Judith A. Newmark

Genetics and imaging to assess oocyte and preimplantation embryo health

Two major criteria are currently used in human assisted reproductive technologies (ART) to evaluate oocyte and preimplantation embryo health: (1) rate of preimplantation embryonic development; and (2) overall morphology. A major gene that regulates the rate of preimplantation development is the preimplantation embryo development (Ped) gene, discovered in our laboratory. In mice, presence of the Ped gene product, Qa-2 protein, results in a fast rate of preimplantation embryonic development, compared with a slow rate of preimplantation embryonic development for embryos that are lacking Qa-2 protein. Moreover, mice that express Qa-2 protein have an overall reproductive advantage that extends beyond the preimplantation period, including higher survival to birth, higher birthweight, and higher survival to weaning. Data are presented that suggest that Qa-2 increases the rate of development of early embryos by acting as a cell-signalling molecule and that phosphatidylinositol-32 kinase is involved in the cell-signalling pathway. The most likely human homologue of Qa-2 has recently been identified as human leukocyte antigen (HLA)-G. Data are presented which show that HLA-G, like Qa-2, is located in lipid rafts, implying that HLA-G also acts as a signalling molecule. In order to better evaluate the second criterion used in ART (i.e. overall morphology), a unique and innovative imaging microscope has been constructed, the Keck 3-D fusion microscope (Keck 3DFM). The Keck 3DFM combines five different microscopic modes into a single platform, allowing multi-modal imaging of the specimen. One of the modes, the quadrature tomographic microscope (QTM), creates digital images of non-stained transparent cells by measuring changes in the index of refraction. Quadrature tomographic microscope images of oocytes and preimplantation mouse embryos are presented for the first time. The digital information from the QTM images should allow the number of cells in a preimplantation embryo to be counted non-invasively. The Keck 3DFM is also being used to assess mitochondrial distribution in mouse oocytes and embryos by using the k-means clustering algorithm. Both the number of cells in preimplantation embryos and mitochondrial distribution are related to oocyte and embryo health. New imaging data obtained from the Keck 3DFM, combined with genetic and biochemical approaches, have the promise of being able to distinguish healthy from unhealthy oocytes and embryos in a non-invasive manner. The goal is to apply the information from our mouse model system to the clinic in order to identify one and only one healthy embryo for transfer back to the mother undergoing an ART procedure. This approach has the potential to increase the success rate of ART and to decrease the high, and undesirable, multiple birth rate presently associated with ART.

Soluble human leukocyte antigen-G and pregnancy success

Non-invasive methods of assessing embryo quality are critical for pregnancy success following IVF or intracytoplasmic sperm injection (ICSI). The addition of new non-invasive morphological and biochemical analyses may further improve pregnancy success, allowing the transfer of a single embryo, thereby reducing the risks involved in multiple births following IVF/ICSI. The presence of a protein, soluble human leukocyte antigen-G (sHLA-G), in embryo cultures has been suggested as a way to non-invasively predict embryo quality and pregnancy success, especially when used in conjunction with current embryo quality assessment methods. Detection of sHLA-G in embryo culture medium has been correlated with pregnancy success in 12 studies, but three studies were not able to detect sHLA-G. This is a review of the literature on sHLA-G detection in IVF/ICSI, and reasons are proposed for the reported discrepancies, as well as guidelines for reporting of data in future studies. Furthermore, it is suggested that the use of an HLA-G transgenic mouse model would advance understanding of the mechanism of action of sHLA-G in preimplantation embryos and its correlation to embryo health and viability. Research on a mouse model, combined with clinical studies, should enable the development of a fast and reliable method for utilizing sHLA-G detection to improve pregnancy success after IVF/ICSI.

