Twinning is a complex event occurring in approximately 1 in 80 spontaneous pregnancies, with an increased incidence of 1 in 4 for babies conceived by in vitro fertilisation (IVF) (1, 2). Variation exists across twinning events and can be differentiated by zygosity (1, 3–6). This term refers to whether twins originate from one fertilised egg, defined as monozygotic, from two eggs fertilised by two different sperm, dizygotic, or a postulated intermediate of these events defined as sesquizygotic (Fig. 1) (1, 3–6). An understanding of twinning events requires knowledge across relevant fields of obstetrics, developmental biology, and clinical and molecular genetics (1).
In the present text, the characteristics and main causes of monozygotic and dizygotic twinning will be examined, and their increased incidence in IVF will be discussed.
2. MONOZYGOTIC TWINS
Monozygotic twins, more commonly known as identical twins, originate from one fertilised egg, producing a single zygote which divides and develops into two independent and genetically identical embryos (8). Although embryos are expected to be genetically identical, monozygotic twins show a high degree of discordance for complex genetic traits and disorders (1, 4). This genetic discordance is attributable to somatic mutations in cells, arising due to differential epigenetic control and stochastic processes over the course of development (4).
Monozygotic twin pairs represent approximately a third of all spontaneous twins, and while this rate remains stable across populations, ethnicity and race, the specific cause of monozygosity in humans has not yet been established (9). However, evidence suggests that monozygotic twinning may be associated with a suboptimal zona pellucida, inner cell mass or the age of the oocyte after ovulation (1, 2). Furthermore, skewed X chromosome inactivation may provide an alternative explanation for the increased incidence of monozygotic twins in humans (10–12).
The inactivation of one X-chromosome during early mammalian female development occurs in order to maintain the correct expression of genes (13). In rare cases, X-linked mutations may influence cell viability, resulting in a preference for the activation of the other X chromosome (12). This event is thought to occur when the initial zygote contains three chromosome sets and loses one to form a diploid zygote (12). The reduction in progenitor pool size results in extreme X-chromosome skewing (10). A similar mechanism is thought to explain the increased incidence of monozygotic twinning in humans, although this concept still remains controversial (10).
As briefly noted, evidence in animals has indicated that damage to the inner cell mass or zona pellucida may also be associated with monozygotic twinning (14). When considering the cyclic nature of human reproduction, the fertilisation of an ‘older’ oocyte with a more delicate zona pellucida could result in the blastocyst splitting in two (1, 14). The cytoplasm of an ‘older’ ovum may not have sufficient nutrient and energy sources, resulting in developmental delay and an increased risk of programming errors (1, 15). Additionally, familial monozygotic twinning may be an inherited defect involving zona pellucida proteins, causing the zona to be more susceptible to the early separation of blastomeres, failure or delay of cell-to-cell recognition and adhesion (9).
Frequency and relevance to Assisted Reproduction Techniques (ARTs)
In 2011 there was approximately a 36% increase in the occurrence of monozygotic twins as a result of ART procedures in the United States (16). While a reduction in the proportion of higher order births followed changes in the regulations surrounding the transfer of three or more embryos during IVF (16), the rate of monozygotic twinning remains higher in ART compared to spontaneous conceptions (17). Specifically, monozygosity following ART has been established to be higher for day 5-6 transfers compared to day 2-3 transfers, whereas intracytoplasmic sperm injection (ICSI) seemed to reduce the incidence of monozygotic twins (18). Although these findings do not consider all potential risk factors influencing the incidence of monozygosity in ART, they assist in the greater understanding and prevention of such cases.
The increased prevalence of monozygotic twinning in both spontaneous pregnancies and in IVF is thought to be associated with a suboptimal or even absent zona pellucida (1, 19, 20). The zona may be damaged as a result of handling or penetration during ICSI (1). In addition, increased age, follicle-stimulating drugs or culture media could induce zona hardening (19). If zona pellucida is absent, damaged or hardens during the early zygote stages when tight junctions between cells have not yet been established, blastomeres could fall apart, thus resulting in regrowth from two points (19, 20). This hypothesis is supported by the fact that dichorionic diamniotic twins occur more frequently than expected in IVF. It also suggests that monozygotic twinning process and subsequent separation of twins observed in IVF happen at an early stage (19, 20).
3. DIZYGOTIC TWINS
Dizygotic twins arise from two separate oocytes ovulated at the same time, followed by independent events of fertilisation by a single spermatozoon (1, 3). Although not common in humans, the ovulation of multiple dominant follicles during the same menstrual cycle may occur due to hypothalamic-pituitary-ovarian axis dysregulation (21–23). Thus, dizygotic twinning depends on a multiple ovulation event, the timing of intercourse and subsequent fertilisation of both oocytes (24).
