Before fertilisation, the mammalian oocyte is arrested at metaphase of the second meiotic division (MII), and it only resumes meiosis upon penetration by a spermatozoon. Approximately 16 to 18 hours after fertilisation, two pronuclei (2PN) form; one is derived from the oocyte and encloses the maternal genome, whereas the other originates from the spermatozoon and contributes the paternal complement (2). The pronuclear stage is followed by the fusion of both pronuclei in a process called syngamy, which results in the diploid zygote (3, 4) (Figure 1). However, dysregulation of any of the processes involved may cause unusual cytogenetic events, and it may occasionally be associated with an abnormal pronuclei number (5). Triploidy is characterised by the presence of an additional chromosome complement, often recognised by the presence of three pronuclei (3PN) (2). 3PN is the most common chromosomal abnormality to impact human gestation, accounting for 17% of all spontaneous miscarriages (4, 6-8). Triploidy is an embryonic lethal event and, as such, it prevents normal embryonic development in most cases. Yet, several live births have been reported (9, 10).
A recent case report of sex-discordant monochorionic twins has challenged the notion that monochorionic twins are definitely monozygotic (1) (see our previous post). This phenomenon is explained by a postulated third type of twinning referred to as sesquizygosity, where individuals share between 50% to 100% of their genetic identity. Unlike monozygotic and dizygotic cases, the underlying mechanisms of this rare event are less extensively defined (1, 11). However, since the advent of advances in ART, the mechanisms involved in triploidy can be further elucidated to provide an explanation for sesquizygosity.
The origin of triploidy may be classified as one of two occurrences: diandry or digyny, resulting in an extra paternal or maternal haploid chromosome set, respectively. Each event comprises numerous postulated mechanisms involving the control of sperm entry into the oocyte, pronuclei formation, mitotic spindle formation and initiation of meiosis.
The proposed mechanisms of triploid embryo formation and mechanisms explaining sesquizygotic twinning are discussed below, considering the relevance and incidence of triploid embryos in IVF.
TRIPLOID INCIDENCE AND RELEVANCE TO ART
In the context of ART, 5% to 7% of all IVF or ICSI cycles result in at least one abnormal pronuclei event (4, 13, 14). In particular, triploidy has been demonstrated to occur at a slightly lower, yet significant rate in ICSI cycles (2.5%) when compared to IVF (8.1%) (3, 15). Such a difference is presumably explained by the fact that ICSI reduces the probability of polyspermic fertilisation (3, 15).
Multiple studies have associated 3PN with low blastocyst formation and implantation rates, which provides a rationale for routine pronuclei observation in ART procedures (16). This clinical laboratory practice serves as a prognostic indicator of embryo competence and normal fertilisation (5, 6, 16). Normally, zygotes with variation from 2PN and two polar bodies are discarded in order to increase the chances of selecting the best embryo for transfer (3, 15). However, it is important to note that visualisation of 2PN at the pronuclear stage does not always eliminate the possibility of triploidy, and triploid embryos may cleave at a rate comparable to normally fertilised embryos (Figure 2) (6, 8, 15). Additionally, while IVF allows for the examination of the pronuclear stage, the incidence of 3PN is presumed to occur in vivo at a similar rate; however, these embryos are usually unable to pass developmental checkpoints and result in miscarriage (17).
Although the factors and mechanisms contributing to triploidy remain controversial, the incidence of 3PN in assisted reproduction cycles has been associated with advanced maternal age or semen abnormalities in some cases (18). Additionally, a link between 3PN and maternal genetic cause, high gonadotropin dose and large oocyte yields during ovarian stimulation has also been established (19, 20). Therefore, the complex relationship between ART and the incidence of triploidy is likely to be of multifactorial aetiology.
POSTULATED MECHANISMS EXPLAINING SESQUIZYGOTIC TWINNING
Diandry is assumed to be the most likely cause of triploid embryos and manifests in approximately 5% of inseminated oocytes (6, 21). Diandric triploidy of dispermic origin refers to the fertilisation of one oocyte by two individual spermatozoa, thus resulting in two paternal pronuclei (Figure 3) (4). This form of triploidy is prevalent in normozoospermic males and may be confirmed by the presence of two Y chromosomes using cytogenetic analyses (22-24). Dispermic fertilisation occurs at a rate of 61.5% and has been demonstrated to account for 86% of triploid embryos following IVF (21).
To prevent polyspermy, the entrance of multiple sperm into the ooplasm, protective mechanisms such as the plasma membrane block and the oocyte cortical granule reaction exist (Figure 1). The plasma membrane block consists of a rapid electrical depolarization of the oolemma, right after its fusion with the sperm membrane (25). While the plasma membrane block has been extensively studied in sea urchins, the cellular signalling events involved in establishing membrane potential in humans remain poorly understood (25-27). The cortical reaction has been largely characterised across animal species and is known to occur due to the oscillation of calcium ions inside the oocyte (26). The subsequent exocytosis of cortical granules triggers a signalling cascade results in the hardening of the zona pellucida, among other features. This event prevents sperm binding after fertilisation, thus providing a definitive block to polyspermy (26, 28).
However, in the case of diandric triploidy these mechanisms fail, with the cause being attributable to multiple factors (4, 21, 29-31). Whereas explanations exist that establish direct connections between increased sperm concentrations and the incidence of polyspermy, alternative studies have shown an approximate 30% increase in polyspermy rate that seems to be associated with the dysregulation between the timing of ovulation and oocyte maturation (4, 24, 25, 31-33). In any case, the link between the incidence of 3PN and poor oocyte quality remains unclear. Consequently, whether the embryo is capable of correcting such errors during early development will require further study (34).
