Minireview

BIOLOGY OF REPRODUCTION (2015) 92(1):19, 1–8 Published online before print 3 December 2014. DOI 10.1095/biolreprod.114.125575

Minireview Polar Bodies in Assisted Reproductive Technology: Current Progress and Future Perspectives1 Yanchang Wei,3 Teng Zhang,3 Ya-Peng Wang,3 Heide Schatten,4 and Qing-Yuan Sun2,3 3 4

State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri

During meiotic cell-cycle progression, unequal divisions take place, resulting in a large oocyte and two diminutive polar bodies. The first polar body contains a subset of bivalent chromosomes, whereas the second polar body contains a haploid set of chromatids. One unique feature of the female gamete is that the polar bodies can provide beneficial information about the genetic background of the oocyte without potentially destroying it. Therefore, polar body biopsies have been applied in preimplantation genetic diagnosis to detect chromosomal or genetic abnormalities that might be inherited by the offspring. Besides the traditional use in preimplantation diagnosis, recent findings suggest additional important roles for polar bodies in assisted reproductive technology. In this paper, we review the new roles of polar bodies in assisted reproductive technology, mainly focusing on single-cell sequencing of the polar body genome to deduce the genomic information of its sibling oocyte and on polar body transfer to prevent the transmission of mtDNA-associated diseases. We also discuss additional potential roles for polar bodies and related key questions in human reproductive health. We believe that further exploration of new roles for polar bodies will contribute to a better understanding of reproductive health and that polar body manipulation and diagnosis will allow production of a greater number of healthy babies. assisted reproductive technology, genomics, human reproduction, mitochondria, mitochondrial disease, oocyte, polar body, preimplantation genetic diagnosis, single-cell sequencing

INTRODUCTION The unequal divisions during the first and the second meiosis result in a large oocyte and two diminutive polar bodies, which contain a redundant set of chromosomes plus a small amount of cytoplasmic organelles. The first polar body (PB1) is extruded after the onset of the luteinizing hormone 1

Supported by the National Basic Research Program of China (2012CB944404, 2011CB944501). Correspondence: Qing-Yuan Sun, State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, #1 Beichen West Rd., Chaoyang District, Beijing 100101, China. E-mail: [email protected] 2

Received: 2 October 2014. First decision: 29 October 2014. Accepted: 2 December 2014. Ó 2015 by the Society for the Study of Reproduction, Inc. eISSN: 1529-7268 http://www.biolreprod.org ISSN: 0006-3363

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surge [1], and extrusion of the PB1 is an important hallmark of oocyte meiotic maturation. The homologous chromosomes become separated between two unequal cytoplasmic masses during this process [2, 3]. In humans, the oocyte is approximately 100-fold larger in volume than the PB1, and it contains an average of 3.14 3 105 mitochondria/oocyte [4, 5]. The PB1 contains membranous material, mitochondria, ribosomes, cortical granules, and other cytoplasmic material [6]. While the oocyte progresses to metaphase II, the spindle usually is not well organized and chromosomes gradually degenerate at late telophase [7]; however, the PB1 can divide off the oocyte. Formation of the second polar body (PB2) occurs after fertilization. Differing from the PB1 that contains bivalent chromosomes, the PB2 contains a haploid set of chromatids. The chromatids in the PB2 are protected by a nuclear envelope that is more persistent than that in the PB1, which disappears within a few hours in some mouse strains and after 20 h in humans [8–10]. Usually, the PB2 is recognizable in late preimplantation development with continued new protein synthesis. Studies in the PO (Pathology, Oxford) strain of mice found that nearly two-thirds of PB2s persisted and remained intact through the early blastocyst stage [11]. Moreover, the distribution of the surviving PB2s was highly nonrandom in early blastocysts [11]. Together with other findings, the evidence indicates that the PB2 may also play a role in the polarity patterning of the developing conceptus [12]. One unique feature of the female gamete is that polar bodies can provide beneficial information about the genetic background of the oocyte without potentially destroying it. Polar body biopsy has been applied in preimplantation genetic diagnosis (PGD) to detect chromosomal or genetic abnormalities that might be inherited by the offspring. The majority of PGD clinics perform biopsies on preimplantation embryos rather than on polar bodies [13]. However, the polar body biopsy may have advantages in certain conditions. Indeed, some couples find polar body biopsy morally attractive, because it does not disrupt the fertilized embryo. Although the polar bodies have limited life spans, they have the potential to support normal development when they are transferred to an enucleated oocyte [14]. Moreover, mitochondria segregation is random during meiosis, which provides an opportunity to use polar bodies for screening of mitochondrial mutations. In most cases of reproductive medicine, polar bodies are neglected other than for PGD [15–17]. They are tiny and contribute to successful development only by allowing diploidy to be established. However, polar bodies have roles beyond traditional PGD. One exciting role for polar bodies is emerging

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tance concerns the safety of polar body biopsy. Clinical studies provide strong evidence that polar body biopsy is safe and does not affect embryo quality [38, 39]. Besides genetic material, the polar body also contains RNAs. Previously, it was shown that mRNAs present in oocytes can also be detected in polar bodies [40]. Importantly, the transcriptome of the PB1 can accurately reflect that of its sibling oocyte in humans [41]. Single-cell transcriptome analysis showed that of the 5431 mRNAs recovered from the PB1, 5256 (;97%) of them shared similar expression levels as its sibling oocyte [41]. This suggests that transcriptional detection and quantification by high-throughput techniques could acquire firsthand information of global gene expression in mature oocytes and thus lead to molecular diagnostic applications.

with the development of new high-throughput genome-wide single-cell sequencing techniques [18–20]. Recently, a study showed that the genome of a single oocyte, including information regarding aneuploidy and genetic variants that may be associated with human disease, can be accurately deduced by sequencing the genome of its sibling polar bodies [19]. Another exciting study recently found that polar body transfer (transfer of the polar body from a patient’s eggs to healthy eggs) can prevent the transmission of mtDNA disease [21]. In this paper, the new roles of polar bodies in assisted reproductive technology are reviewed. Although polar bodies are believed to be genetic discards, they contain genetic information about the oocyte that can help guide the decision about whether a given embryo is healthy. Moreover, they retain potential for contributing to a new life with great potential to prevent the inheritance of mtDNA disease.

