Introduction
This article analyzes the history and validity of the current explanation on the genesis and timing of human monozygotic (MZ) twinning (referred to in the following as the ‘model’). At close examination, the model is less than satisfactory in explaining some important aspects of the timing of MZ twinning. This article is written to invite a re-examination of the question, and also to offer an alternative explanation.
The model
The model, proposed more than a half century ago, enjoys today an unreserved support by embryologists, reproduction scientists and bioethicists. It rests on two basic postulates:
1. MZ twinning is a post-fertilization event resulting from the splitting of an early embryo.
2. The timing of twinning establishes the structure of membranes; in other words, the developmental stage at which splitting takes place determines chorionicity and amnionicity.
In scientific and popular publications alike, the model has crystallized in formalized patterns of verbal and graphic presentation. It follows one of two ways for measuring embryologic time: either the successive stages of early development (2-cell stage, cleavage, morula, early and late blastocyst, bilaminar embryonic disc, primitive streak); or, more usually, the sequence of post-fertilization days. Accordingly, it is said that splitting from day 1 to 3 (i.e. before compaction of the morula) results in the production of dichorionic/diamniotic (DC/DA) twins; splitting between days 5 and 8 (by division of the inner cell mass at the blastocyst stage) results in the production of monochorionic/diamniotic (MC/DA) twins; splitting between days 9 and 12 (by partition of the bilaminar embryonic disc at the late blastocyst stage) results in the production of monochorionic/monoamniotic (MC/MA) twins; and splitting around day 14 or later (when the primitive streak is formed) results in the appearance of conjoined twins (see Note Footnote 1). The standard graphic representation of the model consists in a dual combination of embryological and obstetrical diagrams. Ordinarily, such diagrams show besides the developmental stages at which the embryo splits and the ways the embryonic membranes are affected, a representation of an open uterus depicting twins, placentas, and chorionic and amniotic sacs.
An extensive bibliographical search has shown that the model not only has met with general approval (see Note Footnote 2), but it is put forth in a factual, assertive style, conveying the certitude that it is not a hypothetical explanation, but a sound description of hard facts (see Note Footnote 3). Such pretension is, as I will try to demonstrate, an educated but deceptive guess. The apparent soundness of the model is the result of a curious epistemic evolution that changed an initial and tentative explanation into an accepted and undisputed truth.
The matter is relevant in two ways: first, the timing of MZ twinning is a biological issue; therefore, in the age of evidence-based medicine, we must be interested in knowing in what measure the information we posses on the matter is reliable. Second, the issue has the utmost bioethical relevance, as debates on the beginning of human life and the ethics of in vitro fertilization (IVF) or embryo experimentation have been persistently focused on the timing of MZ twinning.
The building of the model: a short history
The history of the embryology of human twinning is long and complex. Here, attention will be centred on the birth and evolution of the model (see Note Footnote 4; Sobotta, Reference Sobotta1901; Schwalbe, Reference Schwalbe1906; Patterson, Reference Patterson1913).
Although there are some remote precedents, it seems reasonable to choose as the starting point of the model an article published by Corner in 1922, in which he described three pairs of MC twins in the pig. Corner did not hide his discontent over the inadequacy of the then current explanations on the origin of identical twins (see Note Footnote 5; Corner, Reference Corner1922). However, after acknowledging the scarcity of facts and the abundance of conjectures, Corner dared at the end of his article to offer a new hypothesis. ‘. . .we may permit ourselves to indulge in a brief speculation regarding the morphogenesis of human monochorionic twins. . . [H]uman single-ovum twins may be of two types. One. . .arising by duplication of the inner cell mass before formation of the amniotic cavity (pig type) would typically give rise to two embryos in a single chorion with two independent amniotic cavities. . .The second kind of twinning, occurring by duplication of the actual embryonic areas after formation of the amniotic cavity (armadillo type), would produce two embryos within a single amnion.’(see Note Footnote 6; Corner Reference Corner1922) Corner did not consider the timing of MZ twinning of the DC DA type influenced by the then prevailing opinion that DC twins were always dizygotic (DZ), and MZ twins always MC.
The data were not new, but its interpretation supposed a fundamental innovation. Corner introduced for the first time the idea that differences in the structure of the fetal membranes in MC twins were dependent on the moment of partition of the embryo. Henceforth, time became a decisive factor for the understanding of twinning.
