Abstract
Assisted reproductive technologies have been used to achieve pregnancies since the first successful test tube baby was born in 1978. Infertile couples are at an increased risk for multiple miscarriages and the application of current protocols are associated with high first-trimester miscarriage rates. Among the contributing factors of these higher rates is a high incidence of fetal aneuploidy. Numerous studies support that protocols including ovulation-induction, sperm cryostorage, density-gradient centrifugation, and embryo culture can induce genome instability, but the general mechanism is less clear. Application of the genome theory and 4D-Genomics recently led to the establishment of a new paradigm for sexual reproduction; sex primarily constrains genome integrity that defines the biological system rather than just providing genetic diversity at the gene level. We therefore propose that application of assisted reproductive technologies can bypass this sexual reproduction filter as well as potentially induce additional system instability. We have previously demonstrated that a single-cell resolution genomic approach, such as spectral karyotyping to trace stochastic genome level alterations, is effective for pre- and post-natal analysis. We propose that monitoring overall genome alteration at the karyotype level alongside the application of assisted reproductive technologies will improve the efficacy of the techniques while limiting stress-induced genome instability. The development of more single-cell based cytogenomic technologies are needed in order to better understand the system dynamics associated with infertility and the potential impact that assisted reproductive technologies have on genome instability. Importantly, this approach will be useful in studying the potential for diseases to arise as a result of bypassing the filter of sexual reproduction.
The concept of 4D-Genomics
Genes and genomes represent different levels of genetic organization with distinct genetic coding systems [Heng Citation2009; Heng et al. Citation2009; Heng et al. Citation2011a]. According to the traditional gene theory, the DNA sequence codes for all the genetic information necessary for the life of an organism. Information transfers from DNA to RNA to proteins, and this exchange lies in the foundation of modern biology. However, under the genome theory, the information regarding assembly of parts is most likely not stored within the individual gene or genetic locus. DNA only encodes for the parts and some tools of the system (RNAs, proteins, regulatory elements). The complete interactive genetic network is coded by genome topology-mediated self-organization [Heng Citation2009; Citation2010; Heng et al. Citation2010; Heng et al. Citation2011a; Ye et al. Citation2007]. Under genome theory, the genome is not merely the entire DNA sequence or the vehicle of all genes. Rather, the genome context or landscape (the genomic topologic relationship among genes and other sequences within three-dimensional nuclei) defines the genetic system and ensures system inheritance [Heng Citation2009; 2013].
Altered genomes yield altered genetic networks [Heng Citation2009; Heng et al. Citation2011a], and understanding the pattern of genome dynamics provides key information of how the entire genomic system works. The concept of 4D-Genomics was formed based on the genomic reality that genetic information is preserved by the three-dimensional topology of the genome through time [Heng 2013]. This new concept calls for a departure from the less informative 1D gene-defined traditional genetics and recognizes that the genomic topology represents the framework for ‘system inheritance,’ which is distinctly different from ‘part inheritance’ (e.g., genetic information encoded in individual genes).
Main function of sexual reproduction
Application of genome theory and 4D-Genomics recently led to the establishment of a new paradigm for sexual reproduction [Gorelick and Carpinone Citation2009; Gorelick and Heng Citation2011; Heng Citation2007b; Wilkins and Holliday Citation2009]. In contrast to the century-old reasoning that the function of sexual reproduction increases genetic variation, under the new paradigm sexual reproduction mainly acts to reduce genome alterations despite its secondary function of mixing genes. This new thinking is supported by the restoration of genetic, genomic, and epigenetic status that occurs during sexual reproduction (in this context, we consider sexual reproduction as meiosis plus syngamy). In addition, some DNA damage is repaired, and many mutations are avoided. Chromatin marks like cytosine methylation are reset to levels that worked for both parents. This small scale proofreading has significance. Most importantly, however, large chromosomal abnormalities such as number changes and structural rearrangements are purged. Through this mechanism, the karyotype is preserved and so is the identity of a given species. Under normal circumstances, a given species will be maintained despite changing gene mutations and environments over time, as the framework of the genome is the same for a species. Therefore, sexual reproduction actually serves as a conservation force for biology. In other words, sexual reproduction-mediated genome constraint provides overall system stability and filters out variation, particularly large variation, to preserve a genome-mediated system. However and very rarely, when there are different offspring that have the same or similar altered genomes, they can mate. If sexual reproduction is successful and the altered genome favorably persists, a new species can be formed.
