Galia Oron
Introduction
Infertility affects approximately 15% of couples of reproductive age; assisted reproduction technology (ARTs) may help to overcome infertility, resulting in pregnancy and preferably a live birth (1). While the effectiveness and efficiency of ARTs have greatly increased, success rates are far from maximal (2,3). Data from the 2015 Assisted Reproductive Technology National Summary Report, Centers for Disease Control and Prevention, illustrate the current state of ARTs in the United States. In 2015, nationwide, 91,090 ART cycles were initiated using fresh, nondonor eggs or embryos; 80,644 initiated cycles (88.5%) proceeded to the egg retrieval stage; and 59,336 initiated cycles (65%) proceeded to the embryo transfer stage. This indicates that egg retrieval and fertilization steps are successful in most but not all cycles. Unfortunately, only 26,708 (29%) initiated cycles resulted in pregnancy and 21,771 (24%) in live birth deliveries (4).
Clearly there is room for improvement, given the significant emotional stress cost and disruptive nature of each treatment cycle. Efforts to determine the likelihood to succeed rely on key components of ART, including endometrial thickness, best related to receptivity, and manual embryo grading to select the most competent embryo(s) for transfer. Detailed morphologic assessment is routinely used to select embryos with the highest likelihood of implantation, based on cell number, cell symmetry, fragmentation, and other physical components (5–7). Nevertheless, these strict morphological parameters remain subject to various inter- and intraobserver variability. Predictive computerized algorithms installed in time-lapse monitoring systems (TMSs) provide a more objective scoring of embryos, and assist embryologists in selection via continual assessment of morphological changes, by documenting timing of events and length of intervals during embryo development (8–11). However, TMS has not yet proven to result in improved pregnancy and live birth rates (12). The field of ART would benefit from new quantitative diagnostic and therapeutic tools to further improve implantation, pregnancy, and live birth rates (13).
The emergence of new omics technologies such as epigenomics, genomics, transcriptomics, and proteomics have enabled research into new molecular methods for embryo selection, leading to the definition of optimal molecular traits of germ cells, embryos, and endometrium. Taking a broader view of complex biological systems may further expand the complex context of genotype-phenotype interactions in order to determine optimal molecular traits of cells and tissues involved in reproduction (14). This chapter is organized to provide a brief background on the applications of omics technologies to female reproductive target cells and tissues. The applications of omics technologies on spermatozoa and seminal plasma research affecting male infertility are beyond the scope of this chapter.
Oocyte
Oocyte quality is one of the main factors associated with treatment success in ART, and understanding the steps in oocyte development and maturation are crucial for successful treatment (15). Numerous studies focus on criteria for choosing high-quality oocytes, which lead to higher rates of fertilization, early embryo development, and healthy offspring (16,17). Cumulus cells play an important role in oocyte maturation; they communicate with each other and with the oocyte through gap junctions sharing a bidirectional molecular interaction, allowing for metabolic exchange and excreted biomarkers that can reflect oocyte competence levels (18). Omics technologies can provide more information regarding cumulus cell function, oocytes’ quality, and their interactions (19).
Transcriptomics
Morphological assessment of the zona pellucida (ZP) is the gold standard method in oocyte selection in most ART clinics, but this approach is limited by precision boundaries. Studies have shown that differentially expressed candidate genes were respectively overexpressed and underexpressed in cumulus/granulosa cells from oocytes that led to a successful pregnancy, versus oocytes that did not (20,21). ZP properties’ variation is associated with differences in cumulus/granulosa cell gene expression, but ZP morphology is associated with a transcriptomic gene pattern that is not directly related to known gene biomarkers of oocyte development. Further studies using larger lists of candidate markers are required to identify suitable genes highly correlated with ZP morphological criteria, in order to reinforce the accuracy of oocyte selection and potentially increase ART success rates (19).
