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

PURPOSE: 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 non-fertilized 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.

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.

Expression of inhibin/activin subunits and their receptors and binding proteins in human preimplantation embryos.

PURPOSE: Our purpose was to study the role of inhibin/activin during embryogenesis. METHODS: Transcripts of inhibin/activin subunits (alpha, beta A, beta B), activin receptors (types I and II), and follistatin were detected by a reverse transcriptase-polymerase chain reaction in human reproductive cells and preembryos cultured alone or co-cultured with human endometrial cells. RESULTS: Transcripts of alpha, beta A, beta B subunits were all detected in granulosa luteal cells, but only beta A units were detected in endometrial stromal and decidualized cells. In human preimplantation embryos, none of these subunits were detected in embryos from the four-cell to the morula stage and only beta A subunits were detectable in blastocyst embryos. Activin receptors were detectable in all of the studied embryos and cells. Transcripts of beta A, activin receptors, and follistatin were differentially expressed in human preimplantation embryos cultured in vitro and their expressions were significantly enhanced with the presence of endometrial stromal cells. CONCLUSIONS: Our data suggest that there is a possible endometrium-embryo interaction via endometrial activins and preimplantation embryo receptors and that the embryonic expressions of these activins, their receptors, and binding proteins are dependent on embryonic stage.

Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos.

OBJECTIVE: To demonstrate the mechanism by which human endometrial stromal cells improve embryo quality in coculture. DESIGN: Randomized study. SETTING: Academic research center. PATIENT(S): Patients undergoing IVF-ET. INTERVENTION(S): Donated human embryos were cultured randomly either alone (group A) or with human endometrial stromal cells (group B), and the embryonic expression of insulin-like growth factors (IGFs) and their receptors was detected by reverse transcriptase polymerase chain reaction after culture. MAIN OUTCOME MEASURE(S): The embryo frequency distribution of groups A and B before and after culture and the embryonic transcripts of the IGF family genes of the two study groups after culture were compared. RESULT(S): The embryo frequency distribution of the day 3 embryonic stages in groups A and B was not different. However, after culture, a statistically significant difference in blastocyst formation was observed between groups A and B. A significant increase in the expression of IGF-1, IGF-2, the IGF-1 receptor, and the insulin-receptor also was noted. Among the embryos that reached the blastocyst stage, the expression of IGF-1 and the IGF-1 receptor also was significantly different in the two study groups. CONCLUSION(S): Human endometrial stromal cells enhanced the expression of IGFs and their receptors in cocultured human embryos, which may be essential for improving embryo quality.

Immunohistochemical analysis of insulin-like growth factor-binding proteins -1, -2, and -3 in implantation sites of the mouse.

PURPOSE: Our purpose was to analyze potential interactions between the embryo and the maternal endometrial interface in vivo by analyzing immunolocalization of insulin-like growth factor-binding proteins (IGFBPs) -1, -2, and -3 in implantation sites of the mouse. METHODS: Six-week-old B6D2F1 female mice underwent superovulation followed by mating and sacrifice at timed intervals. Formalin-fixed paraffin-embedded tissue was used for avidin-biotin immunocytochemical localization of IGFBPs utilizing standard methodology. RESULTS: Immunostaining at 1.5 days post coitum revealed light staining in the epithelial glandular cells and faint staining in decidual stroma for both IGFBP-1 and IGFBP-2. At 7.5-10.5 days post coitum, there was moderate-dense immunostaining in the decidualized stromal cells at the implantation site for all three IGFBPs, whereas light immunostaining was seen in nonimplantation site decidua. CONCLUSIONS: Compartmentalization of immunostaining for IGFBP-1, -2, and -3 within decidualized stroma suggests that these proteins may be regulated by trophoblastic and/or embryonic signals.

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. METHOD OF STUDY: Thirty donated day 3 embryos were cultured in vitro for an additional 3 days to observe their developmental potential and were semiquantitatively analyzed for the expression of IGF-I, IGF-II, IGF-IR, IGF-IIR, and insulin-R. RESULTS: Our results show that the activity of these gene expressions correlates well with the morphological assessment and that high and more gene expressions were often associated with embryos of high growth potential. CONCLUSION: The IGF system may indeed play an important role in human embryogenesis; IGF gene expressions can be a good indicator of embryonic developmental stage and/or growth potential; finally, the IGF system can serve as a marker for embryo quality.

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.

Endometrial secretory proteins enhance early embryo development.

PURPOSE: To evaluate the role of endometrial stromal cells and their secretory proteins in early embryo development, two-celled CB6F1 mouse embryos were cultured alone or cocultured with human endometrial stromal cells in various culture conditions. RESULTS: The percentage of embryo blastocyst formation, hatching, and outgrowth was significantly greater in (1) coculture with endometrial stromal cells than in a cell-free control when both coculture and control were carried out in protein-free medium or in RPMI 1640 plus 10% fetal calf serum; (2) coculture with hormone (i.e., progesterone plus relaxin)-treated cells than in coculture with hormone-nontreated cells; and (3) media supplemented with isolated endometrial secretory proteins than in media supplemented with BSA (0.35%). Embryo development was not found to be significantly different in coculture and in media supplemented with endometrial secretory protein. CONCLUSION: Our data provides credence to the theory that endometrial stromal cells enhance embryo development by secreting specific proteins that are beneficial to embryo growth in vitro.

