Search

Oocyte quality is a key factor influencing IVF success. The oocyte and surrounding cumulus cells, known collectively as the cumulus oocyte complex (COC), communicate bi-directionally and regulate each other's metabolic function to support oocyte growth and maturation. Many studies have attempted to associate metabolic markers with oocyte quality, including metabolites in follicular fluid or 'spent medium' following maturation, gene expression of cumulus cells and measuring oxygen consumption in medium surrounding COCs. However, these methods fail to provide spatial metabolic information on the separate oocyte and cumulus cell compartments. Optical imaging of the autofluorescent cofactors – reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] and flavin adenine dinucleotide (FAD) – has been put forward as an approach to generate spatially resolved measurements of metabolism within individual cells of the COC. The optical redox ratio (FAD/[NAD(P)H + FAD]), calculated from these cofactors, can act as an indicator of overall metabolic activity in the oocyte and cumulus cell compartments. Confocal microscopy, fluorescence lifetime imaging microscopy (FLIM) and hyperspectral microscopy may be used for this purpose. This review provides an overview of current optical imaging techniques that capture the inner biochemistry within cells of the COC and discusses the potential for such imaging to assess oocyte developmental competence. Infertility is often treated using in vitro fertilisation (IVF). However, patients take home a baby in only ∼30% of initiated cycles. This success rate may be improved by selecting the egg (oocyte) with the highest chance of resulting in a baby. A non-invasive photo of the oocyte and its companion cells can provide information on the inner biochemistry of these cells. Such a photo may provide insight into the health of individual oocytes and lead to improved IVF success. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.10.012 Byline: Hannah M. Brown (a), Kylie R. Dunning (a), Rebecca L. Robker (a), Melanie Pritchard (b), Darryl L. Russell (a) Keywords: Folliculogenesis; Extracellular matrix remodeling; Infertility; Lymphangiogenesis; Protease Abstract: Murine ovarian folliculogenesis commences after birth involving oocyte growth, somatic cell differentiation and structural remodeling of follicle stromal boundaries. The extracellular metalloproteinase ADAMTS-1 has activity against proteoglycans and collagen and is produced by the granulosa cells of ovarian follicles. Mice with ADAMTS-1 gene disruption are subfertile due to an unknown mechanism resulting in severely reduced ovulation. Here we show that ADAMTS-1 is necessary for structural remodeling during ovarian follicle growth. A significant reduction in the number of healthy growing follicles and corresponding follicle dysmorphogenesis commencing at the stage of antrum formation was identified in ADAMTS-1.sup.-/- ovaries. Morphological analysis and immunostaining of basement membrane components identified stages of follicle dysgenesis from focal disruption in ECM integrity to complete loss of follicular structures. Cells expressing the thecal marker Cyp-17 were lost from dysgenic regions, while oocytes and dispersed cells expressing the granulosa cell marker anti-mullerian hormone persisted in ovarian stroma. Furthermore, we found that the ovarian lymphatic system develops coincidentally with follicular development in early postnatal life but is severely delayed in ADAMTS-1.sup.-/- ovaries. These novel roles for ADAMTS-1 in structural maintenance of follicular basement membranes and lymphangiogenesis provide new mechanistic understanding of folliculogenesis, fertility and disease. Author Affiliation: (a) School of Paediatrics and Reproductive Health, Research Centre for Reproductive Health, The University of Adelaide, Adelaide, South Australia, 5005, Australia (b) Centre for Functional Genomics and Human Disease, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia Article History: Received 21 March 2006; Revised 11 October 2006; Accepted 11 October 2006

A new fiber functionalization architecture for single‐fiber imaging and sensing is presented. 5(6)Carboxy‐seminaphthorhodafluor‐2 (a fluorescent pH sensor) is attached to a silk‐binding peptide and the complex added to aqueous silk fibroin protein. These bind with a Kd of 36 µM as determined by a fluorescence polarization assay. The fiber is dip‐coated into the silk and peptide mixture, and scanning electron microscopy images reveal a uniform silk coating on the fiber tip. The coating is stable to repeated washes and does not affect the imaging light emitted from the fiber, which allows concurrent optical coherence tomography (OCT) imaging and pH sensing. Oocytes are metabolically stimulated with CoCl2 to produce lactic acid, and a pH reduction of 0.04 is measured using the probe. The distance between fiber tip and oocyte is monitored by simultaneous OCT acquisitions to precisely position the probe. Lastly, OCT imaging of an ovary revealed the presence/absence of an oocyte within a follicle, an important step toward improving patient outcomes during in vitro fertilization, by limiting the number of invasive follicle punctures required. These results demonstrate the utility of this new coating to enable simultaneous OCT imaging and sensing, which provides significant insight into complex biological systems. [ABSTRACT FROM AUTHOR]

