Glycoprotein 130 promotes human blastocyst development in vitro.

OBJECTIVE: To investigate the efficacy of leukemia inhibitory factor (LIF) and/or glycoprotein 130 (gp130) on in vitro growth of human embryos. DESIGN: Laboratory study. SETTING: University hospital-based IVF clinic. PATIENT(S): A total of 164 frozen embryos that survived thawing were cultured in media supplemented with LIF and/or gp130 or control media. INTERVENTION(S): Morphological development was evaluated by light microscopy. Protein expression profiles of single blastocysts were evaluated using matrix-assisted laser desorption/ionization time of flight-based intact cell mass spectrometry. MAIN OUTCOME MEASURE(S): Embryo development and protein content. RESULT(S): Addition of gp130 to culture media improved blastocyst formation (73% vs. 43%). Addition of LIF to the culture media did not improve embryo development. Protein fingerprint spectra were obtained that revealed significant intensity changes for multiple molecular species including thymosin beta-10, thymosin beta-4, histone H2A, histone H2B, histone H4, ubiquitin, ubiquitin-T, and acyl-CoA binding protein. CONCLUSION(S): Glycoprotein 130, but not LIF, seems to be beneficial for preimplantation embryo development, implicating a physiological role in regulating preimplantation development in humans and thus ought to be included in culture media designed for embryo culture to the blastocyst stage. Furthermore, these findings highlight the great potential of matrix-assisted laser desorption/ionization time of flight mass spectrometry and intact cell mass spectrometry as a versatile tool in reproductive medicine research.

Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells.

PURPOSE: NANOG and OCT4 are required for the maintenance of pluripotency in embryonic stem cells (ESCs). These proteins are also expressed in the inner cell mass (ICM) of the mouse pre-implantation embryo. METHODS: Immunohistochemistry was used to show the presence of NANOG and OCT4 protein, and in situ hybridization was used to localize NANOG mRNA in human embryos from two-cell to blastocyst stage, and in human ESCs (hESCs). RESULTS: Nanog and Oct4 were co-localized in human embryos from morula and blastocyst stages. NANOG mRNA was detected in a group of cells in the morula, in cells of the ICM of blastocysts, and evenly in hESCs. All non-differentiated hESCs expressed NANOG and OCT4 protein. Pluripotent cells expressing NANOG and Oct4 were eccentrically localized, probably in polarized cells in a human compacted morula, which appears to be different from expression in murine embryos. CONCLUSION: In this study, we demonstrate that whole mount in situ hybridization is amenable to localization of mRNAs in human development, as in other species.

A prospective randomized sibling-oocyte study of two media systems for culturing cleavage-stage embryos-impact on fertilization rate.

PURPOSE: Although several media systems have been developed, data from prospective randomised clinical studies are still lacking. In the present study we compared the effects of 2 different media systems on embryo morphology and development at days 2/3 using sibling oocytes. METHODS: In this prospective sibling-split trial, 1206 oocytes from 110 women were divided via alternate allocation to fertilization and culture in media system A (G-IVF (TM) v5 PLUS/ G-1(TM) v5 PLUS) or for fertilization and culture in media system B (Universal IVF medium/EmbryoAssist (TM)). RESULTS: The use of media system A significantly increased the normal fertilization rate (73.5% versus 67.2%; p = 0.030) and embryo utilization rate (55.5% versus 42.9%; p = 0.001), whereas polyploidy and embryo quality were similar in the two groups. CONCLUSION: The different impacts on fertilization and early embryo development between the two commercially available and commonly used media systems show the importance of evaluation of the efficacy of existing sequential culture media and the need to further improve media for in vitro development of human embryos.

The presence of histidine-rich glycoprotein in the female reproductive tract and in embryos.

