User:Algwu/sandbox

Introduction
It is well established that the larger source of aneuploidies in the human embryo stems from oogenesis. There is a spectrum of factors, both intrinsic and exogenous, which can lead to eventual increased chromosome count discrepancies in the final oocyte. The most abundant causes of oogenetic aneuploidy stem from fundamental differences between the gamete-generating processes of oogenesis and spermatogenesis, as well as developmental changes associated with maternal aging. No matter the source, the end result is almost always a type of segregation aberration in either Meiosis 1 or Meiosis 2, which directly induces the observed aneuploidy).

Checkpoint Variations between Oogenesis and Spermatogenesis
Preservation of genomic integrity is of utmost importance to the embryo; thus it is no surprise that gametogenesis includes many points where genetic material is inspected for its spatial position in the cell, its physical condition, and its temporal location in respect to the rest of the genetic material, as well as to the overall cell environment. Because spermatogenesis is continuous and occurs more frequently than oogenesis, it would seem intuitive that evolutionarily, oogenesis would select more aggressively, or at least match male meiosis’ tolerance, for healthy gametes during production. If this were true, it would be observed that male and female meiosis would be equal contributors as sources of embryonic aneuploidies. This, however, is not the case: from early studies of pregnancies, it became clear that most aneuploidies are maternal in origin. Further examination into this observation lead to the reasoning that the checkpoints of oogenesis are perhaps not as severe as those of spermatogenesis-or, along the same lines, oocytes are genetically programmed to be more “robust” than spermatocytes. Studies in mice have shed light on the fact that not only do some genes act differently depending on the gametocyte’s fate(sperm or egg), but that in many cases, changes and pressures on gametogenesis that would render males sterile would not completely halt gamete production in females. Furthermore, it appears that there exist cellular conditions that would violate checkpoints and lead to arrest of spermatogenesis which do not lead to the same in oogenesis. These observations have been examined experimentally by targeting and knocking out genes involved in DNA structure maintenance, DNA repair, and the like and examining the offspring for gamete production and fertility.

Recombination, the process by which genetic material is spatially rearranged, involves having chromosomes crossing over; inducing breaks in them; waiting for strand invasion and spatial rearrangements to finish; and undergoing DNA repair afterwards- all while maintaining overall genome integrity. Every step along the way is controlled to minimize mistakes or permanent damage to the genome. The mechanisms of inducing recombination are actually genetically controlled: studies in cattle and mice ( have revealed genes such as Rec8 which appear to be able to induce the double stranded breaks necessary for recombination to occur . These genes help to ensure that processes such as crossing over and DNA breaks are induced only where and when necessary, keeping the genome stable during recombination. Eukaryotes also have multiple systems in place to check for DNA damage not intentionally caused, such as from UV exposure, aiding in the prevention of any errors during meiosis.  While recombination during meiosis is usually assumed to follow synapsis, there is evidence that there exist regulatory genes that independently control for each event. The survival of female gametes even when mutations are induced in similar genes indicate that despite these many checkpoints, oocytes are able to evade apoptosis and survive even in the presence of damage. Even in cases where both males and females experience sterility/infertility, the percentage of females which are infertile remain markedly lower than the percentage of males which are sterile.

The robustness of female meiosis leads to continuation of production despite disruption, whereas male game production is arrested. This could lead problems as oogenesis continues, including chromosome shattering and micronucleus formation, which is also a source of eventual aneuploidy. While inducing breaks and controlling damage to DNA are important, the precise movement and rearrangement of chromosomes during meiosis is also necessary to ascertain proper segregation of chromosomes. This process has a number of checkpoints as well, one of which is the Spindle Assembly Checkpoints (SAC). Essentially, the SAC ensures that chromosomes are properly attached to the spindle complex before segregation is attempted -otherwise there is a danger of having rogue loose chromosomes in the cell-which would almost surely end in aneuploidy. The attachment of a microtubule to each chromosome’s kinetochore is induced by cyclin; regulation of this interaction during previous meiotic phases is what allows for proper chromosome alignment, segregation, and movement during Meiosis 1. As with recombination, it seems that oocytes can forgo the restrictive properties of the checkpoint even if they do not meet its requirements-for example, having one chromosome unattached to the complex. In fact, some studies have postulated that proper attachment and alignment of chromosomes may not even be a checkpoint in oocyte development. In a 2011 study, it was found that anaphase initiation could begin even in the presence of univalent (unpaired) chromosomes. This does not mean oocytes are formed without regard for genome stability: in the presence of multiple univalent chromosomes (induced by deformation of the meiotic spindle through mutating Mlh 1, oogenesis is indeed arrested. Nonetheless, the higher tolerance of the SAC in female gametogenesis compared to male gametogenesis is a contributing factor to aneuploidies of maternal origin. As previously mentioned, genetic mutations can lead to errors during meiosis, and due to the higher tolerance for mistakes in oogenesis, these problems may remain undetected until the final gametes are produced. The variety of deviations from the norm that can occur is tremendous, from improper crossing over to failure to recombine . In these experiments, the test organism usually has a gene of interest mutated in its germline, and its offspring (which now lack expression of that gene) are examined for any changes (during life or through autopsy). Controls are usually provided through wild-type or non-mutated organisms of the same species, and serve as a baseline for regular growth, development, and expression of the target gene. Germ cells can be karyotyped through microarrays so that the chromosomes can be visualized, or the DNA can be sequenced to detect aneuploidies that may have occurred.