Study Question: Can label-free, non-invasive optical imaging by hyperspectral autofluorescence microscopy discern between euploid and aneuploid cells within the inner cell mass (ICM) of the mouse preimplantation embryo?Summary Answer: Hyperspectral autofluorescence microscopy enables discrimination between euploid and aneuploid ICM in mouse embryos.What Is Known Already: Euploid/aneuploid mosaicism affects up to 17.3% of human blastocyst embryos with trophectoderm biopsy or spent media currently utilized to diagnose aneuploidy and mosaicism in clinical in vitro fertilization. Based on their design, these approaches will fail to diagnose the presence or proportion of aneuploid cells within the foetal lineage ICM of some blastocyst embryos.Study Design, Size, Duration: The impact of aneuploidy on cellular autofluorescence and metabolism of primary human fibroblast cells and mouse embryos was assessed using a fluorescence microscope adapted for imaging with multiple spectral channels (hyperspectral imaging). Primary human fibroblast cells with known ploidy were subjected to hyperspectral imaging to record native cell fluorescence (4-6 independent replicates, euploid n = 467; aneuploid n = 969). For mouse embryos, blastomeres from the eight-cell stage (five independent replicates: control n = 39; reversine n = 44) and chimeric blastocysts (eight independent replicates: control n = 34; reversine n = 34; 1:1 (control:reversine) n = 30 and 1:3 (control:reversine) n = 37) were utilized for hyperspectral imaging. The ICM from control and reversine-treated embryos were mechanically dissected and their karyotype confirmed by whole genome sequencing (n = 13 euploid and n = 9 aneuploid).Participants/materials, Setting, Methods: Two models were employed: (i) primary human fibroblasts with known karyotype and (ii) a mouse model of embryo aneuploidy where mouse embryos were treated with reversine, a reversible spindle assembly checkpoint inhibitor, during the four- to eight-cell division. Individual blastomeres were dissociated from control and reversine-treated eight-cell embryos and either imaged directly or used to generate chimeric blastocysts with differing ratios of control:reversine-treated cells. Individual blastomeres and embryos were interrogated by hyperspectral imaging. Changes in cellular metabolism were determined by quantification of metabolic co-factors (inferred from their autofluorescence signature): NAD(P)H and flavins with the subsequent calculation of the optical redox ratio (ORR: flavins/[NAD(P)H + flavins]). Autofluorescence signals obtained from hyperspectral imaging were examined mathematically to extract features from each cell/blastomere/ICM. This was used to discriminate between different cell populations.Main Results and the Role Of Chance: An increase in the relative abundance of NAD(P)H and decrease in flavins led to a significant reduction in the ORR for aneuploid cells in primary human fibroblasts and reversine-treated mouse blastomeres (P < 0.05). Mathematical analysis of endogenous cell autofluorescence achieved separation between (i) euploid and aneuploid primary human fibroblast cells, (ii) control and reversine-treated mouse blastomeres cells, (iii) control and reversine-treated chimeric blastocysts, (iv) 1:1 and 1:3 chimeric blastocysts and (v) confirmed euploid and aneuploid ICM from mouse blastocysts. The accuracy of these separations was supported by receiver operating characteristic curves with areas under the curve of 0.97, 0.99, 0.87, 0.88 and 0.93, respectively. We believe that the role of chance is low as mathematical features separated euploid from aneuploid in both human fibroblasts and ICM of mouse blastocysts.Large Scale Data: N/A.Limitations, Reasons For Caution: Although we were able to discriminate between euploid and aneuploid ICM in mouse blastocysts, confirmation of this approach in human embryos is required. While we show this approach is safe in mouse, further validation is required in large animal species prior to implementation in a clinical setting.Wider Implications Of the Findings: We have developed an original, accurate and non-invasive optical approach to assess aneuploidy within the ICM of mouse embryos in the absence of fluorescent tags. Hyperspectral autofluorescence imaging was able to discriminate between euploid and aneuploid human fibroblast and mouse blastocysts (ICM). This approach may potentially lead to a new diagnostic for embryo analysis.Study Funding/competing Interest(s): K.R.D. is supported by a Mid-Career Fellowship from the Hospital Research Foundation (C-MCF-58-2019). This study was funded by the Australian Research Council Centre of Excellence for Nanoscale Biophotonics (CE140100003) and the National Health and Medical Research Council (APP2003786). The authors declare that there is no conflict of interest. [ABSTRACT FROM AUTHOR]