Missed connections: recombination and human aneuploidy.

The physical exchange of DNA between homologs, crossing-over, is essential to orchestrate the unique, reductional first meiotic division (MI). In females, the events of meiotic recombination that serve to tether homologs and facilitate their disjunction at MI occur during fetal development, preceding the MI division by several decades in our species. Data from studies in humans and mice demonstrate that placement of recombination sites during fetal development influences the likelihood of an MI nondisjunction event that results in the production of an aneuploid egg. Here we briefly summarize what we know about the relationship between aneuploidy and meiotic recombination and important considerations for the future of human assisted reproduction.

A candidate gene analysis and GWAS for genes associated with maternal nondisjunction of chromosome 21.

Human nondisjunction errors in oocytes are the leading cause of pregnancy loss, and for pregnancies that continue to term, the leading cause of intellectual disabilities and birth defects. For the first time, we have conducted a candidate gene and genome-wide association study to identify genes associated with maternal nondisjunction of chromosome 21 as a first step to understand predisposing factors. A total of 2,186 study participants were genotyped on the HumanOmniExpressExome-8v1-2 array. These participants included 749 live birth offspring with standard trisomy 21 and 1,437 parents. Genotypes from the parents and child were then used to identify mothers with nondisjunction errors derived in the oocyte and to establish the type of error (meiosis I or meiosis II). We performed a unique set of subgroup comparisons designed to leverage our previous work suggesting that the etiologies of meiosis I and meiosis II nondisjunction differ for trisomy 21. For the candidate gene analysis, we selected genes associated with chromosome dynamics early in meiosis and genes associated with human global recombination counts. Several candidate genes showed strong associations with maternal nondisjunction of chromosome 21, demonstrating that genetic variants associated with normal variation in meiotic processes can be risk factors for nondisjunction. The genome-wide analysis also suggested several new potentially associated loci, although follow-up studies using independent samples are required.

Temporal changes in chromosome abnormalities in human spontaneous abortions: Results of 40 years of analysis.

Studies during the past 50 years demonstrate the importance of chromosome abnormalities to the occurrence of early pregnancy loss in humans. Intriguingly, there appears to be considerable variation in the rates of chromosome abnormality, with more recent studies typically reporting higher levels than those reported in early studies of spontaneous abortions. We were interested in examining the basis for these differences and accordingly, we reviewed studies of spontaneous abortions conducted in our laboratories over a 40-year-time span. Our analyses confirm a higher rate of abnormality in more recent series of spontaneous abortions, but indicate that the effect is largely, if not entirely, attributable to changes over time in the maternal age structures of the study populations. © 2016 Wiley Periodicals, Inc.

Estrogenic exposure alters the spermatogonial stem cells in the developing testis, permanently reducing crossover levels in the adult.

Bisphenol A (BPA) and other endocrine disrupting chemicals have been reported to induce negative effects on a wide range of physiological processes, including reproduction. In the female, BPA exposure increases meiotic errors, resulting in the production of chromosomally abnormal eggs. Although numerous studies have reported that estrogenic exposures negatively impact spermatogenesis, a direct link between exposures and meiotic errors in males has not been evaluated. To test the effect of estrogenic chemicals on meiotic chromosome dynamics, we exposed male mice to either BPA or to the strong synthetic estrogen, ethinyl estradiol during neonatal development when the first cells initiate meiosis. Although chromosome pairing and synapsis were unperturbed, exposed outbred CD-1 and inbred C3H/HeJ males had significantly reduced levels of crossovers, or meiotic recombination (as defined by the number of MLH1 foci in pachytene cells) by comparison with placebo. Unexpectedly, the effect was not limited to cells exposed at the time of meiotic entry but was evident in all subsequent waves of meiosis. To determine if the meiotic effects induced by estrogen result from changes to the soma or germline of the testis, we transplanted spermatogonial stem cells from exposed males into the testes of unexposed males. Reduced recombination was evident in meiocytes derived from colonies of transplanted cells. Taken together, our results suggest that brief exogenous estrogenic exposure causes subtle changes to the stem cell pool that result in permanent alterations in spermatogenesis (i.e., reduced recombination in descendent meiocytes) in the adult male.

Evidence for paternal age-related alterations in meiotic chromosome dynamics in the mouse.

Increasing age in a woman is a well-documented risk factor for meiotic errors, but the effect of paternal age is less clear. Although it is generally agreed that spermatogenesis declines with age, the mechanisms that account for this remain unclear. Because meiosis involves a complex and tightly regulated series of processes that include DNA replication, DNA repair, and cell cycle regulation, we postulated that the effects of age might be evident as an increase in the frequency of meiotic errors. Accordingly, we analyzed spermatogenesis in male mice of different ages, examining meiotic chromosome dynamics in spermatocytes at prophase, at metaphase I, and at metaphase II. Our analyses demonstrate that recombination levels are reduced in the first wave of spermatogenesis in juvenile mice but increase in older males. We also observed age-dependent increases in XY chromosome pairing failure at pachytene and in the frequency of prematurely separated autosomal homologs at metaphase I. However, we found no evidence of an age-related increase in aneuploidy at metaphase II, indicating that cells harboring meiotic errors are eliminated by cycle checkpoint mechanisms, regardless of paternal age. Taken together, our data suggest that advancing paternal age affects pairing, synapsis, and recombination between homologous chromosomes--and likely results in reduced sperm counts due to germ cell loss--but is not an important contributor to aneuploidy.

Human aneuploidy: mechanisms and new insights into an age-old problem.

Trisomic and monosomic (aneuploid) embryos account for at least 10% of human pregnancies and, for women nearing the end of their reproductive lifespan, the incidence may exceed 50%. The errors that lead to aneuploidy almost always occur in the oocyte but, despite intensive investigation, the underlying molecular basis has remained elusive. Recent studies of humans and model organisms have shed new light on the complexity of meiotic defects, providing evidence that the age-related increase in errors in the human female is not attributable to a single factor but to an interplay between unique features of oogenesis and a host of endogenous and exogenous factors.

An algorithm for determining the origin of trisomy and the positions of chiasmata from SNP genotype data.

Trisomy causes mental retardation, pregnancy loss, IVF failure, uniparental disomy and several other pathologies, and its accurate detection is thus clinically essential. Most trisomies arise at meiosis I and are associated with increasing maternal age and reduction or alteration in recombination patterns. Investigations into the relationship between trisomy and meiotic recombination have used short tandem repeat markers; however, this approach is limited by the resolution with which the position of crossovers can identified. As cytogenetics enters the post-genomic era, recent work has used array comparative genomic hybridisation (aCGH) to screen for trisomy of all 24 chromosomes, determining chromosome copy number by dosage analysis. However, aCGH has a fundamental drawback for studying the aetiology of trisomy since neither the parent and phase of origin nor uniparental disomy can be ascertained. The development of SNP microarrays has made it possible to analyse multiple loci for sequence variation, and the proprietary software provided can determine the presence of aneuploidy by algorithms based on fluorescence intensity. To the best of our knowledge, however, such software is not equipped to determine the phase of origin of the error or the position of any chiasmata. In this study, therefore, we present an algorithm to determine the parent of origin, the phase of origin and the location of chiasmata in a series of nine "trisomy triplets" (i.e. samples derived from father, mother and their trisomic foetus). Novel adaptations of well-established principles are applied along with a simple algorithm written in Microsoft Excel for visualisation of the results. Such analysis has a range of applications in preimplantation and prenatal diagnosis.

Predicting meiotic pathways in human fetal oogenesis.

Gene function prediction has proven valuable in formulating testable hypotheses. It is particularly useful for exploring biological processes that are experimentally intractable, such as meiotic initiation and progression in the human fetal ovary. In this study, we developed the first functional gene network for the human fetal ovary, HFOnet, by probabilistically integrating multiple genomic features using a naïve Bayesian model. We demonstrated that this network could accurately recapture known functional connections between genes, as well as predict new connections. Our findings suggest that known meiosis-specific genes (i.e., with functions only in meiotic processes in the germ cells) make either no or a few functional connections but are highly clustered with neighbor genes. In contrast, known nonspecific meiotic genes (i.e., with functions in both meiotic and nonmeiotic processes in the germ cells and somatic cells) exhibit numerous connections but low clustering coefficients, indicating their role as central modulators of diverse pathways, including those in meiosis. We also predicted novel genes that may be involved in meiotic initiation and DNA repair. This global functional network provides a much-needed framework for exploring gene functions and pathway components in early human female meiosis that are difficult to tackle by traditional in vivo mammalian genetics.

Meiotic recombination in human oocytes.

Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1) in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of "vulnerable" crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.

The bisphenol A experience: a primer for the analysis of environmental effects on mammalian reproduction.

It is increasingly evident that environmental factors are a veritable Pandora's box from which new concerns and complications continue to emerge. Although previously considered the domain of toxicologists, it is now clear that an understanding of the effects of the environment on reproduction requires a far broader range of expertise and that, at least for endocrine-disrupting chemicals, many of the tenets of classical toxicology need to be revisited. Indeed, because of the wide range of reproductive effects induced by these chemicals, interest among reproductive biologists has grown rapidly: in 2000, the program for the annual Society for the Study of Reproduction meeting included a single minisymposium on the fetal origins of adult disease, one platform session on endocrine disruption, and 23 toxicology poster presentations. In contrast, environmental factors featured prominently at the 2009 meeting, with strong representation in the plenary, minisymposia, platform, and poster sessions. Clearly, a lot has happened in a decade, and environmental issues have become an increasingly important research focus for reproductive biologists. In this review, we summarize some of the inherent difficulties in assessing environmental effects on reproductive performance, focusing on the endocrine disruptor bisphenol A (BPA) to illustrate important emerging concerns. In addition, because the BPA experience serves as a prototype for scientific activism, public education, and advocacy, these issues are also discussed.

Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice.

In eukaryotes, diploid cells give rise to haploid cells via meiosis, a program of two cell divisions preceded by one round of DNA replication. Although key molecular components of the meiotic apparatus are highly conserved among eukaryotes, the mechanisms responsible for initiating the meiotic program have diverged substantially among eukaryotes. This raises a related question in animals with two distinct sexes: Within a given species, are similar or different mechanisms of meiotic initiation used in the male and female germ lines? In mammals, this question is underscored by dramatic differences in the timing of meiotic initiation in males and females. Stra8 is a vertebrate-specific, cytoplasmic factor expressed by germ cells in response to retinoic acid. We previously demonstrated that Stra8 gene function is required for meiotic initiation in mouse embryonic ovaries. Here we report that, on an inbred C57BL/6 genetic background, the same factor is also required for meiotic initiation in germ cells of juvenile mouse testes. In juvenile C57BL/6 males lacking Stra8 gene function, the early mitotic development of germ cells appears to be undisturbed. However, these cells then fail to undergo the morphological changes that define meiotic prophase, and they do not display the molecular hallmarks of meiotic chromosome cohesion, synapsis and recombination. We conclude that, in mice, Stra8 regulates meiotic initiation in both spermatogenesis and oogenesis. Taken together with previous observations, our present findings indicate that, in both the male and female germ lines, meiosis is initiated through retinoic acid induction of Stra8.

Proteins involved in meiotic recombination: a role in male infertility?

Meiotic recombination results in the formation of crossovers, by which genetic information is exchanged between homologous chromosomes during prophase I of meiosis. Recombination is a complex process involving many proteins. Alterations in the genes involved in recombination may result in infertility. Molecular studies have improved our understanding of the roles and mechanisms of the proteins and protein complexes involved in recombination, some of which have function in mitotic cells as well as meiotic cells. Human gene sequencing studies have been performed for some of these genes and have provided further information on the phenotypes observed in some infertile individuals. However, further studies are needed to help elucidate the particular role(s) of a given protein and to increase our understanding of these protein systems. This review will focus on our current understanding of proteins involved in meiotic recombination from a genomic perspective, summarizing our current understanding of known mutations and single nucleotide polymorphisms that may affect male fertility by altering meiotic recombination.

Human female meiosis: what makes a good egg go bad?

Critical events of oogenesis occur during three distinct developmental stages: meiotic initiation in the fetal ovary, follicle formation in the perinatal period, and oocyte growth and maturation in the adult. Evidence from studies in humans and mice suggests that the genetic quality of the egg may be influenced by events at each of these stages. Recent experimental studies add additional complexity, suggesting that environmental influences might adversely affect all three stages. Thus, understanding the molecular control of oogenesis during these critical developmental windows will not only contribute to an understanding of human aneuploidy, but also provide a means of assessing potential effects of environmental exposures on human reproductive health.

The origin of trisomy 13.

Trisomy 13 is one of the most common trisomies in clinically recognized pregnancies and one of the few trisomies identified in liveborns, yet relatively little is known about the errors that lead to trisomy 13. Accordingly, we initiated studies to investigate the origin of the extra chromosome in 78 cases of trisomy 13. Our results indicate that the majority of cases (>91%) are maternal in origin and, similar to other autosomal trisomies, the extra chromosome is typically due to errors in meiosis I. Surprisingly, however, a large number of errors also occur during maternal meiosis II ( approximately 37%), distinguishing trisomy 13 from other acrocentric and most nonacrocentric chromosomes. As with other trisomies, failure to recombine is an important contributor to nondisjunction of chromosome 13.

The Mre11 complex influences DNA repair, synapsis, and crossing over in murine meiosis.