Such analyses not only addressed the issue of the costs of meiosis and sexual reproduction, a longstanding topic of debate [Gorelick and Heng Citation2011; Heng Citation2007b; Treisman and Dawkins Citation1976; Uyenoyama Citation1984; Williams Citation1992], but also shed new light on evolutionary theory. Interestingly, the correlation between infertility and elevated chromosomal abnormality has contributed to this important realization.
During fertilization for example, the competition among sperm and tightly regulated sperm-egg interaction both serve to eliminate or reduce genomic diversity. This is supported by a previous study that focused on whether zona pellucida-bound spermatozoa (characterized as having normal morphology, good motility, and the ability to tightly bind to the zona pellucida) exhibited lower aneuploidy rates than spermatozoa selected based only on good motility [Van Dyk et al. Citation2000]. Capturing of zona-bound spermatozoa was performed using the hemizona assay [Oehninger et al. Citation1991], and fluorescence in-situ hybridization (FISH) analysis was performed to determine aneuploidy incidence. There was a reduced frequency of spermatozoa with chromosome aneuploidy and diploidy bound to hemizona compared to samples selected based on motility only. The importance of the physiological sperm-egg interaction can also be illustrated by the high rate of aneuploidy detected in miscarriages following in vitro fertilization (IVF) (41%) and intracytoplasmic sperm injection (ICSI) (76%) [Lathi and Milki Citation2004]. It is also known that there is elevated chromosomal abnormality in sperm from infertile patients [Calogero et al. Citation2001].
Early in development a drastically altered genome would generally be aborted, and indeed the majority of spontaneously aborted early embryos display chromosomal abnormalities. These spontaneous abortions serve as another means of reducing genomic diversity. Approximately 50% of early (<15 weeks) losses are chromosomally abnormal, whereas 20% of later (15-24 weeks) losses display cytogenetic aberrations [Warburton et al. 1986]. However, with the utilization of more sensitive visualization methods such as FISH and spectral karyotyping (SKY), we anticipate that an even greater proportion of these miscarriages will be identified as carrying genome aberrations.
To summarize, many altered germ line cells will not become functional sperm or eggs, particularly when chromosomal abnormalities are involved. In contrast, point mutations can often survive the meiotic process. Altered sperm and eggs will generally not be fertilized. Even if fertilization occurs, maternally and paternally transmitted chromosomal aberrations can lead to pregnancy loss, developmental defects, infant mortality, infertility, and genetic diseases in offspring [Marchetti and Wyrobek Citation2005]. These events will further preclude the transmission of an altered genome, reducing the overall probability of genome diversity or its ability to evolve. Therefore, sexual reproduction provides multiple barriers that maintain order at the genome or chromosomal level.
Implications of the main function of sex – cautions to consider for assisted reproductive technologies
Approximately 10-15% of all recognized pregnancies in the general population result in first-trimester, spontaneous loss [Bettio et al. Citation2008]. Infertile couples are at an increased risk of repeated miscarriage, and miscarriage rates from pregnancies achieved after assisted reproductive technologies (ART) are reported within the range of 36-69% [Nayak et al. Citation2011]. Contributing factors of these higher rates of miscarriage include endometrial dysfunction, advanced maternal age, embryo culture conditions, the usage of ovulation-inducing drugs, and the high incidence of fetal aneuploidy. Aneuploidy rates of first trimester losses range from approximately 40-80% (), and a 2011 comparative genomic hybridization (CGH) study of failed pregnancies achieved using ART showed an aneuploidy rate of 83% [Nayak et al. Citation2011].
Table 1. Aneuploidy frequency after assisted reproductive technologies in past studies.
