene sequence by specific primers for sexing, W1282X and AF508 mutation in cystic fibrosis, sickle cell mutation in human beta-globin, and steroid 21 hydroxylase gene (CYP21) in various conditions. Group I: Blastomeres (n=30) were used to perform 5 sequential PCRs. Group II: Blastomeres (n=10) were used to perform PRINS prior to 5 sequential PCRs. Group III: Blastomeres ( n = l l ) were stained with hematoxylin before performing 5 sequential PCRs. Group IV: Blastomeres (n=7) were used to pre-amplify whole DNA by DOP-PCR before performing PCR. Results: A high percentage (96.7%) of blastomeres were fixed before PCR was performed. The amplification efficiency of 4 experimental groups were shown in the following table: