Polar bodies in assisted reproductive technology

BOR Papers in Press. Published on December 3, 2014 as DOI:10.1095/biolreprod.114.125575
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Polar bodies in assisted reproductive technology: current progress and future perspectives1
Yanchang Wei,3 Teng Zhang,3 Ya-Peng Wang,3 Heide Schatten,4 Qing-Yuan Sun2,3
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of
Sciences, Beijing, China
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Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri
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Running title: New roles of polar bodies in reproduction
Keywords: polar body; oocyte; assisted reproductive technology; preimplantation genetic
diagnosis; single-cell sequencing; mitochondrial disease
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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].
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ABSTRACT
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 to the offspring. Besides the traditional use in
preimplantation diagnosis, recent emerging findings suggest that there are 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 deducing the genomic information of its sibling oocyte, and 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 more healthy babies.
INTRODUCTION
The unequal divisions during the first and the second meiosis result in a large oocyte and two
diminutive polar bodies (PBs), 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 (LH) 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 contains an average of 3.14 × 105 mitochondria per oocyte [4,
5]. The PB1 contains membranous material, mitochondria, ribosomes, cortical granules, as well
as other cytoplasmic material [6]. While the oocyte progresses to metaphase II (MII), the spindle
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Copyright 2014 by The Society for the Study of Reproduction.
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usually is not well organized, and chromosomes gradually degenerate at late telophase [7],
although the PB1 can divide off the oocyte.
Formation of the second polar body (PB2) occurs after fertilization. Different from PB1 that
contains bivalent chromosomes, PB2 contains a haploid set of chromatids. The chromatids in
PB2 are protected by a nuclear envelope that is more persistent than that in PB1, which
disappears within a few hours in some mouse strains and after 20 hours in humans [8-10].
Usually, 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
PB2’s persisted and remained intact through the early blastocyst stage [11]. Moreover, the
distribution of the surviving PB2 was highly non-random in early blastocysts [11]. Together with
other findings, the evidence indicates that PB2 may also play a role in the polarity patterning of
the developing conceptus [12].
One unique feature of the female gamete is that PBs can provide beneficial information about
the genetic background of the oocyte without potentially destroying it. PB biopsy has been
applied in preimplantation genetic diagnosis (PGD) to detect chromosomal or genetic
abnormalities that might be inherited to the offspring. The majority of PGD clinics perform
biopsies on preimplantation embryos rather than PBs [13]. However, the PB biopsy may have
advantages in certain conditions. Indeed, since the use of the technique, some couples found PB
biopsy morally attractive because it does not disrupt the fertilized embryo. Although the PBs
have limited life spans, they have the potential to support normal development when they are
transferred to an enucleated oocyte [14]. Moreover, since mitochondria segregation is random
during meiosis, it provides an opportunity to use PBs for screening of mitochondrial mutations.
PBs are usually neglected in most cases of reproductive medicine, other than for
preimplantation genetic diagnosis (PGD) [15-17]. They are tiny and contribute to successful
development only by allowing diploidy to be established. However, there are additional roles for
PBs other than just the traditional PGD. An exciting role for PBs is emerging with the
development of new high throughput genome-wide single-cell sequencing techniques [18-20].
Recently, a study reported 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 PBs [19]. Another exciting study recently
reported 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 review, the new roles of PBs in
assisted reproductive technology are reviewed. Although PBs are believed to be genetic discards,
they contain genetic information about the oocyte which can help guide the decision of whether a
given embryo is healthy or not. Moreover, they retain potential for contributing to a new life with
great potential to prevent the inheritance of mtDNA disease.
TRADITIONAL ROLES OF POLAR BODIES IN ASSISTED REPRODUCTIVE
TECHNOLOGY
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 of natural pregnancy loss and IVF failure is chromosomal aneuploidy [22, 23].
Maternal meiotic errors explain most of the chromosomal aneuploidies [24-26], and about eighty
percent 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
non-invasive diagnostic protocol for the indirect genetic analysis of the oocyte, which allows to
predict the related genetic material of the maternal contribution to the early embryos [27]. The
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first polar body includes the counterpart of the genetic materials present in the oocyte. The
second polar body may be used to validate the chromosome defects and those raised during the
second meiosis, as well as detecting 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 for many years [29].
Polymerase chain reaction (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 1,000 fold compared to that using traditional approaches such as fluorescent probes [30].
However, it is limited by the 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].
Since the analysis of PBs 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 first polar body can
result in normal embryo development, which may be discarded inadvertently [35]. It has been
reported previously that the 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 importance 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 had been shown
that mRNAs present in oocytes can also be detected in polar bodies [40]. Importantly, the
transcriptome of the first polar body can accurately reflect that of its sibling oocyte in humans
[41]. Single-cell transcriptome analysis showed that of the 5,431 mRNAs recovered from the
first polar body, 5,256 of them (~97%) shared similar expression levels as its sibling oocyte [41].
This suggests that transcriptional detection and quantification by high throughput techniques
could acquire first-hand information of global gene expression in mature oocytes, and thus lead
to molecular diagnostic applications.
NEW ROLES FOR THE POLAR BODY TO DEDUCE THE GENOME OF THE
OOCYTE
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]. This study tested over 1000 oocytes for single gene defects by using PCR.
A total of 237 unaffected oocytes were pre-selected 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 PB1 and PB2 is a way to avoid undetected ADO.
Advances in whole-genome and whole-transcriptome amplification make sequencing of the
minute amounts of DNA and RNA within a single cell possible [18, 44, 45]. This new technique
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will offer a window into 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 whole-genome single-cell
sequencing analysis. Two years ago, single-cell 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, PBs can be safely removed from the oocyte and
used for preimplantation genetic diagnosis or screening (PGD or PGS) in IVF, with the aim to
select a healthy oocyte. Previously, the main methods for PGD or PGS included fluorescence in
situ hybridization (FISH) [46], comprehensive chromosome screening (CCS) of aneuploidy [47],
single nucleotide polymorphism (SNP) array [48, 49], array based comparative genomic
hybridization (CGH) [50, 51], and quantitative real-time PCR [52]. However, these methods
cannot detect aneuploidy and single nucleotide variants (SNVs, which is related to Mendelian
disease) at the same time. Very recently, an exciting study reported single-cell genomic analysis
on human oocytes [19].
Before that, the nonuniformity of single-cell whole-genome amplification limited its use. In
that study, Hou et al. utilized whole genomic analysis of single human oocytes by multiple
annealing and looping-based amplification cycle (MALBAC)-based sequencing technique [19].
In principle, with the information of the donor’s haplotypes, the female pronucleus can be easily
deduced from that of PB1 and PB2. The deduction is based on the fact that the total genomes of
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 (Figure 1). Previous studies found that human oocytes have a higher frequency
of crossover than that of sperm [53, 54]. However, these studies have mainly relied on genetic
linkage analysis based on family pedigree, which may 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 and his colleagues phased the
genomes with detected SNPs and carried out the first crossover map of human oocytes at high
resolution [19]. 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 non-sister
chromatids can affect the probability of those chromatids being involved in other crossovers in
the same meiosis [57, 58]. By simultaneously sequencing PB1 and PB2 and the female
pronucleus, Hou and his colleagues resolved this issue and found an expected crossover
interference and a weak chromatid interference [19]. Together, their findings showed that the
genome of the oocyte pronucleus which included information related to aneuploidy and SNP in
disease-associated alleles, can be accurately deduced by sequencing the genomes of 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 PBs uniquely offers the possibility of identifying rare genetic variants, which may
be potentially as important as disease related SNPs.
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The genome coverage for single oocyte sequencing at 1 × is ~32%, whereas this rate for a
single sperm cell is ~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 PB
biopsy has certain advantages especially if aneuploidy or the disease-associated allele is inherited
from the mother. On the one hand, PB biopsy is carried out on oocytes rather than 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 that freezing of embryos may enhance
implantation and reduce IVF associated obstetrical complications [63, 64]. On the other hand, the
PB biopsy removes only the redundant genetic material that is dispensable for subsequent
development, whereas embryo biopsy removes cells from the developing embryo. However,
there are also disadvantages of PB biopsy. It cannot detect aneuploidy or mutations inherited
from the father, and it can also not 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. Single oocyte analysis by next-generation sequencing is costly,
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 PBs 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 (indels). In the vast ocean of genetic variants, what would be the information each one
carries? Which of them would be effectively used to predict the developmental potential and/or
disease susceptibility? How would these signals be separated by noise? Single-cell sequencing
technology with MALBAC and other approaches represents only the beginning. There will be a
long way to go for the new single-cell sequencing technique in the application of reproductive
biology and reproductive medicine.
NEW ROLES FOR THE POLAR BODY IN PREVENTING TRANSMISSION OF
INHERITED MITOCHONDRIAL DISEASES
Almost all mitochondrial DNA is maternally inherited in mammals, although there are few
exceptions in other organisms [65, 66]. Since the mitochondrial genome encodes the 13
polypeptides plus tRNAs and rRNAs which are essential for oxidative phosphorylation,
mitochondrial diseases are usually devastating although 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 clinically present mtDNA disorders are 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 there are some characteristics of mitochondrial
genetics including ‘bottleneck’ segregation [72-74] and selective replication [75, 76] which may
bring about difficulty for the detection of heteroplasmy levels [77, 78], evidence suggests that
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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 US Food and Drug Administration (FDA) and the UK Human Fertilization and
Embryology Authority (HFEA) have been committed to the development of new strategies to
prevent mtDNA disease. Mitochondrial replacement (MR) 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 carry-over (< 2.0%) in early embryos [83]. However, one disadvantage of the PNT is
that the manipulation which requires both donor and the recipient’s fertilized eggs discards half
of the embryos. Another technique - spindle-chromosome transfer (ST) has been achieved in
both nonhuman primate and early human embryos to eliminate the maternal inheritance of
mtDNA mutations [84-87]. These findings suggested minimal mutated mtDNA carry-over in
nonhuman primate offspring and human preimplantation embryos. However, the spindle is very
sensitive to micromanipulation, which frequently induces premature activation of oocytes and
results in karyotype abnormalities [84].