Genome instability has a negative correlation with standard semen parameters. Sperm DNA damage is significantly high in patients showing oligospermia (low sperm count) and severe morphological abnormalities [Fortunato et al. Citation2012; Moskovtsev et al. Citation2009]. Correlations were noted between DNA normality and sperm concentration, motility, rapid motility, normal morphology, and head defects [Varghese et al. Citation2009]. A highly statistically significant negative correlation was found between the percentage of normal sperm with fragmented DNA and embryo quality [Avendano et al. Citation2010]. Fertilization success is associated with the sperm genome, as a negative correlation was observed between fertilization rates and sperm DNA damage, while low sperm DNA damage has been associated with successful pregnancy [Bakos et al. Citation2008]. These findings imply that genomic instability negatively impacts fertility.
Numerous studies support that current ART can result in higher frequencies of genome instability. In preparation of IVF, follicle-stimulating drugs are administered for oocyte harvest. Exposure of the developing oocyte to supraphysiological concentrations of gonadotrophins may disturb oocyte maturation and the completion of meiosis leading to chromosomal aneuploidy within oocytes and/or embryos [Hodges et al. Citation2002]. In vitro-matured oocytes exposed to high concentrations of follicle-stimulating hormone (FSH) showed accelerated nuclear maturation and increased aneuploidy [Roberts et al. Citation2005]. Higher FSH doses have been associated with embryos with meiotic cell division errors [Katz-Jaffe et al. Citation2005]. Lower aneuploidy rates in embryos are observed following mild ovarian stimulation [Baart et al. Citation2007].
Higher genomic instability after follicle-stimulating drug exposure has been observed in mouse model systems under certain ovulation stimulation protocols. When compared to zygotes derived from spontaneous ovulation, mouse zygotes after ovarian stimulation showed an increased rate of chromosomal aberrations in the female pronucleus and compromised embryo development [Vogel and Spielmann Citation1992]. Mouse embryos originating from stimulated females showed a fourfold increase in the frequency of sister chromatid exchange than embryos from spontaneous ovulations, suggestive of induced-DNA lesion by ovarian stimulation [Elbling and Colot Citation1985]. It must be noted that these alterations are the result of certain ovarian stimulation protocols, however, and are not normally observed after routine superovulation procedures in transgenic mice facilities.
Gamete care and storage techniques used alongside ART may also induce genome instability. Density-gradient centrifugation can have detrimental effects on sperm DNA quality [Zini et al. Citation2000]. The increase of sperm DNA fragmentation after cryopreservation in liquid nitrogen and thawing is well documented [Riel et al. Citation2011; Thomson et al. Citation2009; Zribi et al. Citation2010]. A recent study of sperm cryostorage techniques found that chromatin decondensation was related to both the type of technique used and the duration of storage [Fortunato et al. Citation2012]. In another study, when compared to fresh samples, cryopreservation in liquid nitrogen and thawing resulted in statistically significant decreases in viability, DNA integrity, and the proportion of motile spermatozoa [Gianaroli et al. 2012]. Loss of motility and viability were also associated with lyophilization, however DNA integrity was maintained. The type of cryostorage technique used for oocyte preservation is important as well, as conventional slow-freezing when compared to vitrification (combining ultrarapid cooling with minimum volume and high cryoprotectant concentration) [Kuwayama et al. Citation2005] resulted in a higher percentage (39.1% to 17.7%) of spindle assembly and chromosome alignment abnormalities and lower oocyte survival rate (61.0% to 91.8%) [Cao et al. Citation2009].
We propose that through the application of artificial reproductive technologies, the filter of sexual reproduction that promotes genome and overall system stability is bypassed. Under normal circumstances, sperm containing aberrant genome packages would not be capable of reaching the egg and delivering the genomic payload due to a variety of potential morphological abnormalities. However, ART can unintentionally select these sperm cells for conception. These are, in the case of IVF, placed in culture to fertilize oocytes, far away from the obstacle course that the reproductive tract provides. Further, poor motility and inability to penetrate the oocyte zona pellucida are not issues with ICSI, as sperm are directly injected into oocytes.
