H.-C. Liu

Improved pregnancy outcome with gonadotropin releasing hormone agonist (GnRH-a) stimulation is due to the improvement in oocyte quantity rather than quality.

The impact of gonadotropin releasing hormone agonist (GnRH-a) on the quality and quantity of oocytes harvested in in vitro fertilization—embryo transfer (IVF-ET) patients was studied by comparing the results for patients stimulated with gonadotropin alone and with gonadotropin plus GnRH-a. Adding GnRH-a significantly improved the viable pregnancies per transfer and reduced the spontaneous abortions, which seemed to improve oocyte quality. However, when oocyte quality was evaluated by the fertilization rate and the implantation and delivery rates per embryos transferred, there were no significant difference in the results, indicating that GnRH-a did not improve the oocyte quality. On the other hand, GnRH-a significantly increased the average number of oocytes harvested, fertilized, and transferred, and this increased number of oocytes transferred has been demonstrated to increase pregnancy and multiple-pregnancy rates. Multiple pregnancy with more embryos implanted would significantly reduce the abortion rate. Abortion rates decreased inversely to the number of embryos implanted. Our data strongly suggest that the efficacy of GnRH-a on IVF-ET patients was due more to the quantity increase than the quality of embryos transferred.

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.

Tumor necrosis factor is present in maternal sera and embryo culture fluids during in vitro fertilization

The incidence of tumor necrosis factor alpha (TNF) in sera of women undergoing in vitro fertilization (IVF) and in embryo culture fluids was evaluated using an enzyme-linked immunosorbent assay. Just prior to embryo transfer to the uterus, 24 of 49 maternal sera (49.0%) contained TNF. The incidence and range of TNF concentrations (84-920 pg/ml) did not differ between women with eventual successful pregnancies and women who subsequently suffered preclinical or clinical abortions. At 8 days post-embryo transfer, 24 of 56 sera (42.9%) contained TNF. Again, the occurrence of TNF was of no predictive value for the eventual outcome of the pregnancy. TNF was also detected in culture fluids from the in vitro fertilized eggs of 12 of 49 women (24.5%). In 9 women, TNF was detected in each of the embryo culture fluids tested. As was the case for sera, the presence or absence of TNF in the culture fluids was unrelated to pregnancy outcome. In 21 patients, paired sera and culture fluids were analyzed. In 9 of 10 women with TNF in their culture fluids, TNF was also present in the corresponding serum. Of 12 women with serum TNF, 9 also had TNF in their culture fluids. Since the culture fluids contained 10% maternal sera, it appeared that in most cases TNF in the culture fluids was derived, at least in part, from the serum. However, in 6 of 9 women TNF levels in the cultures exceeded levels in the corresponding sera. TNF was also identified in 2 of 10 culture fluids in which Plasmanate was substituted for serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Mouse Ovarian Tissue Cryopreservation Has Only a Minor Effect on In Vitro Follicular Maturation and Gene Expression

AbstractPurpose: To establish a protocol for ovarian tissue cryopreservation which can retain fertility potential after thawing and to evaluate the impact of cryopreservation on development and gene expression during folliculogenesis. Methods: A controlled randomized study in a clinical and academic research setting in a university medical center was conducted to study cryopreservation and in vitro maturation (IVM) of mouse ovarian follicles. Preantral follicles isolated from either fresh (Group A) or cryopreserved (Group B) murine ovarian tissues were used to test their fertility potential by in vitro culture–in vitro maturation (IVC-IVM). Expression of Graafian follicles derived from both groups were detected by DNA microarray techniques for comparison. Results: Although there were no significant differences in IVM outcomes and follicular gene expression between the two experimental groups, cryopreservation appears to induce the expression of heat shock proteins, DNA-damage-inducible protein 45 and death-related apoptosis genes (i.e., Fas and Fas-ligand). Conclusion: Cryopreservation may trigger biological events not amenable to normal cell function and follicular development. However, neither follicular development nor gene expression was dramatically changed after cryopreservation. These data suggest that although our current cryopreservation techniques yield competent follicles and mature oocytes, subtle changes observed in gene expression imply that the present cryopreservation techniques need to be further refined.

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.

Microsurgical fertilization procedures: The absence of stringent criteria for patient selection

Subzonal sperm insertion and partial zona dissection were applied in 250 in vitro fertilization cycles in couples (n =200) with abnormal semen analyses; 61 clinical pregnancies were established (24% per egg retrieval). Patients were selected without using minimal cutoff criteria. The study included patients with 0% normal sperm forms (strict criteria), no motile sperm (but some live cells), and sperm counts which could be assessed only after centrifugation. Patients were categorized into three subsets. Group A (n =116 cycles) failed to fertilize in a previous cycle. Group B (n =40) was excluded from IVF due to the severity of sperm profiles, such as a maximum of 2% normal forms. Group C (n =94) constitutes those patients for whom a standard cycle could possibly result in failure. Monospermic fertilization rates were 18% (A), 19% (B), and 24% (C). The incidences of embryo replacement were 63% (A), 53% (B), and 69% (C). Rates of clinical pregnancy were 22% (A), 23% (B), and 28% (C). The presence of one, two, or three semen abnormalities did not correlate with the outcome of microsurgical fertilization. Twenty-two percent of patients with combined oligoasthenoteratozoospermia became pregnant. Moreover, ongoing pregnancies were established in instances with 0% normal sperm forms and no progressively motile spermatozoa. It is concluded that stringent cutoff criteria may not be necessary when both partial zona dissection and subzonal sperm insertion are performed efficiently.

Application of complementary DNA microarray (DNA chip) technology in the study of gene expression profiles during folliculogenesis

OBJECTIVE Using oligonucleotide microarray (DNA chip)-based hybridization analysis to gain a comprehensive view of gene expression and regulation involved in folliculogenesis. DESIGN Prospective randomized study. SETTING Academic institution. ANIMAL(S) B6D2F1 female mice. INTERVENTION(S) Superovulation. MAIN OUTCOME MEASURE(S) Preantral follicles isolated from day 14 B6D2F-1 mice were stimulated in vitro to form Graafian follicles. Total RNA extracted from the mouse preantral and Graafian follicles were reverse transcribed, labeled with digoxigenin-11-dUTP, and then hybridized with Clontech Atlas mouse cDNA expression arrays for comparison. RESULT(S) Of 588 known studied genes, 39 and 61 were detected in preantral follicles and in Graafian follicles, respectively, and 17 were highly expressed consistently in both preantral and Graafian follicles. Performing clustering analysis, we found that 15 detected genes were down-regulated and 46 were up-regulated as the follicles advanced to mature stages. CONCLUSION(S) We have successfully developed a sensitive DNA chip technology that is able to simultaneously and quantitatively study gene expression profiles in a small number of follicles (1.5-15 follicles). Several folliculogenesis-related genes have been identified. Some of these genes were expressed, indicating that they may be essential for follicle growth and maturation, whereas others were up-regulated only during late follicular development, indicating stage-specific roles.

Granulocyte Macrophage-Colony Stimulating Factor Production by Autologous Endometrial Co-Culture Is Associated with Outcome for In Vitro Fertilization Patients with a History of Multiple Implantation Failures

PROBLEM: To determine whether granulocyte macrophage (GM)‐colony stimulating factor (CSF) produced by autologous endometrial co‐culture was associated with outcome in 53 patients with a history of multiple in vitro fertilization failures.

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.