NEW ROLES FOR THE POLAR BODY TO DEDUCE THE GENOME OF THE OOCYTE

TRADITIONAL ROLES OF POLAR BODIES IN ASSISTED REPRODUCTIVE TECHNOLOGY

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Polar body biopsy was initially used for detecting maternal original single gene defects. One of the most comprehensive studies of polar body detection of single gene defects was reported by Rechitsky et al. [42]. That study tested more than 1000 oocytes for single gene defects by using PCR. A total of 237 unaffected oocytes were preselected for transfer back to 114 patients, resulting in 34 unaffected pregnancies and the birth of 23 healthy children. Allele dropout (ADO) is one of the greatest concerns for misdiagnosis in PGD, which occurs in the heterozygous condition when only one of the two alleles amplifies [43]. The ADO rate in this study was 7.8%. An additional disadvantage of polar body testing for the diagnosis of mendelian diseases is crossover or genetic recombination. Testing both the PB1 and PB2 is a way to avoid undetected ADO. Advances in whole-genome and whole-transcriptome amplification make sequencing the minute amounts of DNA and RNA within a single cell possible [18, 44, 45]. This new technique will offer a new tool for the development of assisted reproductive techniques. Human germ cells undergo homologous recombination of paternal and maternal genomes. This results in crossovers in individual chromosomes and contributes to genetic diversity in evolution. Therefore, each human germ cell has a unique genome, and this necessitates wholegenome single-cell sequencing analysis. Two years ago, singlecell sequencing has been applied to human sperm [18, 45]. More recently, it has been achieved in a single oocyte [19]. As mentioned previously, a unique feature of the oocyte is that the PB1 and PB2 are dispensable for embryonic and subsequent development but contain the potential to provide beneficial information about the genetic background of the oocyte. Therefore, polar bodies can be safely removed from the oocyte and used for PGD or PGS in IVF, with the aim to select a healthy oocyte. Previously, the main methods for PGD or PGS included FISH [46], comprehensive chromosome screening of aneuploidy [47], single-nucleotide polymorphism (SNP) array [48, 49], array-based CGH [50, 51], and quantitative real-time PCR [52]. However, these methods cannot detect aneuploidy and single-nucleotide variants (SNVs), which are related to mendelian disease, at the same time. Very recently, an exciting publication reported single-cell genome analysis on human oocytes [19]. Before that, the nonuniformity of single-cell whole-genome amplification limited its use. Hou et al. [19] utilized wholegenome analysis of single human oocytes by multiple annealing and looping-based amplification cycle (MALBAC)-based sequencing technique. In principle, with the

Since the birth of the first in vitro fertilization (IVF) baby in 1978, how to improve the outcomes of assisted reproductive technologies has been the focus of reproductive medicine. The major reason for natural pregnancy loss and IVF failure is chromosomal aneuploidy [22, 23]. Maternal meiotic errors explain most of the chromosomal aneuploidies [24–26], and about 80% of chromosomal aneuploidies observed in embryos may be caused by chromosome segregation errors during the first meiosis of oocytes. Polar body diagnosis (PBD) provides a noninvasive diagnostic protocol for the indirect genetic analysis of the oocyte, which allows prediction of the related genetic material of the maternal contribution to the early embryos [27]. The PB1 includes the counterpart of the genetic materials present in the oocyte. The PB2 may be used to validate the chromosome defects and those raised during the second meiosis as well as to detect crossovers between homologous chromosomes. Since the first report of PBD by Verlinsky et al. [28], numerous studies using a variety of approaches have been employed to detect chromosomal errors in polar bodies. Fluorescence in situ hybridization (FISH), which employs fluorescently labeled probes binding to their target sequences, has been introduced for the detection of chromosomal aneuploidy [29]. PCR has been used for the diagnosis of single-gene-associated abnormalities. The sensitivity based on PCR approaches has been demonstrated to be increased by 1000-fold compared to that using traditional approaches, such as fluorescent probes [30]. However, it is limited by amplification failure and contamination. Comparative genomic hybridization (CGH) has become a widely used method for its high throughput potential and superior speed [31]. CGH screens the whole genome of the polar body, but polyploidy and balanced translocations cannot be identified by this technique [32, 33]. Because the analysis of polar bodies does not always reflect the status of oocytes and embryos, the value of polar body biopsy has remained controversial [34]. Consistent with this information, it has been shown that about half of the single chromatid errors detected in the PB1 can result in development of a normal embryo, which may be discarded inadvertently [35]. It has been reported that polar body analysis by array CGH could accurately reflect the maternal meiotic origin of aneuploidy in cleavage-stage embryos [36]. However, another study showed that preimplantation genetic screening (PGS) based on polar bodies poorly predicted the embryo’s ploidy and reproductive potential [37]. Another question of impor-

NEW ROLES OF POLAR BODIES IN REPRODUCTION

FIG. 1. Schematic charts for genome analyses of single human oocytes. During the meiotic process of human oocytes, homologous recombination occurs before chromosome segregation. The PB1 and PB2, which are dispensable for embryo development, are used for whole-genome singlecell sequencing. Based on the principle that the total genomes of the PB1, PB2, and female pronucleus from the same oocyte are two copies of both maternal and paternal DNA, the genome of the oocyte can be accurately deduced from the genomes of the PB1 and PB2. If the genomic information shows aneuploidy or disease-associated alleles, it is inappropriate to perform embryo transfer. Otherwise, embryo transfer will result in a healthy baby.

information of the donor’s haplotypes, the female pronucleus can be easily deduced from that of the PB1 and PB2. The deduction is based on the fact that the total genomes of the PB1, PB2, and female pronucleus from the same oocyte are two copies of both maternal and paternal DNA. Therefore, by sequencing the PB1 and PB2, the alleles of the female pronucleus can be deduced (Fig. 1). Previous studies showed that human oocytes have a higher frequency of crossover than that of sperm [53, 54]. However, those studies relied mainly on genetic linkage analysis based on family pedigree, which may 3

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be affected by the selection [53, 54]. The cytological assay of homologous recombination in human oocytes can detect the crossover number and distribution at single-cell levels, but the resolution is very low [54]. By sequencing the triads of the PB1 and PB2 and the oocyte’s pronuclei, Hou et al. [19] phased the genomes with detected SNPs and carried out the first crossover mapping of human oocytes at high resolution. The nonrandomly distributed crossovers can implicate the crossover interference along the genome [55, 56]. The chromatid interference is another type of genetic interference. It refers to a situation in which the occurrence of a crossover between any two nonsister chromatids can affect the probability of those chromatids being involved in other crossovers in the same meiosis [57, 58]. By simultaneously sequencing the PB1 and PB2 and the female pronucleus, Hou et al. [19] resolved this issue and found an expected crossover interference and a weak chromatid interference. Taken together, these findings showed that the genome of the oocyte pronucleus, which included information related to aneuploidy and SNP in diseaseassociated alleles, can be accurately deduced by sequencing the genomes of the PB1 and PB2. Future studies will need to determine whether whole-genome analysis of single human oocytes based on the MALBAC technique will enable accurate and cost-effective selection of high-quality embryos for transfer. Moreover, increasing evidence suggests that many human diseases may result from recent emergence of rare genetic variations [59–62]. Whole-genome sequencing of polar bodies uniquely offers the possibility of identifying rare genetic variants, which may be potentially as important as diseaserelated SNPs. The genome coverage for single oocyte sequencing at 13 is approximately 32%, whereas this rate for a single sperm cell is approximately 20% [18]. This may be caused by the looser chromatin structures in oocytes compared to sperm. Although embryo biopsies are more widely used in PGD or PGS, the polar body biopsy has certain advantages, especially if aneuploidy or the disease-associated allele is inherited from the mother. On the one hand, polar body biopsy is carried out on oocytes rather than on embryos; thus, more time is available for sequencing and analysis. This may avoid freezing of embryos. However, it should be noted that recent evidence suggests freezing of embryos may enhance implantation and reduce IVF-associated obstetrical complications [63, 64]. On the other hand, the polar body biopsy removes only the redundant genetic material that is dispensable for subsequent development, whereas embryo biopsy removes cells from the developing embryo. However, polar body biopsy has disadvantages as well. It cannot detect aneuploidy or mutations inherited from the father, and it also cannot detect the aneuploidy and mutations arising from mitosis. In principle, the MALBAC sequencing method can also be applied to blastocysts, considering the genetic variants that are associated with human disease or aneuploidy may come from the father or embryonic mitosis. The cost of single-oocyte analysis by nextgeneration sequencing is comparable or even less than the CGH array. With the sequencing costs decreasing rapidly, single-oocyte evaluation will gain advantages in the future. While identification of genetic variants in polar bodies with the single-cell whole-genome sequencing approach provides opportunities for deducing the genotype of the oocyte and prediction of disease susceptibility, it also poses a major challenge. Individual oocytes will differ by millions of single nucleotides as well as by thousands of copy number variants and insertions and deletions. In the vast ocean of genetic variants, what would be the information each one carries? Which of them could be effectively used to predict the

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NEW ROLES FOR THE POLAR BODY IN PREVENTING TRANSMISSION OF INHERITED MITOCHONDRIAL DISEASES With few exceptions, mtDNA is maternally inherited in mammals [65, 66]. Because the mitochondrial genome encodes the 13 polypeptides plus tRNAs and rRNAs, which are essential for oxidative phosphorylation, mitochondrial diseases are usually devastating but very rare [67]. Because escape of free electrons from the electron-transport chain produces reactive oxygen species, the mitochondrial genome is more susceptible to damage than the nuclear genome [68]. A common feature of maternally inherited mitochondrial disease is heteroplasmy, which refers to mixing of the mutated and wild-type mtDNA in the same cell. The heteroplasmy level affects the clinical phenotypes. In humans, patients with more than 60% mutated mtDNA may develop severe systemic disease, such as cancer, diabetes, heart disease, blindness, deafness, liver failure, infertility, and migraine [67, 69, 70]. The incidence of clinically present mtDNA disorders is at least 1 in 10 000 individuals, but the frequency of the pathogenic mtDNA mutation is approximately 1 in 200 of the general population [71]. At present, inherited mitochondrial diseases are incurable, and most of the treatments are supportive. Although some characteristics of mitochondrial genetics, including ‘‘bottleneck’’ segregation [72–74] and selective replication [75, 76], may bring about difficulty in the detection of heteroplasmy levels [77, 78], evidence suggests that PGD can be used to accurately examine mtDNA heteroplasmy in human preimplantation embryos [79]. However, clinical experience remains limited at present, and whether PGD always represents an effective solution remains uncertain [78, 80]. Thus, new approaches that can prevent the transmission of mtDNA from mother to offspring are highly desirable. The U.S. Food and Drug Administration and the U.K. Human Fertilization and Embryology Authority have been committed to the development of new strategies to prevent mtDNA disease. Mitochondrial replacement, which refers to transferring the nuclear genome from the patient’s oocyte to the enucleated healthy oocyte, is a hopeful and promising technique [81]. In mice, previous studies showed that pronuclear transfer (PNT) between zygotes can correct mtDNA-related phenotypes [82]. However, PNT-generated mice possessed 6%–21% heteroplasmic mtDNA (average, 11%) at the weaned stage, and the average increase was 12% to possess 5%–44% heteroplasmic mtDNA (average, 23%) at Day 300 after birth [82]. In humans, previous studies showed that PNT between zygotes resulted in minor donor mtDNA carryover (,2.0%) in early embryos [83]. However, one disadvantage of PNT is that the manipulation, which requires both donor and recipient fertilized eggs, discards half of the embryos. Another technique, spindle-chromosome transfer, has been performed in both nonhuman primate and early human embryos to eliminate the maternal inheritance of mtDNA mutations [84–87]. These findings suggest minimal mutated mtDNA carryover in nonhuman primate offspring and human preimplantation embryos. However, the spindle is very sensitive to micromanipulation, which frequently induces 4