In addition to circumventing the filter of sexual reproduction, ART can potentially induce additional genome instability within gamete and embryonic systems. Supraphysiological levels of FSH as well as cryostorage, sperm centrifugation, and culture conditions can all negatively impact the genomic system. ART may also induce global instability at the epigenetic level, as supported by aberrant imprinted methylation arising from ART and, in addition, the timing of ART coincides with critical epigenetic events during gametogenesis and early embryogenesis including epigenetic reprogramming and maternal/paternal pronuclear genome modifications [Denomme and Mann 2012]. This implies that even if conception and pregnancy are achieved through ART, this success may come with stress-induced genomic alterations and later health complications in the embryo or later in life. In order to improve the efficacy of reproductive technologies, appropriate monitoring is needed to observe genome system instability. This will ensure that ART are administered safely, reducing potential health burdens associated with genomic instability.
Interestingly, ART-associated somatic cell instability is less likely to last for many generations. Even if the children of ART may be predisposed to potential medical conditions due to genome instability, the sexual filter of natural reproduction will purify the genome when these individuals mature and reproduce. As a result, potential side effects of ART to the genome will not likely affect future generations. Based on this viewpoint, ART should be considered as a powerful means to repopulating endangered species, where the key is to increase genomes, and nature takes care of the next step through genome constraint and preservation.
Various methods are needed to monitor genome integrity during ART applications
With the realization that sexual reproduction serves as a filter that eliminates significant genome alteration, it is necessary to monitor the stability of the genome following ART. Further information regarding stochastic structural alterations is urgently needed, as higher frequencies of non-clonal chromosomal aberrations (NCCAs) have been linked with genome instability, disease conditions, and drug resistance [Heng et al. Citation2006c; Heng Citation2007a; Citation2010; Heng et al. Citation2011a; Ye et al. Citation2009]. NCCAs include all random structural and numerical aberrations, including aneuploidy and reciprocal translocations. Previously, the significance of NCCAs has been largely ignored as they have been treated as genetic ‘noise’ [Heng et al. Citation2004; Heng et al. Citation2006a; Heng et al. Citation2006b; Heng et al. 2013]. Pre- and post-natal NCCA frequencies of infertile couples, live births, and products of conception should be assessed and compared with the general fertile population to analyze genomic instability following ART. In addition, it is important to study the potential link between unstable genomes after ART and increased rates of common diseases.
We have previously demonstrated SKY as an effective approach for pre- and post-natal karyotype analysis [Heng et al. Citation2003]. SKY is a molecular cytogenetic technique used to simultaneously visualize chromosomes at the single cell level by labeling chromosome-specific DNA with different fluorophores, resulting in each pair of chromosomes assigned a different color [Ye et al. Citation2006]. We propose that SKY analysis can provide a wealth of information to infertility cases by focusing on patients at an individual cell based, genome-level resolution that can reveal phenomena (e.g., reciprocal translocations, polyploidy) not detectable by other methods (i.e., CGH). This strategy can influence the selection of an optimal ART approach for patients. Proper genomic monitoring before, during, and after ART applications with SKY can improve the efficacy of current and developing ART by observing any induced genomic instability.
We also need to evaluate existing reproductive techniques, as they have the potential to apply stress to the genome system. Culture conditions for in vitro fertilization represent such an example. Our group recently characterized chromosome fragmentation (C-frag), a form of mitotic cell death distinct from apoptosis, mitotic catastrophe, and premature chromosome condensation (PCC) [Stevens et al. Citation2007; Stevens et al. Citation2010; Stevens et al. Citation2011]. This type of cell death occurs during metaphase where condensed chromosomes are progressively degraded. Our group was also the first to identify genome chaos, described as a rapid karyotypic shattering followed by immediate shuffling and assembly of complex chromosomes [Heng et al. Citation2006c; Heng et al. Citation2011a]. Both C-frag and genome chaos are phenomena observed in cell culture conditions and induced by a wide variety of stresses. These include temperature change and hyperoxic environments; exposure to these stresses occur during in vitro fertilization procedures. Gamete harvesting and care protocols should also be monitored as current techniques stress the genome within gametes. Centrifugation and cryostorage procedures should all be reassessed in an effort to maximize their efficacies while minimizing stress-induced genome instability. Hormone stimulation protocols may induce chromosomal abnormalities during in vitro fertilization [Munne et al. Citation1997], and these must be reviewed and monitored as well to limit genome instability. External stresses during applications of ART might serve as a common mechanism for genome instability for select individuals. As illustrated by our evolutionary mechanism of common disease [Heng Citation2010; Heng et al. Citation2011a; Heng et al. Citation2011b], many different factors can be linked to system stress and genome instability.