Phase-subtraction cell-counting method for live mouse embryos beyond the eight-cell stage

Since 1978 in vitro fertilization (IVF) procedures have resulted in the birth of over 3 million babies. Yet in 2005, IVF procedures had a live birth rate of only 34%, with 32% of these births resulting in multiple pregnancies. These multiple pregnancies were directly attributed to the transfer of multiple embryos to increase the probability that a single, healthy embryo was included. The predominantly accepted noninvasive viability markers for embryos created by IVF are (1) number of cells at specific time points during development and (2) overall morphology of the embryo. Currently, it is difficult to count the number of cells beyond the eight-cell stage noninvasively. We report a nontoxic cell-counting method capable of counting cell numbers ranging from 8 to 26 in live mouse embryos. This method is derived from the fusion of differential interference contrast and optical quadrature microscopy and is verified by epifluorescence images of Hoechst-stained nuclei. The phase-subtraction cell-counting method is the first accurate, nontoxic technique to count cells through the morula stage in mouse embryos and may enhance the use of cell number as a viability marker if adopted for use with human embryos in the IVF clinic.

Expression of major histocompatibility complex class I proteins and their antigen processing chaperones in mouse embryonic stem cells from fertilized and parthenogenetic embryos

Embryonic stem (ES) cells are pluripotent cells with the potential to differentiate into cells or tissues that may be used for transplantation therapy. Parthenogenetic ES (pES) cells have been recently derived from both mouse and human oocytes and hold promise as a cell source that is histocompatible to the oocyte donor. Because of the importance of major histocompatibility complex (MHC) antigens in mediating tissue rejection or acceptance, we examined levels of mRNA and protein expression of MHC class I proteins, as well as several MHC class I antigen processing and presentation chaperones in mouse ES cells derived from both fertilized (fES) and parthenogenetic (pES) embryos. We found that H-2K, Qa-2, TAP1, TAP2, and tapasin mRNAs were all expressed at low levels in undifferentiated and differentiating ES cells and were significantly upregulated in response to interferon-gamma (IFN-gamma) treatment following 14 days of differentiation. Likewise, expression of H-2K(b) and H-2K(k) proteins were upregulated to detectable levels by IFN-gamma after 14 days of differentiation, but Qa-2 protein expression remained low or absent. We also found that MHC class I, TAP1, TAP2, and tapasin mRNAs were all expressed at very low levels in ES cells compared with T cells, suggesting transcriptional regulation of these genes in ES cells. Calnexin, a chaperone molecule involved in other pathways than MHC expression, had mRNA levels that were similar in ES cells and T cells and was not upregulated by IFN-gamma in ES cells. Overall, ES cells derived from fertilized embryos and parthenogenetic embryos displayed remarkably similar patterns of gene expression at the mRNA and protein levels. The similarity between the fES and pES cell lines with regard to expression of MHC class I and antigen-processing machinery provides evidence for the potential usefulness of pES cells in transplantation therapy.

Combining optical quadrature and differential interference contrast to facilitate embryonic cell counting with fluorescence imaging for confirmation

The Multifunctional Staring Mode Microscope was developed to permit three modes of imaging for cell counting in mouse embryos: Optical Quadrature, Differential Interference Contrast (DIC), and Fluorescence Imaging. The Optical Quadrature Microscope, consisting of a modified Mach-Zender Interferometer, uses a 632.8 nm laser to measure the amplitude and phase of the signal beam that travels through the embryo. Four cameras, preceded by multiple beamsplitters, are used to read the four interferograms, which are then combined to produce an image of the complex electric field amplitude. The phase of the complex amplitude is then unwrapped using a 2-D phase unwrap algorithm and images of optical path length are produced. To combine the additional modes of DIC and Fluorescence Imaging with the Optical Quadrature Microscope, a 632.8 nm narrow bandpass beamsplitter was placed at the output of the microscope. This allows the laser light to continue through the Mach-Zender while all other wavelengths are reflected at 90 degrees to another camera. This was effective in combining the three modes as the fluorescence wavelength for the Hoechst stain is well below the bandpass window of the beamsplitter. Both live and fixed samples have been successfully imaged in all three modes. Accuracy in cell counting was achieved by using the DIC image for detecting cell boundaries and the Optical Quadrature image for phase mapping to determine where cells overlap. The final results were verified by Hoechst fluorescence imaging to count the individual nuclei. Algorithms are currently being refined so larger cell counts can be done more efficiently.