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premature activation of oocytes and results in karyotype abnormalities [84]. Recently, a study found that the PB1 and PB2 can be used as donor genomes to replace the genome of the recipient oocytes to prevent inherited mtDNA disease [21]. The polar body contains few mitochondria but, theoretically, shares the same genome as the oocyte. Moreover, as discussed previously, single-cell genome analysis of human oocytes indicates that the PB1 and PB2 potentially have the same genome as their corresponding oocyte [19, 36]. Indeed, polar body transfer may have several advantages. First, the PB1 or PB2 contains few mitochondria but carries the entire genome [4, 88]. Therefore, a minimal carryover of donor mtDNA is expected in reconstructed embryos and offspring generated by polar body transfer. Second, the PB1 or PB2 are separated from the oocyte and can be easily manipulated without chromosome damage. Finally, each donor egg has a PB1, a PB2, and a maternal pronucleus. All can be used as donor nuclei and thus significantly increase the efficiency of the use of donor eggs. In a recent study, Wang et al. [21] compared the effects of four types of genome transfer in mice: PB1 transfer, PB2 transfer, spindle-chromosome transfer, and PNT (Fig. 2). All types of reconstructed embryos supported normal development and production of live offspring. PB1 transfer generated undetectable levels of mtDNA heteroplasmy in all offspring. The easy visualization and convenient manipulation of the PB1 suggests the feasibility of PB1 transfer as an effective approach. Indeed, the manipulation of PB1 transfer is more similar to that of intracytoplasmic sperm injection, which requires only a single step [89]. Similarly, PB2 transfer also generated offspring with little or even undetectable levels of mtDNA heteroplasmy [21]. However, the disadvantage of PB2 transfer is that the recipient egg should be female pronucleus-enucleated, because the PB2 only includes a haploid set of the maternal genome. This will pose technical challenges. The spindle-chromosome transfer produced offspring with low to medium levels of mtDNA heteroplasmy [21]. Because the human spindle is smaller than that of the mouse, human spindle-chromosome transfer is expected to result in less variant carryover. The PNT-derived offspring had the highest level (23.7%) of donor mtDNA carryover. This may be primarily caused by zygotic activation, which leads to mtDNA amplification around the pronucleus [72, 90], and is consistent with previous reports of a similar level of mtDNA heteroplasmy after PNT [82, 91]. Taken together, these data suggest that polar body transfer results in minimal donor mtDNA carryover compared to other methods. Importantly, the F2 generation derived from polar body transfer still maintains the minimal donor mtDNA variants [21]. Furthermore, the array CGH study in single oocytes indicates that in a normal oocyte, both the PB1 and the spindlechromosome complex have a diploid genome with no alterations whereas the PB2 has the same haploid genome as the female pronucleus [21]. This is consistent with previous studies showing that the polar bodies and the oocyte potentially share the same genomic landscape [19, 36]. Considered together, these findings indicate that polar body transfer may have great potential in the near future to prevent inheritance of mitochondrial disease. While polar body transfer offers beneficial protection from mitochondrial diseases, it also poses major challenges and risks. First, maternal inheritance of mitochondria represents a natural selection in that the mitochondrial gene pool can only be shaped via the female germline [65]. Polar body transfer alters this process and changes the mitochondrial gene pool of humans. Because mitochondrial replacement involves germline genetic modification, it can be inheritable and can possibly

developmental potential and/or disease susceptibility? How would these signals be separated from noise? Single-cell sequencing technology with MALBAC and other approaches represents only the beginning. There is a long way to go for the new single-cell sequencing technique in the application of reproductive biology and reproductive medicine.


Downloaded from www.biolreprod.org. FIG. 2. Comparison of four types of genome transfer to prevent transmission of mitochondrial disease. Different colors (red and blue) within the mitochondria indicate different mitochondrial genotypes. All types of reconstructed embryos support normal development and can produce live offspring for at least two generations. PB1 transfer results in undetectable levels of mtDNA variants in offspring for at least two generations. Spindle-chromosome transfer leads to low to medium levels of mtDNA heteroplasmy in both the first and second generation of offspring. PB2 transfer generated offspring with undetectable to very low donor mtDNA variants. PNT generated offspring with the highest donor mtDNA carryover.

affect future generations. Second, coordinated mitochondrialnuclear genome interactions have become highly specific during evolution [92]. Mitochondrial replacement would disrupt such highly specific and coordinated interactions due to the incompatibility between unmatched nuclear and mitochondrial genomes [93]. Third, polar body transfer may induce epigenetic alterations in offspring and subsequent generations. Assisted reproductive technology has been reported to be associated with an increased incidence of epigenetic disorders, such as Angelman syndrome and Beckwith-Wiedemann syndrome [94]. Somatic cell nuclear transfer has been well-characterized for epigenetic reprogramming errors [95]. Whether polar body transfer increases the risk of epigenetic disorders in offspring and subsequent generations requires further investigation. It will be important to study

epigenomic patterns of human preimplantation embryos generated by polar body transfer to confirm the consistency of epigenetic models between those generated by polar body transfer and normal ones. It will also be helpful to analyze epigenetic profiling in different tissues of offspring derived from polar body transfer. QUESTIONS AND FUTURE PERSPECTIVES These emerging findings support the idea that besides traditional roles in PBD, polar bodies may play multiple important roles in assisted reproductive technology, including deducing the genome of the oocyte by polar body single-cell sequencing and preventing transmission of mtDNA disease by polar body transfer. These findings provide encouragement to 5

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with development of the high-throughput sequencing technique, single-cell-based epigenomic profiling will be helpful to further resolve this issue. If the polar body does carry the same epigenetic information as its sibling oocyte, there may be important implications for human reproductive health. As indicated above, gametic epigenetics is very important in determining the health states and nongenetic disease susceptibility of offspring. Identification of epigenetic marks in oocytes that represent the good or bad information that mothers are going to transmit to their offspring is a potential strategy to prevent nongenetic diseases. The rapid improvement of high-throughput sequencing makes the genome-wide identification of such markers feasible. Once we have established the epigenetic fingerprint in oocytes, nongenetic diseases can be deduced from the epigenetics of its sibling polar body. Such epigenetic diagnosis holds great promise to predict susceptibility to certain nongenetic diseases, and it will be helpful in the prevention of certain epigeneticassociated disorders. FIG. 3. Key questions associated with polar bodies remain unresolved. The polar body contains both genetic material and epigenetic information (e.g., DNA methylation, noncoding RNAs, and chromatin proteins). Many fundamental questions are associated with polar bodies that require further clarification: 1) Whether the polar body has the same incidence of DNA mutation and DNA damage as that of its sibling oocyte, 2) whether the polar body carries the same epigenetic information as that of its sibling oocyte, 3) whether the polar body transfer leads to epigenetic alterations in offspring and subsequent generations, and 4) whether epigenetic marks can be identified in polar bodies to predict and prevent the transmission of specific nongenetic diseases.