During development, the triploid state of the embryo may be maintained through the segregation of chromosomes resulting in a mixoploid embryo, consisting of diploid and triploid blastomeres (2n/3n) (Figure 2) (14). Alternatively, one haploid chromosome set may be eliminated; in this case, either a normal diploid zygote or a tripolar spindle may form. As a consequence of tripolar spindle formation, gross aneuploidy results, and embryonic development is unable to continue in most cases (35). However, the occasional survival of trisomics and uniparental disomies depend on mixoploidy and the proportion of diploid and triploid cells present in the embryo (35).
In the case of sesquizygosity, chimeric triploid-derived 2n embryonic cell populations may develop into mosaic/chimaeras or twins (35). This division of the zygote following dispermic fertilisation is described by a cytogenetic phenomenon defined as postzygotic diploidization of triploids (PDT), where individuals present with common maternal and distinct paternal genomes, yet display more similarity for some traits than seen in dizygotic twins (35). Thus, the most likely karyotype of sesquizygotic individuals would be 69, XXX, XXY, because cells of uniparental lineage are usually outcompeted by cells of biparental lineage; however, in very rare cases triploid embryos may convey XYY (Figure 3) (1, 23). Preimplantation Genetic Testing (PGT) may provide further insight into the intermediate between monozygotic and dizygotic twinning events (35), assessing for genotypic differences among circulating foetal DNA; whereas the absence of such differences is indicative of monozygotic twins, their presence suggests dizygosity (21). Since mosaic/chimeric and triploid embryos add to the complexity of PGT analysis, sesquizygosity further complicates this issue. Consequently, each sesquizygotic twinning event should be considered on a case-by-case basis.
Diplospermic fertilisation occurs due to the fertilisation of one oocyte by a binucleate or diploid sperm (Figure 3) (37). This form of diandric triploidy is attributable to nondisjunction during either meiotic division of spermatogenesis, resulting in a sperm containing a diploid set of chromosomes (37, 38). While triploidy of diplospermic origin accounts for a portion of triploid embryos (8.3%), genetic analyses of 3PN zygotes following ICSI established that the presence of binucleate sperm occurs at a higher rate in oligosazoospermic, cryptoazoospermic and azoospermic males (33.3%), compared to approximately 0.3% in normozoospermic males (4, 37, 39). Initially, these prevalence rates may not seem to be of major concern, considering that binucleate sperm are usually incapable of fertilisation and of producing a viable zygote (4). Nevertheless, binucleate sperm may occasionally fertilise oocytes before completion of the first meiotic division or in the context of ICSI, where sperm are able to bypass the oocyte’s natural sperm selection mechanisms and form a triploid embryo (40-42).
Parthenogenetic oocyte activation
Diandry may also be the result of parthenogenetic oocyte activation, where embryo development commences without paternal genetic contribution, thus forming two diploid blastomeres (Figure 4) (43). Parthenogenetic activation is thought to result from a failed cytokinesis event, where in the absence of complete mitosis the genome of the cell is duplicated (4, 44, 45). In this circumstance, the fertilisation of a diploid two-blastomere embryo by two individual spermatozoa results in a triploid embryo (46). While this postulated mechanism provides a likely explanation for triploidy, studies in mouse models suggest that parthenogenetically activated oocytes are not capable of efficient sperm chromatin remodelling, and so they are unable to support zygote development (47).
Triploidy of digynic origin refers to an extra maternal haploid set resulting from the fertilisation of a haploid oocyte by a spermatozoon. Digynic triploidy is predominant among early miscarriages and has been attributed to 50-60% of all spontaneous abortions (8). Triploidy in this instance is explained by failures during either meiotic division or as a consequence of polar body retention (Figure 5). Equal ratios of XXX and XXY with the absence of XYY cells imply embryos are of digynic origin (21).
Endoreduplication of the female nucleus
Endoreduplication of the female nucleus results in a ‘giant cell’ with a single haploid nucleus, and has been shown to accompany abnormal pronuclei formation (Figure 5) (45, 48). In this case, errors during the first or second meiotic division may explain digynic triploidy (49). Although triploidy of digynic origin due to endoreduplication is not entirely understood, the activation of complex downstream enzyme pathways as a requirement for the progression of meiotic development has been well-established (50). Consequently, the dysregulation of genetic or molecular factors involved in any stage of this process may result in post-meiotic cell errors, causing second polar body retention and subsequent triploidy (51).
Second polar body retention
Alternate explanations for digynic triploidy may be associated with second polar body retention (Figure 5). Studies using pericentromeric markers in triploid cell lines have demonstrated that this anomaly contributes to the majority of digynic triploid embryo cases (3%), and may be associated with failure during the second meiotic division, or due to disruption of the meiotic spindle following ICSI (10, 15). Several reports have associated second meiotic arrest with age-related infertility or errors during spindle formation (52, 53). However, due to the ethical issues surrounding the study of human fertilisation, the causative mechanisms of second polar body retention can only be observed during intervals of the ART procedures. As a result, these issues remain not well understood.
Twinning events provide insight into the cellular mechanisms occurring during embryogenesis that may influence triploidy. While in the context of ART well-established clinical and laboratory aspects may prevent the incidence of 3PN, in vitro rates may differ from in vivo rates due to various conditions for fertilisation, including treatment of infertile couples who may be predisposed to abnormal fertilisation events (17).
Whereas triploid embryos are usually an embryonic lethal event, extraordinary cases of dispermic fertilisation may provide explanations for the phenomenon of sesquizygotic twinning. As sesquizygosity is a rare event, ascertainment bias and difficulty defining criteria for such cases exist. However, a greater understanding of the aetiology and mechanisms involved in twinning events may aid management and possible prevention of such cases in the future.
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