If both ovulated oocytes are fertilised by spermatozoa from one male, dizygotic twins are expected to share 50% of their genes from each parent, like any other pair of siblings (1). However, there are several reported cases of dizygotic twins with different paternal genetic contributions, which may be explained by superfetation. In this circumstance, two separate fertilisation and implantation events occur in subsequent ovulation cycles; in these cases, a second zygote implants in the uterus, where pregnancy had already been achieved previously (25, 26).
Dizygotic twinning has been associated with higher concentrations of maternal follicle-stimulating hormone (FSH) (23, 27). As FSH levels change depending on age, geographical location, season, body mass index (BMI) and ethnic origin, the frequency of spontaneous dizygotic twinning varies widely among population groups (1). FSH levels increase with BMI and/or age, with the most significant increment at the age of 37. Additionally, due to the circadian regulation of FSH, levels fluctuate in response to variation of daylight and to seasonal and geographical effects (27, 28). A high frequency of dizygotic pregnancies has been observed in equatorial Africa and during the summer months in North Finland and Japan (15, 21).
Predisposition to dizygotic twinning pregnancies may be inherited through both autosomal recessive, autosomal dominant, and paternally derived inheritance patterns (29, 30). It has been reported that genes associated with dizygotic twinning are linked to chromosome 3 and are carried by 7–15% of the population (31). Inheritance of these genes may confer a presumptive selective advantage by producing more offspring; however, it is correspondingly associated with a high trade-off risk associated with multiple pregnancy (32).
Other theoretical mechanisms of dizygotic twinning exist, such as polar body twinning, where fertilisation of the oocyte and polar body occur by separate spermatozoa (1). However, this occurrence has been reported only once, and no viable offspring were produced (33).
Frequency and relevance to ARTs
Since 1980, accompanying the prevalence of ARTs involving ovulation stimulation drugs, there has been a dramatic rise in dizygotic twinning rate (15, 34). In the context of IVF, dizygotic twinning is more likely to take place when more than one embryo is transferred into the uterus (1, 35), given the chances of survival.
4. PLACENTATION IN TWINS
Placental development differs depending on zygosity, and its connection with perinatal mortality has been previously reported in a variety of studies (3, 5, 6). Timing of embryo division is thought to influence placental development in monozygotic twins. Whereas dizygotic twins exhibit dichorionic diamniotic placentation, defined as separate placentas and membranes, 1-2% of dizygotic twins exhibit one placenta and associated membranes, referred to as monochorionic monoamniotic placentation. In contrast, 70-75% of monozygotic twins share one placenta with monochorionic diamniotic membranes, while the remaining 25% presents with dichorionic diamniotic placentation (Fig. 2, 3) (1).
For these reasons, in 1973, clinicians Benirschke and Chung proposed the examination of placental membranes in order to determine zygosity, type of chorion and presence of anomalies in multiple pregnancies (3). Current practice outlined in the National Institute for Health and Care Excellence (NICE) guidelines for twin and triplet pregnancy involves antenatal screening and diagnostic monitoring for multiple pregnancies (36). For practical reasons and until proven otherwise, a monochorionic placenta is assessed as a monozygotic twin pregnancy. In the case of dichorionic placentas, if twins appear to seem similar at birth, biochemical studies such as blood types, enzyme polymorphisms and Human Leukocyte Antigens (HLA) are completed to determine monozygosity (1, 6).
Placentation and membrane type allow for the estimation of the timing of twinning events and the percentage of surviving twin pregnancies (Table 1) (6). Monochorionic monoamniotic placentation of all types accompanies a high risk of vascular compromise due to umbilical cord twisting (5). Such imbalance may result in twin reversed arterial perfusion sequence; this is a rare variation of twin-twin transfusion syndrome in which the blood systems of the twins are combined, causing severe malformation and disruptive congenital anomalies (1, 5). Additionally, the sharing of hematopoietic stem cells due to vascular connections may result in microchimerism between dizygotic twins and mosaicism, should genetic discordance arise between monozygotic twins (35).
Twinning events provide insight into the cellular mechanisms occurring during embryogenesis. However, the long-held notion that twins are either monochorionic or dizygotic has been challenged by a recent rare case report indicating the existence of sesquizygosity, a third type of twinning (37). The mechanisms involved in sesquizygosity, its relevance to triploidy and implications in ART will be discussed in a future post.
Multiple pregnancies whether monozygotic, dizygotic or sesquizygotic are associated with various risks and complications for both the mother and foetuses, which in some cases may even result in stillbirth. A greater understanding of the aetiology and mechanisms involved in twinning events may aid management and possible prevention of such cases. As for ART, the goal of reducing the incidence of multiple pregnancies has been pushing forward the efforts to move definitively to the leading strategy of single embryo transfer.
Check part II: Sesquizygosity.
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