Increased interleukin-2 production in response to human type I collagen stimulation in patients with systemic sclerosis.

Peripheral blood mononuclear cells (PBMC) from patients with systemic sclerosis (SSc) produced increased amounts of interleukin-2 (IL-2), in a dose-dependent manner, in response to stimulation with human type I collagen, whereas PBMC from normal subjects did not. At a dose of 50 micrograms human type I collagen/10(6) PBMC, PBMC from SSc patients (n = 17) produced 8 times as much IL-2 as did PBMC from 16 normal subjects (P less than 0.005) and 3 times as much as did PBMC from a group of 13 rheumatoid arthritis patients (P less than 0.05). In contrast, IL-2 production by PBMC after nonspecific stimulation with the mitogen, phytohemagglutinin, did not differ among the SSc, rheumatoid arthritis, and normal control groups. Cell depletion experiments indicated that the IL-2-producing cells in SSc patients are CD4+. Thus, SSc patients have CD4 cells that are specifically sensitized to human type I collagen and can produce increased levels of IL-2. Measurement of IL-2 production stimulated by human type I collagen may be useful in evaluating disease activity, and further investigation of this process may contribute to the delineation of the pathogenesis of SSc.

Differential effects of human alpha and gamma interferon on mixed lymphocyte culture and on T-cell-mediated cytotoxicity. Inhibition of proliferation but not of IL-2 production by alpha interferons.

We compared the effects of natural and recombinant (r) alpha (IFN-alpha) and gamma (IFN-gamma) interferons on the proliferative responses of human peripheral blood mononuclear cells to mitogens and allogeneic cells in mixed lymphocyte culture (MLC) and on the generation of specific T-cell-mediated cytotoxicity. In 14 of 19 donors, natural IFN-gamma and rIFN-gamma had no significant effect on the proliferative responses to mitogens or allogeneic cells in MLC, even at very high IFN-gamma concentrations (10,000 U/ml). In the remaining 5 donors, a statistically significant (p less than 0.001) enhancement by 49 +/- 8% of the proliferative responses was observed. In contrast, natural IFN-alpha and rIFN-alpha 2 significantly inhibited (p less than 0.001) proliferative responses to mitogens and to allogeneic cells, even at concentrations as low as 10 U/ml, in agreement with previous reports. Although natural and recombinant IFN-alpha significantly inhibited these proliferative responses, they did not affect interleukin-2 (IL-2) production in these cultures, suggesting that they inhibit proliferation by a mechanism that does not involve inhibition of IL-2 production. rIFN-gamma did not affect the generation of specific cytotoxicity in MLC, although it was significantly enhanced by natural IFN-alpha and rIFN-alpha 2. Additionally, we compared the ability of human rIFN-alpha subtypes to inhibit proliferative responses to allogeneic cells in MLC. rIFN-alpha 2, rIFN-alpha 4, and rIFN alpha 7 displayed the most potent inhibitory activity of allogeneic responses and were active at concentrations as low as 0.3-0.6 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor bearer T cells suppress Bacillus Calmette-Guérin-potentiated antitumor responses. III. Identification of an auxiliary efferent suppressor-T-cell population.

T cells (Ts-eff) induced in BALB/c mice by subcutaneous (sc) growth of syngeneic Meth A tumors can adoptively suppress the effector phase of delayed-type hypersensitivity (DTH) in Bacillus Calmette-Guérin (BCG)-primed and unprimed recipients which have been sensitized with irradiated Meth A cells but they do not inhibit the augmented DTH response in recipients inoculated with cyclophosphamide (CY) 2 days prior to sensitization. By reconstituting CY-treated immunized recipients with selected spleen cell populations, it has been demonstrated that Ts-eff suppress DTH by interacting with a second or auxiliary suppressor cell population present in immune but not normal spleens. These auxiliary suppressor cells (Ts-aux) are Thy+, Lyt 1-2+ and I-J+, phenotypically similar to Ts-eff. Their activity is not influenced by B-cell depletion. Unlike Ts-eff, Ts-aux do not bear receptors specific for Meth A cells. Ts-aux and Ts-eff share similar sensitivity to irradiation and high dose (100 mg/kg) CY but unlike Ts-eff, Ts-aux are cortisone sensitive, nondividing, nonadherent cells which are absent from the thymus. The phenotype and mechanism of action of Ts-aux resemble those of the auxiliary or Ts3 cells defined in models of contact sensitivity, DTH to simple haptens, and in vitro antibody responses.