Recently a study reported that 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 PB1 and PB2
potentially have the same genome as their corresponding oocyte [19, 36]. Indeed, PB transfer
may have several advantages. First, PB1 or PB2 contain few mitochondria but carry the entire
genome [4, 88]. Therefore, a minimal carry-over of donor mtDNA is expected in reconstructed
embryos and offspring generated by PB transfer. Second, 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 and his colleagues
compared the effects of four types of genome transfer in mice; PB1 transfer, PB2 transfer,
spindle-chromosome transfer and pronuclear transfer (Figure 2) [21]. 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
manipulation convenience 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 (ICSI), which requires only a single step [89]. Similarly, the 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 pronucleusenucleated, 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]. Since the human spindle is smaller than that of the mouse,
human spindle-chromosome transfer is expected to result in less variant carry-over. The
pronuclear transfer-derived offspring had the highest level (23.7%) of donor mtDNA carry-over.
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This may be primarily caused by zygotic activation which leads to mtDNA amplification around
the pronucleus [72, 90]. This is consistent with previous findings which also reported a similar
level of mtDNA heteroplasmy after pronuclear transfer [82, 91]. Together, these data suggest
that polar body transfer results in minimal donor mtDNA carry-over compared to other methods.
Importantly, the F2 generation derived from PB transfer still maintains the minimal donor
mtDNA variants [21]. Furthermore, the array comparative genome hybridization study in single
oocytes indicates that in a normal oocyte, both the PB1 and the spindle-chromosome 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 which showed that the PBs and
the oocyte potentially share the same genomic landscape [19, 36]. 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. Since
mitochondrial replacement involves germline genetic modification, it can be inheritable and can
possibly affect future generations. Second, coordinated mitochondrial-nuclear 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 would 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 would
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 prevention of transmission of
mtDNA disease by polar body transfer. These findings are encouraging to further explore more
potential roles of polar bodies in human reproductive health. However, this also raises many
fundamental questions which require further clarifications (Figure 3).
A question of central importance is whether the incidence of DNA mutations and DNA
impairment (such as double strand breaks) in polar bodies is identical to the sibling oocyte
(Figure 3). Or whether there is a possibility that the abandoned polar bodies carry genomes with
more defects, such as higher incidence of DNA mutations. Detailed analysis of single oocyte
sequencing data would be useful to resolve this question.
Another key question concerns whether the polar body carries the same epigenomes as that of
its sibling oocyte (Figure 3). Epigenomic alterations include a series of chromatin and DNA
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modifications, such as cytosine methylation and histone modification. Other epigenetic
regulations involve non-coding RNAs (such as siRNAs, microRNAs 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
exhibits 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 with the development of the high throughput
sequencing technique, single-cell-based epigenomic profiling would be helpful to further resolve
this issue.
If the polar body does carry the same epigenetic information as that of 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 non-genetic disease
susceptibility of offspring. Identification of epigenetic marks in oocytes which represent the
good or bad information that mothers are going to transmit to their offspring, is a potential
strategy to prevent non-genetic 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, non-genetic diseases can be deduced from the epigenetics of its
sibling polar body. Such epigenetic diagnosis will hold great promise to predict susceptibility to
certain non-genetic diseases, and will also be helpful in the prevention of certain epigeneticassociated disorders.
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Figure legends
Figure 1. Schematic charts for genome analyses of single human oocytes. During the meiotic
process of human oocytes, homologous recombination occurs before chromosome segregation.
The first polar body (PB1) and second polar body (PB2), which are dispensable for embryo
development, are used for whole-genome single-cell sequencing. Based on the principle that the
total genomes of 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 diseaseassociated alleles, it is inappropriate to perform embryo transfer. Otherwise, embryo transfer will
result in a healthy baby.
Figure 2. Comparison of four types of genome transfers to prevent transmission of
mitochondrial disease. From top to bottom, PB1 transfer, spindle-chromosome transfer, PB2
transfer and pronuclear transfer, respectively. 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. Pronuclear transfer generated offspring with the highest donor
mtDNA carry-over.
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Figure 3. Key questions associated with polar bodies remain unresolved. The polar body
contains both genetic material and epigenetic information (such as DNA methylation, noncoding RNAs and chromatin proteins). There are many fundamental questions associated with
polar bodies which requires 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; 4) Whether
epigenetic marks can be identified in polar bodies to predict and prevent the transmission of
specific non-genetic diseases.
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Figure 1
Figure 2
Figure 3

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