Metabolomics
The follicular fluid (FF) is the in vivo microenvironment in which the oocyte develops, containing metabolites excreted by the oocytes and granulosa cells that are essential for follicular growth and oocyte maturation. The metabolic profile of the oocyte’s FF is theoretically supposed to reflect the oocyte quality and correlate with the reproductive potential of the embryo. Studies are still controversial regarding spectroscopic analysis of metabolites to predict implantation, due to numerous limitations including mixture of FF with flushing medium containing its own metabolites, contamination of FF from the previously aspirated follicle, and the need for single embryo transfer in order to correlate the metabolic analysis of the FF with the oocyte that results in embryo implantation (22–24).
Embryo
Genomics
Preimplantation genetic screening (PGS) is used to identify embryos with chromosomal aberrations by removing 1–2 blastomeres from an eight-cell embryo, or a number of trophectoderm cells from a blastocyst-stage embryo. In contrast to preimplantation genetic diagnosis (PGD), PGS patients do not present an inherited genetic disorder, and PGS is most commonly applied in cases of advanced maternal age, repeated implantation failure, recurrent abortions, and severe male factor infertility.
In recent years, novel array-based technology have been introduced in PGS, including comparative genetic hybridization (CGH), single nucleotide polymorphism (SNP) arrays (25), and next-generation sequencing (NGS) (26,27). In CGH, the embryonic and control DNA samples are amplified separately using a whole genome amplification approach fluorescently labeled (embryonic DNA in green and control DNA in red), then mixed to compete for hybridization of complementary sequences. A computer calculates the ratio of green-to-red fluorescence, with gains in red indicating deficient embryonic DNA, and gains in green indicating an extra copy of that region in embryonic DNA. CGH-based technology cannot detect polyploidies such as triploidies, balanced translocations or inversions, point mutations intragenic insertions or deletions, or triplet repeats and is mostly used to diagnose chromosomal aneuploidies and imbalanced translocations.
The SNP can be used to distinguish one chromosome from another in the same individual, and one person from another since SNPs are regions of the genome in which a single nucleotide in the sequence varies within the population. SNP arrays assess how many copies of each chromosome were inherited by an embryo. Both CGH and SNP arrays are readily used to select healthy euploid embryos in an attempt to improve in vitro fertilization (IVF) outcome (28–30). There was great hope that routine implementation of PGS will increase implantation rates, increase live birth rates, and reduce miscarriage rates; however, this is still not supported by the current literature (31). One explanation is that trophectoderm mosaicism leads to discarding embryos with the potential for a normal pregnancy had they been transferred (32). CGH, SNP arrays, and quantitative polymerase chain reaction (qPCR) lack the capacity to detect mosaicism in a single trophectoderm biopsy. The introduction of next-generation sequencing (NGS) allows assessment of embryo mosaicism, and it remains to be seen if this will change the reproductive performance of ART (26,27).
Studies confirming that amplifiable DNA present in blastocoele fluid samples harvested during the vitrification process opens up the future possibility of a promising alternative source of DNA for genetic testing of the embryo, without actual removal of cells from the developing embryo (33–35).
Metabolomics
The endometabolome corresponding to metabolites present inside cells/blastomere requires an invasive procedure. However, the exometabolome or secretome evaluates metabolites from the extracellular environment, such as follicular fluid or oocyte/embryo culture medium. D’Alessandro et al. used mass spectrometry in an attempt to characterize the metabolomic profile of blastocoele fluid withdrawn from a blastocyst cavity prior to cryopreservation, with the purpose of providing metabolite information to support estimations of implantation (36).
Culture medium
Genomics
Shamonki et al. were the first to show the presence of free embryonic DNA in extended culture medium media, collected from embryos grown from day 3 cleavage stage to day 5/6 blastocyst (37). While the prospect of genetic screening of an embryo without invasive removal of cells is appealing as it eliminates any risk of damage to the embryo, there is poor concordance between free-cell DNA in culture medium and trophectoderm biopsy, due to a high level of maternal DNA contamination as well as trophectoderm mosaicism (38–40).
Transcriptomics
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