We propose that monitoring genome stability alongside the application of assisted reproductive technologies will improve the efficacy of the techniques while limiting stress-induced genome instability as a result of these techniques. We can apply these genome analyses to the patients and test embryonic stages to reveal rates of genome alterations. In a recent study, for example, PCR-based comprehensive chromosome screening prior to single embryo transfer was shown to increase ongoing pregnancy rates and decrease miscarriage rates [Forman et al. Citation2012]. Selection of the appropriate genetic technique is key, as focusing solely on individual gene mutation profiles with genetic techniques such as DNA microarrays or even deep sequencing will not provide a comprehensive picture of the overall system stability. We propose that a holistic, single-cell resolution approach, such as measuring NCCA frequencies, will informatively illustrate the genome dynamics associated with infertility.
It has been previously proposed that usage of ART may represent a stressful event that could be associated with future health problems, as the animal evidence regarding the negative effects of embryo culture or stress to the preimplantation period is striking [Rinaudo and Lamb Citation2008]. Pregnant rats fed a low-protein diet give rise to offspring of low birth weight with life-long hypertension [Langley-Evans et al. Citation1999]. Low birth weight has also been linked to predisposition to cardiovascular diseases, diabetes, and stroke. A previous in vitro study of mouse embryos supported that in vitro embryo culture affects both fetal and placental weight during development [Delle Piane et al. Citation2010]. In vitro culture of sheep or cattle embryos has been associated with abnormal skeletal and organ development [Walker et al. Citation2000]. Previous studies support that mouse embryo in vitro culture and gamete manipulation result in long-term developmental and behavioral consequences, including anxiety and memory deficiencies [Ecker et al. Citation2004; Fernandez-Gonzalez et al. Citation2004]. Future studies focusing on genome dynamics of individuals following ART are necessary to trace the potential relationship of increased genome instability and consequential, potentially higher levels of common diseases and disorders.
In order to better understand the system dynamics associated with infertility and the impact that ART may have on genome instability, more single-cell based technologies are needed. As we previously demonstrated, many current genomic technologies that profile mixed cell populations often illustrate clonal genetic alterations while ignoring non-clonal genetic alterations, a true index of population diversity [Heng et al. Citation2006c]. Despite the valuable information it can provide, routine parental and fetal karyotype-level assessments are not yet implemented in current protocols. We call upon investigators to actively discuss this 4D-Genomics approach and apply this new thinking towards the development of new monitoring and assisted reproductive technologies.
Abbreviations
| FISH: | = | fluorescence in situ hybridization |
| IVF: | = | in vitro fertilization |
| ICSI: | = | intracytoplasmic sperm injection |
| SKY: | = | spectral karyotyping |
| ART: | = | assisted reproductive technologies |
| CGH: | = | comparative genomic hybridization |
| FSH: | = | follicle-stimulating hormone |
| NCCA: | = | non-clonal chromosomal aberration |
| C-frag: | = | chromosome fragmentation |
| PCC: | = | premature chromosome condensation. |
Declaration of interest: This work was partially supported by grants to HHQH from the United States Department of Defense (GW093028), SeeDNA Inc, the National Chronic Fatigue and Immune Dysfunction Syndrome Foundation, and the Nancy Taylor Foundation for Chronic Diseases. The authors report no declarations of interest.
Author contributions: Wrote the manuscript: SDH, HHQH. All other authors contributed extensively to discussion of the concept presented in this paper.
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