Histone H2AX serves as an example of the variety of consequences a mutation could incur. It was previously known that Histone H2AX was phosphorylated in the presence of double stranded breaks. In a study performed comparing H2AX-negative mice with non-H2AX deficient mice, it was observed that mice with the H2AX knockout exhibited various levels of infertility, immune deficiency, and growth retardation. The knockout mice exhibited deficient DNA repair; this was especially evident when examining germ cells. Knockout male mice had spermatocytes arrested at Meiosis 1, most likely due to a reduced ability to repair double stranded breaks. Interestingly, unlike their male counterparts, knockout female mice remained fertile and were able to produce litters with wild type counterparts, though it was noted that the litter size was smaller. This result lends support to the hypothesis that spermatogenesis has a lower threshold for error than oogenesis before the gametocytes are prevented from developing further. It was later concluded that Histone H2AX served as a critical component of double stranded repair by serving as a center for recruiting DNA repair elements to the site of the break. The table below shows a sampling of the variety of genetic mutations which may lead to incurred errors (but not necessarily arrest development) in oocytes; the natural gene function; and the effects of the mutation.

Table 2: Genes Associated with Meiotic Division and Effects of their Mutations

Besides the gametogenic process being intrinsically error-prone, physical constraints specific to oogenesis can directly increase chances of chromosome errors. Chromosome reduction during gametogenesis leads to partial degradation of centrioles during spermatogenesis, and complete degradation of centrioles in oogenesis. Centrioles are structures in the cell usually important for microtubule organization and cell divisions, but it appears that oocytes have evolved to be able to undergo an acentrosomal mode of spindle organization coordinated by hepatoma up-regulated protein, or Hurp. While oocytes are perfectly capable of undergoing meiosis and division without centrioles, their absence nonetheless can lead to a physically compromised spindle that is elongated and may not be attached to all chromosomes. These irregular spindles are not enough to trigger the SAC checkpoint, and oocytes will often continue to anaphase unimpeded.

The Effects of Aging
Aging is correlated with a marked increase of aneuploidies and errors, such as failure of chromosome to properly align during meiosis. This is and observed trend consistent among a host of organisms, including mice, and yeast. The causes are various, including altered gene expression, accumulation of DNA damage and increased inefficiency in DNA repair mechanisms , and the length of time oocytes are paused between Meiosis 1 and Meiosis 2 in humans. Cohesin degradation is a well-supported cause of increased meiotic errors due to aging. Cohesins are coil shaped, hinged proteins that link chromosomes together during mitosis and meiosis; they take the form of subunits which form rings. During mitosis, when pairs of chromosomes are lined up and only one division occurs, cohesin can be released all at once after metaphase and initiate anaphasal chromosome separation. During meiosis however, where there are two divisions, some of the cohesin must be preserved between Meiosis 1 and Meiosis 2-specifically, the cohesins linking sister chromatids together. Thus, the cohesin must be released sequentially, even though the gap between degenerations may be decades. The maintenance of the connections made by cohesin is necessary to preserve spindle integrity during meiosis: keeping chromosomes/chromatids attached until separation is completed so that lining up is properly managed-that being said, proper cleavage of cohesin is also necessary to release chromosomes at the right time. There is evidence to show that due to the length time between Meiosis 1 and Meiosis 2, the remaining cohesin is degraded and weakened before the meiotic cycle begins again. This can take the form of loosened rings (inferred from increased distance between pairs of chromatids), weaker links (shown through increased breakages), or altered gene expression (seen from studying cells of varying ages). Weakened cohesin increases the chance of premature chromosome segregation, causing lagging or unattached chromosomes which are then missegregated, causing aneuploidies. The length of time that passes between Meiosis 1 and Meiosis 2, combined with cohesin degradation, is may be the reason that Meiosis 2 seems to be more susceptible to errors than Meiosis 1. This is despite the fact that the possible categories of errors that can occur during Meiosis 1 (which includes inaccuracies between homologue chromosomes as well as sister chromatids) is more than the types of mistakes that can occur during Meiosis 2 (which only concerns sister chromatids). However, analogous to this, whole chromosome aneuploidies are almost exclusively associated with Meiosis 1, where premature separation of chromosomes during Metaphase 1 lead to missegregation of an entire chromosome. Thus while it was previously believed that whole-chromosome nondisjunction was the more common form of aneuploidy in human embryos, comparative genomic hybridization has since shown that single chromatid aneuploidies (which stem from events during Meiosis 2) are over 11 times as common. Gene expression of cohesin-forming proteins during meiosis also changes as aging occurs. For example, Rec8, a gene implicated with influencing recombination rate in cattle, codes for a component of cohesin in mice. Using live cell imaging, expression of Rec8 was found to be reduced in older mice than in younger mice. . Immunofluorescent staining also revealed that while overall expression of Rec8 was not reduced, Rec8 associated with chromosomes was severely reduced.