REFERENCES

further explore even more potential roles of polar bodies in human reproductive health. However, they also raises many fundamental questions that require additional clarifications (Fig. 3). A question of central importance is whether the incidence of DNA mutations and DNA impairment (e.g., double-strand breaks) in polar bodies is identical to that in the sibling oocyte (Fig. 3), or whether a possibility exists that the abandoned polar bodies carry genomes with more defects (e.g., higher incidence of DNA mutations). Detailed analysis of singleoocyte sequencing data will be useful to resolve this question. Another key question concerns whether the polar body carries the same epigenomes as its sibling oocyte (Fig. 3). Epigenomic alterations include a series of chromatin and DNA modifications, such as cytosine methylation and histone modification. Other epigenetic regulations involve noncoding RNAs (e.g., siRNAs, miRNAs, and piRNAs) and regulation by a higher-level organization of the chromatin. Increasing evidence suggests that epigenetic information can be inherited between generations [96–98]. Moreover, our increased knowledge of epigenetic reprogramming indicates that epigenetic modifications are not always completely erased between generations [99–102]. If the epigenetic information is not identical between the polar body and its sibling oocyte, an important concern of polar body transfer is the epigenetic problem. Partial inheritance of epigenetic marks on certain genes involved in significant phenotypes may induce unexpected patterns of inheritance between generations. Although the results from polar body transfers showed that the reconstructed embryos did not exhibit a significant decrease in developmental potential, whether polar body transfer induces epigenetic alterations in offspring requires further investigation. Recently, epigenomic profiling has been achieved in a few human preimplantation embryos [103] and even single mouse embryonic stem cells [104]. We believe that 6

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1. Cahill DJ, Wardle PG, Harlow CR, Hull MG. Onset of the preovulatory luteinizing hormone surge: diurnal timing and critical follicular prerequisites. Fertil Steril 1998; 70:56–59. 2. Sun QY, Schatten H. Regulation of dynamic events by microfilaments during oocyte maturation and fertilization. Reproduction 2006; 131: 193–205. 3. Messinger SM, Albertini DF. Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte. J Cell Sci 1991; 100(Pt 2):289–298. 4. Steuerwald N, Barritt JA, Adler R, Malter H, Schimmel T, Cohen J, Brenner CA. Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR. Zygote 2000; 8:209–215. 5. Fabritius AS, Ellefson ML, McNally FJ. Nuclear and spindle positioning during oocyte meiosis. Curr Opin Cell Biol 2011; 23:78–84. 6. Montag M, Koster M, Strowitzki T, Toth B. Polar body biopsy. Fertil Steril 2013; 100:603–607. 7. Tsai MC, Takeuchi T, Bedford JM, Reis MM, Rosenwaks Z, Palermo GD. Alternative sources of gametes: reality or science fiction? Hum Reprod 2000; 15:988–998. 8. Eichenlaub-Ritter U, Schmiady H, Kentenich H, Soewarto D. Recurrent failure in polar body formation and premature chromosome condensation in oocytes from a human patient: indicators of asynchrony in nuclear and cytoplasmic maturation. Hum Reprod 1995; 10:2343–2349. 9. Ebner T, Yaman C, Moser M, Sommergruber M, Feichtinger O, Tews G. Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum Reprod 2000; 15:427–430. 10. Ebner T, Moser M, Sommergruber M, Yaman C, Pfleger U, Tews G. First polar body morphology and blastocyst formation rate in ICSI patients. Hum Reprod 2002; 17:2415–2418. 11. Gardner RL. The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal-vegetal axis of the zygote in the mouse. Development 1997; 124:289–301. 12. Ciemerych MA, Mesnard D, Zernicka-Goetz M. Animal and vegetal poles of the mouse egg predict the polarity of the embryonic axis, yet are nonessential for development. Development 2000; 127:3467–3474. 13. Scott KL, Hong KH, Scott RT Jr. Selecting the optimal time to perform biopsy for preimplantation genetic testing. Fertil Steril 2013; 100: 608–614. 14. Wakayama T, Yanagimachi R. The first polar body can be used for the production of normal offspring in mice. Biol Reprod 1998; 59:100–104. 15. Verlinsky Y, Kuliev A. Preimplantation Genetics. New York: Plenum Press; 1991. 16. Verlinsky Y, Kuliev A. Preimplantation Diagnosis of Genetic Diseases: A New Technique in Assisted Reproduction. New York: Wiley-Liss; 1993. 17. Kuliev A, Rechitsky S, Verlinsky O, Verlinsky Y. Atlas of Preimplantation Genetic Diagnosis. New York: Parthenon; 2000. 18. Lu S, Zong C, Fan W, Yang M, Li J, Chapman AR, Zhu P, Hu X, Xu L, Yan L, Bai F, Qiao J, et al. Probing meiotic recombination and


19.

20.

21.

22. 23.

24. 25.

26.

27.

29.

30. 31.

32. 33.

34.

35.

36.

37.

38. 39.

40.

41.

42.

43.

44.

45.

46. 47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58. 59.

60.

61.

62.

63.