Determination of the number of cells in preimplantation embryos by using noninvasive optical quadrature microscopy in conjunction with differential interference contrast microscopy.

The number of cells in a preimplantation embryo is directly correlated to the health and viability of the embryo. There are currently no methods to count the number of cells in late-stage preimplantation embryos noninvasively. We assessed the ability of optical quadrature microscopy (OQM) to count the number of cells in mouse preimplantation embryos noninvasively. First, to test for possible light toxicity, we exposed two-cell mouse embryos to OQM and differential interference contrast (DIC) microscopy and assessed their ability to develop to the blastocyst stage. We found no inhibition of development from either mode of microscopy for up to 2 h of light exposure. We also imaged eight-cell to morula stage mouse preimplantation embryos by OQM nd developed two methods for counting the number of cells. The contour signature method (CSM) used OQM images alone and the phase subtraction method (PSM) used both OQM and DIC images. We compared both methods to standard cell counting techniques and found that the PSM was superior to all other noninvasive cell counting methods. Our work on mouse embryos should be applicable to human embryos. The ability to correctly count the number of cells in human preimplantation embryos could lead to the transfer of fewer embryos in in vitro fertilization (IVF) clinics and consequently a lower rate of high-risk multiple-infant births.

Quantitative phase measurements using a quadrature tomographic microscope

The Quadrature Tomographic Microscope measures the amplitude and phase of an image. This information allows the user to see contrast features not available in other microscopes, and is critical to any three-dimensional reconstruction. We report on development and use of test objects to measure the accuracy and repeatability of phase measurements. A simple binary phase grating, a series of glass beads, and preimplantation mouse embryos were used in these experiments. The gratings were fabricated on high-quality fused-silica substrates whose transmission phase error was determined to be less than one-tenth wave error across their 25 mm diameter before fabrication. The phase step of the binary phase grating was measured using both the optical quadrature technique and the usual fringe-counting techniques applied to the raw data. Phase unwrapping techniques were validated by measuring the diameter of glass beads of a known size. Results are presented showing that the phase measurements agree with each other, with the known data, and with the spatial resolution in preimplantation mouse embryos. More complicated objects will be fabricated in the future to validate 3-D imaging techniques.

Analysis of the sex ratio in preimplantation embryos from B6.K1 and B6.K2 Ped gene congenic mice

Purpose: The mouse preimplantation embryo development (Ped) gene product, Qa-2, which is the homolog of human HLA-G, influences the rate of preimplantation embryonic development and overall reproductive success. The sex ratio in preimplantation embryos from Ped gene congenic mice was examined in order to determine whether embryo sex is a confounding factor in the control of the rate of preimplantation development.Methods: B6.K1 (Ped slow) and B6.K2 (Ped fast) congenic mice differ only in the absence (B6.K1) or presence (B6.K2) of the genes encoding Qa-2 protein. We analyzed the sex of B6.K1 (n=221) and B6.K2 (n=260) preimplantation embryos by using Real-Time PCR with primers specific for the X and Y chromosomes.Results: We found that there was no statistically significant difference in the ratio of male to female preimplantation embryos in either strain.Conclusions: We conclude that the sex of the embryos is not a confounding factor that affects the Ped gene control of the rate of preimplantation development. Therefore, the Ped gene is entirely responsible for mediating the faster development of B6.K2 embryos compared to B6.K1 embryos.

Accurate cell counts in live mouse embryos using optical quadrature and differential interference contrast microscopy

Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

The CenSSIS image database

The CenSSIS image database is a scientific database that enables effective data management and collaboration to accelerate fundamental research. This paper describes the design and use of a state-of-the-art relational image database management system, accessible through a standard Web-browser interface. The application utilizes a robust security architecture and is designed for efficient data submission. Our database query engine provides complex query capabilities to facilitate fast and efficient data retrieval. The system offers a highly extensible metadata schema, with the option of storing data within a hierarchical format.