A well-regulated angiogenesis is crucial for proper embryo implantation, embryogenesis, and pregnancy development. Monitoring the presence and distribution of angiogenic regulators in the female reproductive tract and in the early embryo is important for a broader understanding of the molecular aspects of fertility, embryogenesis, and pregnancy. Histidine-rich glycoprotein (HRG) is a glycoprotein involved in angiogenesis. Its presence in the female reproductive tract or in embryos has not previously been studied. Follicular fluid, culture medium, and embryos were obtained from patients undergoing in vitro fertilization (IVF). Biopsies from inner genitalia and placenta were collected at surgery. Histidine-rich glycoprotein presence was investigated by immunohistochemistry and Western blot. Polymerase chain reaction (PCR) was used to determine HRG expression in tissues or by embryos. We identified HRG in follicular fluid, the female reproductive tract, and placenta, as well as in the embryos. Moreover, HRG expression was observed in blastocysts. Thus, the angiogenic properties of HRG might affect fertility.

Hyaluronan-enriched transfer medium in cleavage-stage frozen-thawed embryo transfers increases implantation rate without improvement of delivery rate.

OBJECTIVE: To investigate the efficacy of hyaluronan-enriched transfer media in cleavage-stage frozen embryo transfer cycles. DESIGN: Two commercially available transfer media were prospectively compared in an observational study. SETTING: Hospital-based in vitro fertilization clinic. PATIENT(S): Patients (n = 425) undergoing frozen-thawed embryo transfer (FET). The embryos transferred were included in either a study group (high hyaluronic acid [HA], n = 199) or a control group (low HA, n = 226). INTERVENTION(S): Delivery rate per FET; positive hCG rate, biochemical pregnancy rate, clinical pregnancy rate, implantation rate, and clinical abortion rate were secondary outcomes. RESULT(S): The use of HA in the transfer media significantly increased the positive hCG rate (37.2% vs. 25.2%) and implantation rate (23.1% vs. 15.8%) without increasing the delivery rate (21.6% vs. 21.2%). More subjects in the study group with a positive hCG test experienced biochemical pregnancy (28.4% vs. 8.9%). CONCLUSION(S): Addition of HA to transfer media seems to favor attachment of early embryos in FETs without increasing the delivery rate.

Transcriptome profiling of human pre-implantation development.

BACKGROUND: Preimplantation development is a crucial step in early human development. However, the molecular basis of human preimplantation development is not well known. METHODOLOGY: By applying microarray on 397 human oocytes and embryos at six developmental stages, we studied the transcription dynamics during human preimplantation development. PRINCIPAL FINDINGS: We found that the preimplantation development consisted of two main transitions: from metaphase-II oocyte to 4-cell embryo where mainly the maternal genes were expressed, and from 8-cell embryo to blastocyst with down-regulation of the maternal genes and up-regulation of embryonic genes. Human preimplantation development proved relatively autonomous. Genes predominantly expressed in oocytes and embryos are well conserved during evolution. SIGNIFICANCE: Our database and findings provide fundamental resources for understanding

Expression analysis of the NLRP gene family suggests a role in human preimplantation development.

BACKGROUND: The NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing) family, also referred to as NALP family, is well known for its roles in apoptosis and inflammation. Several NLRPs have been indicated as being involved in reproduction as well. METHODOLOGY: We studied, using the unique human gametes and embryo materials, the expression of the NLRP family in human gametes and preimplantation embryos at different developmental stages, and compared the expression levels between normal and abnormal embryos using real-time PCR. PRINCIPAL FINDINGS: Among 14 members of the NLRP family, twelve were detected in human oocytes and preimplantation embryos, whereas seven were detected in spermatozoa. Eight NLRPs (NLRP4, 5, 8, 9, 11, 12, 13, and 14) showed a similar expression pattern: their expression levels were high in oocytes and then decreased progressively in embryos, resulting in a very low level in day 5 embryos. However, NLRP2 and NLRP7 showed a different expression pattern: their expression decreased from oocytes to the lowest level by day 3, but increased again by day 5. The expression levels of NLRP5, 9, and 12 were lower in day 1 abnormal embryos but higher in day3 and day5 arrested embryos, when compared with normal embryos at the same stages. NLRP7 was down-regulated in day 1 and day 5 abnormal embryos but over-expressed in day3 arrested embryos. CONCLUSIONS: According to our results, different NLRPs possibly work in a stage-dependent manner during human preimplantation development.