7

Verlinsky Y. Reliability of preimplantation diagnosis for single gene disorders. Mol Cell Endocrinol 2001; 183(suppl 1):S65–S68. Thornhill AR, McGrath JA, Eady RA, Braude PR, Handyside AH. A comparison of different lysis buffers to assess allele dropout from single cells for preimplantation genetic diagnosis. Prenat Diagn 2001; 21: 490–497. Wang J, Fan HC, Behr B, Quake SR. Genome-wide single-cell analysis of recombination activity and de novo mutation rates in human sperm. Cell 2012; 150:402–412. Zong C, Lu S, Chapman AR, Xie XS. Genome-wide detection of singlenucleotide and copy-number variations of a single human cell. Science 2012; 338:1622–1626. Harper JC, Delhanty JD. FISH in preimplantation diagnosis. Methods Mol Med 1996; 5:259–268. Wu MY, Chao KH, Chen CD, Chang LJ, Chen SU, Yang YS. Current status of comprehensive chromosome screening for elective singleembryo transfer. Obstet Gynecol Int 2014; 2014:581783. Nannya Y, Sanada M, Nakazaki K, Hosoya N, Wang L, Hangaishi A, Kurokawa M, Chiba S, Bailey DK, Kennedy GC, Ogawa S. A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays. Cancer Res 2005; 65: 6071–6079. Wong KK, Tsang YT, Shen J, Cheng RS, Chang YM, Man TK, Lau CC. Allelic imbalance analysis by high-density single-nucleotide polymorphic allele (SNP) array with whole genome amplified DNA. Nucleic Acids Res 2004; 32:e69. Geraedts J, Montag M, Magli MC, Repping S, Handyside A, Staessen C, Harper J, Schmutzler A, Collins J, Goossens V, van der Ven H, Vesela K, et al. Polar body array CGH for prediction of the status of the corresponding oocyte. Part I: clinical results. Hum Reprod 2011; 26: 3173–3180. Magli MC, Montag M, Koster M, Muzi L, Geraedts J, Collins J, Goossens V, Handyside AH, Harper J, Repping S, Schmutzler A, Vesela K, et al. Polar body array CGH for prediction of the status of the corresponding oocyte. Part II: technical aspects. Hum Reprod 2011; 26: 3181–3185. Treff NR, Tao X, Ferry KM, Su J, Taylor D, Scott RT Jr. Development and validation of an accurate quantitative real-time polymerase chain reaction-based assay for human blastocyst comprehensive chromosomal aneuploidy screening. Fertil Steril 2012; 97:819–824. Broman KW, Murray JC, Sheffield VC, White RL, Weber JL. Comprehensive human genetic maps: individual and sex-specific variation in recombination. Am J Hum Genet 1998; 63:861–869. Cheng EY, Hunt PA, Naluai-Cecchini TA, Fligner CL, Fujimoto VY, Pasternack TL, Schwartz JM, Steinauer JE, Woodruff TJ, Cherry SM, Hansen TA, Vallente RU, et al. Meiotic recombination in human oocytes. PLoS Genet 2009; 5:e1000661. Jeffreys AJ, Murray J, Neumann R. High-resolution mapping of crossovers in human sperm defines a minisatellite-associated recombination hotspot. Mol Cell 1998; 2:267–273. Fung JC, Rockmill B, Odell M, Roeder GS. Imposition of crossover interference through the nonrandom distribution of synapsis initiation complexes. Cell 2004; 116:795–802. Chen SY, Tsubouchi T, Rockmill B, Sandler JS, Richards DR, Vader G, Hochwagen A, Roeder GS, Fung JC. Global analysis of the meiotic crossover landscape. Dev Cell 2008; 15:401–415. Zhao H, McPeek MS, Speed TP. Statistical analysis of chromatid interference. Genetics 1995; 139:1057–1065. Marth GT, Yu F, Indap AR, Garimella K, Gravel S, Leong WF, TylerSmith C, Bainbridge M, Blackwell T, Zheng-Bradley X, Chen Y, Challis D, et al. The functional spectrum of low-frequency coding variation. Genome Biol 2011; 12:R84. Mu XJ, Lu ZJ, Kong Y, Lam HY, Gerstein MB. Analysis of genomic variation in non-coding elements using population-scale sequencing data from the 1000 Genomes Project. Nucleic Acids Res 2011; 39: 7058–7076. Tennessen JA, Bigham AW, O’Connor TD, Fu W, Kenny EE, Gravel S, McGee S, Do R, Liu X, Jun G, Kang HM, Jordan D, et al. Evolution and functional impact of rare coding variation from deep sequencing of human exomes. Science 2012; 337:64–69. Nelson MR, Wegmann D, Ehm MG, Kessner D, St Jean P, Verzilli C, Shen J, Tang Z, Bacanu SA, Fraser D, Warren L, Aponte J, et al. An abundance of rare functional variants in 202 drug target genes sequenced in 14 002 people. Science 2012; 337:100–104. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C. Freezeall can be a superior therapy to another fresh cycle in patients with prior

Article 19


28.