BPA and Exogenous Hormone-like Effects on Oogenesis
Though many of the mistakes in meiosis are age inherent and naturally occurring, external influences can also increase the odds of such errors happening. In particular, Bisphenol-A (BPA) has alters spindle formation and causes permanent inhibitory effects on meiosis in cells. BPA is known to be a weak estrogenic imitator, and in that way could interfere with regular cellular processes. Moreover, continuous exposure to BPA reveals an increase in arrested oocytes (due to checkpoint activation), as well as increased numbers of misaligned chromosomes and spindle malformations. It was also found that BPA could disturb oogenesis at environmentally relevant levels, and that it could leech out of plastics. More broadly, molecular look-alikes that could interfere with hormonal processes can disrupt initiation, maintenance, and conclusion of the meiotic cycle. This effect is especially amplified in oogenesis due to the natural meiotic arrest put in place in the middle of the cycle. It is also known that aneuploidy levels in embryos are elevated when induced ovulation for in-vitro fertilization (IVF) is used for pregnancies. The exact reasons for this, as well as mechanisms for this, are still being researched but there are theories that the hormones used in the procedures somehow increase chromosomal anomalies. For example, when PMSG was administered to female mice, there was a proportional increase of polyploidy embryos based on the dosage of the gonadotrophin administered. Lower gonadotrophin doses are directly associated with lower rates of aneuploidy.

Techniques Used in Detecting Oogeneic Aneuploidy
There have been many investigative tools used in tracking levels and rates of aneuploidies, as well as when and how they occur. The first studies of embryonic aneuploidies used fluorescent in-situ hybridization to detect aneuploidies, and this technique is still sometimes used in screening embryos in artificial reproduction. The great variability of this technique, however, limits its applications greatly. In addition, the Practice Committee of the Society for Assisted Reproductive Technology has reported that FISH can be inaccurate and report falsely high numbers of aneuploidies when, in fact, the embryo is fine. FISH may also not a reliable screening method due to false positives, and the fact that clinical trials have showed that successful live birth rates have not increased even with FISH usage. Microarray analysis of chromosomes is a highly accurate and revealing method of detecting aneuploidies also used in pre-implantation diagnosis: it allows for detection of aneuploidies of not only whole chromosome rearrangements, but also partial chromosome aneuploidies. It is also relatively inexpensive to use, making it favorable for clinical use. Array CGH of polar bodies by comparative genomic hybridization has also been shown to accurately be able to predict maternally originated aneuploidies, with over a 90% accuracy. Clinical trials have also shown that live birth rates have increased with CGH screening, lending to its credibility. Single nucleotide polymorphism arrays, used for molecular karyotyping, makes it easy to accurately analyze large segments of the genome with remarkable specificity and resolution. The resolution of SNP arrays is so great that it allows for the parental origin of the aneuploidy to be identified as well. However, its high costs and complex protocol make its use relatively limited. Through genetic analysis of embryos, live cell imaging, and observations of the effects of genetic mutations, we are now more accurately be able to describe the functions of each step of oogenesis. Further research is being done on more genetic controls on the spatiotemporal arrangement of the genome, the tolerance for error of meiotic checkpoints (as well as when these checkpoints exist and what they check for) , and methods for accurately predicting embryonic aneuploidy. While the nature of assisted reproductive technology seems to necessitate the higher rate of aneuploidy in oocytes (due to artificial interference with hormonal concentrations), we can nonetheless account for this by discovering and cataloguing the oogenic process: fine-tuning assisted reproduction may be possible in the future. For example, because of the BPA revelation, an exogenous contributor to meiotic error was removed from the equation. By increasing insight into the mechanisms of meiosis and buffering support systems for oocytes, we can take advantage of what we know and even induce oocyte formation outside of the body.