Culture and vitrification of human preembryos

Despite improvements in stimulation protocols, culture media formulations and laboratory protocols, the success rates in human IVF remain disappointingly low. The ability to successfully cryopreserve supernumerary embryos in a given IVF cycle without losing significant embryo viability is essential to maximize the cumulative benefit of a given treatment cycle. Therefore, studies on culture, cryopreservation and gene expression of human embryos fertilized in vitro were performed. In these studies the impact of culture media on fertilization of human oocytes in vitro was investigated. Furthermore, the impact of growth factor supplementation to in vitro culture media and embryo survival and cryodamage after vitrification were studied. Using in situ hybridization and immunohistochemistry methods, the expression of genes in the human Fallopian tube, endometrium, and pre-implantation embryos and in human embryonic stem cells (hES) cells was studied. The findings can be summarized as follows: in vitro culture media has impact on normal fertilization. Supplementation of growth factors to in vitro culture media implicates a physiological role in regulating pre-implantation development. Vitrification of embryos is an effective way of cryopreservation. In situ hybridization, immunohistochemical and matrix assisted laser desorption/ionization time of flight mass spectrometry methods are versatile tools in reproductive medicine research. These findings will help to identify markers for embryo development and characterisation of hESC. Furthermore, knowledge obtained will give us tools to improve formulations of culture and cryopreservation media, which in turn might increase the overall results in IVF treatment and maximise the usage of hESC

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

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 understandin

Glycoprotein 130 promotes human blastocyst development in vitro

Objective: To investigate the efficacy of leukemia inhibitory factor (LIF) and/or glycoprotein 130 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 Intervention(s): Morphological development was evaluated by light microscopy. Protein expression 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 Conclusion(s): Glycoprotein 130, but not LIF, seems to be beneficial for preimplantation embry

RT-PCR detection of NLRPs in human oocytes and embryos showed two expression patterns.

<p>A: <i>NLRP4, 5, 8, 9, 11,12, 13 and 14</i> were highly expressed in oocytes and then gradually decreased in embryos with a very low level in day5 (D5) embryos. B: <i>NLRP2</i> and <i>NLRP</i>7 progressively decreased from oocytes to day 3(D3) embryos, then showed a sharp increase in D5. Error bars = SEM (standard error of the mean).</p

Detection of of <i>NLRPs</i> in human gametes and embryos by PCR.

<p>PCR products were about 70 bp in GV, MI, MII oocytes, day2 (D2), day 3(D3), day 5(D5) embryos and spermatozoa (S). M: marker. <i>NLRP1</i> was detected in GV, MI, MII oocytes and D2 embryos, but not in later stages. <i>NLRP2, 4, 7, 11, 12 and 14</i> were detected in all samples. <i>NLRP3</i> was only detected in GV, MI oocytes and spermatozoa. <i>NLRP5, 8, 9, 13</i> were detected in all oocytes and embryos, but not in spermatozoa. <i>NLPR6</i> was only detected in spermatozoa and <i>NLRP10</i> was not detected in any gametes or embryos (figure not shown).</p

Microarray detection of <i>NLRPs</i> in human oocytes and embryos showed two different expression patterns.

<p>A: <i>NLRP4, 5, 8, 9, 11,12, 13 and14</i> were highly expressed in oocytes and then gradually decreased in embryos with a very low level in day5 (D5) embryos. B: <i>NLRP2</i> and <i>NLRP</i>7 progressively decreased from oocytes to day 3(D3) embryos then showed a sharp increase in D5. Error bars = SEM (standard error of the mean).</p

Expression of NLRP5, 9, 12, 7 in day 3 (D3) and day5 (D5) abnormal embryos.

<p>D3 stop dev. =  Embryos stop develop at D3; D5 stop dev. =  Embryos stop develop at D5. A: the expression levels of <i>NLRP5, 9, 12 and 7</i> in development arrested D3 embryos were higher than in normal D3 embryos. B: the expression levels of <i>NLRP5, 9 and 12</i> in development arrested D5 embryos were much higher than in normal D5 embryos, but the expression level of <i>NLRP 7</i> in development arrested D5 embryos was much lower than in normal ones. Error bars = SEM (standard error of the mean).</p

Primers for NLRPs used in this study

<p>Primers for NLRPs used in this study</p