aneuploidy of single sperm cells by whole-genome sequencing. Science 2012; 338:1627–1630. Hou Y, Fan W, Yan L, Li R, Lian Y, Huang J, Li J, Xu L, Tang F, Xie XS, Qiao J. Genome analyses of single human oocytes. Cell 2013; 155: 1492–1506. Streets AM, Zhang X, Cao C, Pang Y, Wu X, Xiong L, Yang L, Fu Y, Zhao L, Tang F, Huang Y. Microfluidic single-cell whole-transcriptome sequencing. Proc Natl Acad Sci U S A 2014; 111:7048–7053. Wang T, Sha H, Ji D, Zhang HL, Chen D, Cao Y, Zhu J. Polar body genome transfer for preventing the transmission of inherited mitochondrial diseases. Cell 2014; 157:1591–1604. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2001; 2:280–291. Spandorfer SD, Davis OK, Barmat LI, Chung PH, Rosenwaks Z. Relationship between maternal age and aneuploidy in in vitro fertilization pregnancy loss. Fertil Steril 2004; 81:1265–1269. Hassold T, Hall H, Hunt P. The origin of human aneuploidy: where we have been, where we are going. Hum Mol Genet 2007; 16:R203–R208. Qiao J, Wang ZB, Feng HL, Miao YL, Wang Q, Yu Y, Wei YC, Yan J, Wang WH, Shen W, Sun SC, Schatten H, et al. The root of reduced fertility in aged women and possible therapeutic options: current status and future prospects. Mol Aspects Med 2014; 38:54–85. Wang ZB, Schatten H, Sun QY. Why is chromosome segregation error in oocytes increased with maternal aging? Physiology (Bethesda) 2011; 26: 314–325. Montag M, van der Ven K, Rosing B, van der Ven H. Polar body biopsy: a viable alternative to preimplantation genetic diagnosis and screening. Reprod Biomed Online 2009; 18(suppl 1):6–11. Verlinsky Y, Ginsberg N, Lifchez A, Valle J, Moise J, Strom CM. Analysis of the first polar body: preconception genetic diagnosis. Hum Reprod 1990; 5:826–829. Verlinsky Y, Cieslak J, Ivakhnenko V, Evsikov S, Wolf G, White M, Lifchez A, Kaplan B, Moise J, Valle J, Ginsberg N, Strom C, et al. Preimplantation diagnosis of common aneuploidies by the first- and second-polar body FISH analysis. J Assist Reprod Genet 1998; 15: 285–289. Sermon K. Current concepts in preimplantation genetic diagnosis (PGD): a molecular biologist’s view. Hum Reprod Update 2002; 8:11–20. Wells D, Alfarawati S, Fragouli E. Use of comprehensive chromosomal screening for embryo assessment: microarrays and CGH. Mol Hum Reprod 2008; 14:703–710. Harper JC, Sengupta SB. Preimplantation genetic diagnosis: state of the art 2011. Hum Genet 2012; 131:175–186. Gabriel AS, Thornhill AR, Ottolini CS, Gordon A, Brown AP, Taylor J, Bennett K, Handyside A, Griffin DK. Array comparative genomic hybridization on first polar bodies suggests that non-disjunction is not the predominant mechanism leading to aneuploidy in humans. J Med Genet 2011; 48:433–437. Harper J, Coonen E, De Rycke M, Fiorentino F, Geraedts J, Goossens V, Harton G, Moutou C, Pehlivan Budak T, Renwick P, Sengupta S, Traeger-Synodinos J, et al. What next for preimplantation genetic screening (PGS)? A position statement from the ESHRE PGD Consortium Steering Committee. Hum Reprod 2010; 25:821–823. Scott RT Jr, Treff NR, Stevens J, Forman EJ, Hong KH, Katz-Jaffe MG, Schoolcraft WB. Delivery of a chromosomally normal child from an oocyte with reciprocal aneuploid polar bodies. J Assist Reprod Genet 2012; 29:533–537. Christopikou D, Tsorva E, Economou K, Shelley P, Davies S, Mastrominas M, Handyside AH. Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age. Hum Reprod 2013; 28:1426–1434. Salvaggio CN, Forman EJ, Garnsey HM, Treff NR, Scott RT Jr. Polar body based aneuploidy screening is poorly predictive of embryo ploidy and reproductive potential. J Assist Reprod Genet 2014; 31:1221–1226. Kuliev A, Rechitsky S. Polar body-based preimplantation genetic diagnosis for mendelian disorders. Mol Hum Reprod 2011; 17:275–285. Levin I, Almog B, Shwartz T, Gold V, Ben-Yosef D, Shaubi M, Amit A, Malcov M. Effects of laser polar-body biopsy on embryo quality. Fertil Steril 2012; 97:1085–1088. Klatsky PC, Wessel GM, Carson SA. Detection and quantification of mRNA in single human polar bodies: a minimally invasive test of gene expression during oogenesis. Mol Hum Reprod 2010; 16:938–943. Reich A, Klatsky P, Carson S, Wessel G. The transcriptome of a human polar body accurately reflects its sibling oocyte. J Biol Chem 2011; 286: 40743–40749. Rechitsky S, Verlinsky O, Amet T, Rechitsky M, Kouliev T, Strom C,

WEI ET AL.

64. 65. 66. 67. 68. 69.

70. 71. 72.

74. 75.

76. 77. 78.

79. 80.

81. 82.

83.

84.

85.

86.

87.

88.

89. 90.

91.

92. 93. 94. 95.

96.

97.

98. 99.

100.

101.

102.

103.

104.

8

M, Turnbull DM. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 2010; 465:82–85. Paull D, Emmanuele V, Weiss KA, Treff N, Stewart L, Hua H, Zimmer M, Kahler DJ, Goland RS, Noggle SA, Prosser R, Hirano M, et al. Nuclear genome transfer in human oocytes eliminates mitochondrial DNA variants. Nature 2013; 493:632–637. Tachibana M, Amato P, Sparman M, Woodward J, Sanchis DM, Ma H, Gutierrez NM, Tippner-Hedges R, Kang E, Lee HS, Ramsey C, Masterson K, et al. Towards germline gene therapy of inherited mitochondrial diseases. Nature 2013; 493:627–631. Tachibana M, Sparman M, Sritanaudomchai H, Ma H, Clepper L, Woodward J, Li Y, Ramsey C, Kolotushkina O, Mitalipov S. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature 2009; 461:367–372. Liu L, Oldenbourg R, Trimarchi JR, Keefe DL. A reliable, noninvasive technique for spindle imaging and enucleation of mammalian oocytes. Nat Biotechnol 2000; 18:223–225. Dalton CM, Carroll J. Biased inheritance of mitochondria during asymmetric cell division in the mouse oocyte. J Cell Sci 2013; 126: 2955–2964. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52:709–720. Santos TA, El Shourbagy S, St John JC. Mitochondrial content reflects oocyte variability and fertilization outcome. Fertil Steril 2006; 85: 584–591. Meirelles FV, Smith LC. Mitochondrial genotype segregation in a mouse heteroplasmic lineage produced by embryonic karyoplast transplantation. Genetics 1997; 145:445–451. Woodson JD, Chory J. Coordination of gene expression between organellar and nuclear genomes. Nat Rev Genet 2008; 9:383–395. Reinhardt K, Dowling DK, Morrow EH. Medicine. Mitochondrial replacement, evolution, and the clinic. Science 2013; 341:1345–1346. Manipalviratn S, DeCherney A, Segars J. Imprinting disorders and assisted reproductive technology. Fertil Steril 2009; 91:305–315. Yang X, Smith SL, Tian XC, Lewin HA, Renard JP, Wakayama T. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet 2007; 39:295–302. Wei Y, Yang CR, Wei YP, Zhao ZA, Hou Y, Schatten H, Sun QY. Paternally induced transgenerational inheritance of susceptibility to diabetes in mammals. Proc Natl Acad Sci U S A 2014; 111:1873–1878. Daxinger L, Whitelaw E. Understanding transgenerational epigenetic inheritance via the gametes in mammals. Nat Rev Genet 2012; 13: 153–162. Heard E, Martienssen RA. Transgenerational epigenetic inheritance: myths and mechanisms. Cell 2014; 157:95–109. Borgel J, Guibert S, Li Y, Chiba H, Schubeler D, Sasaki H, Forne T, Weber M. Targets and dynamics of promoter DNA methylation during early mouse development. Nat Genet 2010; 42:1093–1100. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. Distinctive chromatin in human sperm packages genes for embryo development. Nature 2009; 460:473–478. Smallwood SA, Tomizawa S, Krueger F, Ruf N, Carli N, SegondsPichon A, Sato S, Hata K, Andrews SR, Kelsey G. Dynamic CpG island methylation landscape in oocytes and preimplantation embryos. Nat Genet 2011; 43:811–814. Wang L, Zhang J, Duan J, Gao X, Zhu W, Lu X, Yang L, Li G, Ci W, Li W, Zhou Q, Aluru N, et al. Programming and inheritance of parental DNA methylomes in mammals. Cell 2014; 157:979–991. Smith ZD, Chan MM, Humm KC, Karnik R, Mekhoubad S, Regev A, Eggan K, Meissner A. DNA methylation dynamics of the human preimplantation embryo. Nature 2014; 511:611–615. Guo H, Zhu P, Wu X, Li X, Wen L, Tang F. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing. Genome Res 2013; 23: 2126–2135.