The Mre11 complex (consisting of MRE11, RAD50, and NBS1/Xrs2) is required for double-strand break (DSB) formation, processing, and checkpoint signaling during meiotic cell division in S. cerevisiae. Whereas studies of Mre11 complex mutants in S. pombe and A. thaliana indicate that the complex has other essential meiotic roles , relatively little is known regarding the functions of the complex downstream of meiotic break formation and processing or its role in meiosis in higher eukaryotes. We analyzed meiotic events in mice harboring hypomorphic Mre11 and Nbs1 mutations which, unlike null mutants, support viability . Our studies revealed defects in the temporal progression of meiotic prophase, incomplete and aberrant synapsis of homologous chromosomes, persistence of strand exchange proteins, and alterations in both the frequency and placement of MLH1 foci, a marker of crossovers. A unique sex-dependent effect on MLH1 foci and chiasmata numbers was observed: males exhibited an increase and females a decrease in recombination levels. Thus, our findings implicate the Mre11 complex in meiotic DNA repair and synapsis in mammals and indicate that the complex may contribute to the establishment of normal sex-specific differences in meiosis.

Bisphenol A exposure in utero disrupts early oogenesis in the mouse.

Estrogen plays an essential role in the growth and maturation of the mammalian oocyte, and recent studies suggest that it also influences follicle formation in the neonatal ovary. In the course of studies designed to assess the effect of the estrogenic chemical bisphenol A (BPA) on mammalian oogenesis, we uncovered an estrogenic effect at an even earlier stage of oocyte development--at the onset of meiosis in the fetal ovary. Pregnant mice were treated with low, environmentally relevant doses of BPA during mid-gestation to assess the effect of BPA on the developing ovary. Oocytes from exposed female fetuses displayed gross aberrations in meiotic prophase, including synaptic defects and increased levels of recombination. In the mature female, these aberrations were translated into an increase in aneuploid eggs and embryos. Surprisingly, we observed the same constellation of meiotic defects in fetal ovaries of mice homozygous for a targeted disruption of ERbeta, one of the two known estrogen receptors. This, coupled with the finding that BPA exposure elicited no additional effects in ERbeta null females, suggests that BPA exerts its effect on the early oocyte by interfering with the actions of ERbeta. Together, our results show that BPA can influence early meiotic events and, importantly, indicate that the oocyte itself may be directly responsive to estrogen during early oogenesis. This raises concern that brief exposures during fetal development to substances that mimic or antagonize the effects of estrogen may adversely influence oocyte development in the exposed female fetus.

The synaptonemal complex and meiotic recombination in humans: new approaches to old questions.

Meiotic prophase serves as an arena for the interplay of two important cellular activities, meiotic recombination and synapsis of homologous chromosomes. Synapsis is mediated by the synaptonemal complex (SC), originally characterized as a structure linked to pairing of meiotic chromosomes (Moses (1958) J Biophys Biochem Cytol 4:633-638). In 1975, the first electron micrographs of human pachytene stage SCs were presented (Moses et al. (1975) Science 187:363-365) and over the next 15 years the importance of the SC to normal meiotic progression in human males and females was established (Jhanwar and Chaganti (1980) Hum Genet 54:405-408; Pathak and Elder (1980) Hum Genet 54:171-175; Solari (1980) Chromosoma 81:315-337; Speed (1984) Hum Genet 66:176-180; Wallace and Hulten (1985) Ann Hum Genet 49(Pt 3):215-226). Further, these studies made it clear that abnormalities in the assembly or maintenance of the SC were an important contributor to human infertility (Chaganti et al. (1980) Am J Hum Genet 32:833-848; Vidal et al. (1982) Hum Genet 60:301-304; Bojko (1983) Carlsberg Res Commun 48:285-305; Bojko (1985) Carlsberg Res Commun 50:43-72; Templado et al. (1984) Hum Genet 67:162-165; Navarro et al. (1986) Hum Reprod 1:523-527; Garcia et al. (1989) Hum Genet 2:147-53). However, the utility of these early studies was limited by lack of information on the structural composition of the SC and the identity of other SC-associated proteins. Fortunately, studies of the past 15 years have gone a long way toward remedying this problem. In this minireview, we highlight the most important of these advances as they pertain to human meiosis, focusing on temporal aspects of SC assembly, the relationship between the SC and meiotic recombination, and the contribution of SC abnormalities to human infertility.

SMC1beta-deficient female mice provide evidence that cohesins are a missing link in age-related nondisjunction.

Mitotic chromosome segregation is facilitated by the cohesin complex, which maintains physical connections between sister chromatids until anaphase. Meiotic cell division is considerably more complex, as cohesion must be released sequentially to facilitate orderly segregation of chromosomes at both meiosis I and meiosis II. This necessitates meiosis-specific cohesin components; recent studies in rodents suggest that these influence chromosome behavior during both cell division and meiotic prophase. To elucidate the role of the meiosis-specific cohesin SMC1beta (encoded by Smc1l2) in oogenesis, we carried out meiotic studies of female SMC1beta-deficient mice. Our results provide the first direct evidence that SMC1beta acts as a chiasma binder in mammals, stabilizing sites of exchange until anaphase. Additionally, our observations support the hypothesis that deficient cohesion is an underlying cause of human age-related aneuploidy.