Article 19


73.

fresh blastocyst implantation failure. Reprod Biomed Online 2014; 29: 286–290. Shapiro BS, Daneshmand ST, Garner FC, Aguirre M, Hudson C. Clinical rationale for cryopreservation of entire embryo cohorts in lieu of fresh transfer. Fertil Steril 2014; 102:3–9. Hutchison CA III, Newbold JE, Potter SS, Edgell MH. Maternal inheritance of mammalian mitochondrial DNA. Nature 1974; 251: 536–538. Basse CW. Mitochondrial inheritance in fungi. Curr Opin Microbiol 2010; 13:712–719. Poulton J, Chiaratti MR, Meirelles FV, Kennedy S, Wells D, Holt IJ. Transmission of mitochondrial DNA diseases and ways to prevent them. PLoS Genet 2010; 6:e1001066. Wallace DC. Mitochondrial DNA sequence variation in human evolution and disease. Proc Natl Acad Sci U S A 1994; 91:8739–8746. Brown DT, Herbert M, Lamb VK, Chinnery PF, Taylor RW, Lightowlers RN, Craven L, Cree L, Gardner JL, Turnbull DM. Transmission of mitochondrial DNA disorders: possibilities for the future. Lancet 2006; 368:87–89. Schon EA, DiMauro S, Hirano M. Human mitochondrial DNA: roles of inherited and somatic mutations. Nat Rev Genet 2012; 13:878–890. Chinnery PF, Elliott HR, Hudson G, Samuels DC, Relton CL. Epigenetics, epidemiology and mitochondrial DNA diseases. Int J Epidemiol 2012; 41:177–187. Cao L, Shitara H, Horii T, Nagao Y, Imai H, Abe K, Hara T, Hayashi J, Yonekawa H. The mitochondrial bottleneck occurs without reduction of mtDNA content in female mouse germ cells. Nat Genet 2007; 39: 386–390. Cree LM, Samuels DC, de Sousa Lopes SC, Rajasimha HK, Wonnapinij P, Mann JR, Dahl HH, Chinnery PF. A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes. Nat Genet 2008; 40:249–254. Wai T, Teoli D, Shoubridge EA. The mitochondrial DNA genetic bottleneck results from replication of a subpopulation of genomes. Nat Genet 2008; 40:1484–1488. Fan W, Waymire KG, Narula N, Li P, Rocher C, Coskun PE, Vannan MA, Narula J, Macgregor GR, Wallace DC. A mouse model of mitochondrial disease reveals germline selection against severe mtDNA mutations. Science 2008; 319:958–962. Stewart JB, Freyer C, Elson JL, Wredenberg A, Cansu Z, Trifunovic A, Larsson NG. Strong purifying selection in transmission of mammalian mitochondrial DNA. PLoS Biol 2008; 6:e10. Bredenoord AL, Dondorp W, Pennings G, De Die-Smulders CE, De Wert G. PGD to reduce reproductive risk: the case of mitochondrial DNA disorders. Hum Reprod 2008; 23:2392–2401. Steffann J, Frydman N, Gigarel N, Burlet P, Ray PF, Fanchin R, Feyereisen E, Kerbrat V, Tachdjian G, Bonnefont JP, Frydman R, Munnich A. Analysis of mtDNA variant segregation during early human embryonic development: a tool for successful NARP preimplantation diagnosis. J Med Genet 2006; 43:244–247. Treff NR, Campos J, Tao X, Levy B, Ferry KM, Scott RT Jr. Blastocyst preimplantation genetic diagnosis (PGD) of a mitochondrial DNA disorder. Fertil Steril 2012; 98:1236–1240. Monnot S, Gigarel N, Samuels DC, Burlet P, Hesters L, Frydman N, Frydman R, Kerbrat V, Funalot B, Martinovic J, Benachi A, Feingold J, et al. Segregation of mtDNA throughout human embryofetal development: m.3243A.G as a model system. Hum Mutat 2011; 32:116–125. Wolf DP, Mitalipov S. Mitochondrial replacement therapies can circumvent mtDNA-based disease transmission. Cell Metab 2014; 20: 6–8. Sato A, Kono T, Nakada K, Ishikawa K, Inoue S, Yonekawa H, Hayashi J. Gene therapy for progeny of mito-mice carrying pathogenic mtDNA by nuclear transplantation. Proc Natl Acad Sci U S A 2005; 102: 16765–16770. Craven L, Tuppen HA, Greggains GD, Harbottle SJ, Murphy JL, Cree LM, Murdoch AP, Chinnery PF, Taylor RW, Lightowlers RN, Herbert