Before Corner, the timing of the twinning was considered irrelevant, because, as things were then understood, the exclusive condition for MC twinning was the presence of two embryonic areas within a single blastodermic vesicle. To justify amnionicity, a ‘topological’ reasoning was then adduced: if the two embryonic areas within the blastocyst were sufficiently apart, each could develop separately its own amnion, with the result of MC DA twins; if they were near together, a common amnion would result, and, therefore, MC MA twins (see Note Footnote 7; Schultze, Reference Schultze1897; Bumm, Reference Bumm1902; Wilder, Reference Wilder1904; Newman, Reference Newman1917). In Corner's proposal, the old ‘geometrical’ view ought to be abandoned, so that timing could get the leading role in the explanation of MC twinning. But, regretfully, the immediate impact of the 1922 article by Corner was minimal (see Note Footnote 8; Streeter, Reference Streeter1924; Grosser, Reference Grosser1927; Hughes, Reference Hughes1927; Klein, Reference Klein1927; Potter, Reference Potter1927).
The next important step in the building of the model came in the 1920s, when the statement ‘zygosity equals chorionicity’ was demonstrated false. Siemens, applying his method of polysymptomatic (dermatologic) similarity diagnosis to the study of twins, established firmly that many DC twins were MZ, and not DZ (see Note Footnote 9; Siemens, Reference Siemens1925). Siemens’ findings, soon confirmed by others, forced some important amendments to Corner's model. That was the task of von Verschuer. In 1932, he expanded the model to include DC DA twins among the MZ. Verschuer attributed the origin of the newly characterized type to a partition of the embryo during early cleavage stages, before the beginning of the differentiation into embryoblast and trophoblast (Verschuer, Reference Verschuer1932). The timing of twinning began now with the appearance of the two first blastomeres.
In 1947, the model moved forward. Until then, the dating of the twinning event was referred to embryological stages of imprecise chronology (splitting of blastomeres, formation of two inner cell masses within the blastocyst, splitting of the germ disc, formation of two primitive streaks). Coulton, Hertig and Long, based on Hertig's observation that, in the human embryo, the amnion forms between the 7th and the 13th day after fertilization (Hertig, Reference Hertig1945), offered an estimation of the time of splitting for MA twinning. They reasoned that if the germ disc had split before the formation of the amnion [on day 7], presumably two amnions would then have formed; alternatively, in the case of MA twins, the split ought to occur after that day, but before day 13½, when the primitive streak initiates its development (Coulton et al., Reference Coulton, Hertig and Long1947). In this way, the first, although incomplete, daily calendar was established for MA twinning. The age of the embryo, measured as post-fertilization days, became hereafter a meaningful element for the description and understanding of MZ twinning. Hertig's article had a wide repercussion in the literature (see Note Footnote 10; Conybeare, Reference Conybeare1954; Hanes, Reference Hanes1954).
In 1955, Corner completed his model (Corner, Reference Corner1955). In the Baer Lecture of the Chicago Gynecological Society he delivered the previous year, Corner updated his ideas on the embryological theory of human twinning, considered the support it received from recent findings, and presented some illustrative cases he chose mostly from the Carnegie collection (see Note Footnote 11). Corner emphasized the theoretical character of the model: ‘Embryologists and obstetricians have built up on paper the morphological theory of single-ovum twinning, tracing the various ways in which one egg cell might ultimately develop into two embryos. All this information is in the textbooks; it has chiefly been worked out by deduction from the arrangement of the placentas and fetal membranes as seen at birth or in fetal life. Embryonic specimens early enough to provide direct corroboration of the theory are rare’ (see Note Footnote 12; Corner, Reference Corner1955).
To provide the necessary background of early human embryology for the subsequent analysis of the three critical stages of twinning of the finished model he presented, Corner offered a series of five microphotographs of the stages of development corresponding to the times at which twinning may occur: the 2-cell stage and the morula (about days 2 to 4 after ovulation); the blastocyst (about day 5); the implanted embryo, pre-villous stage (about day 11) and a later embryo (about day 17) (see Note Footnote 13).
Then he analyzed each of the three critical stages. First, he discussed the twinning by separation of the early blastomeres, which he thought highly probable, but which can never be absolutely proven by inspection of the products of conception. His conclusion was that ‘unless the age arrives of ‘test-tube babies’ and of the experimental embryology. . .this type of human twinning must remain a plausible conjecture’ (see Note Footnote 14; Corner, Reference Corner1955). Second, the twinning at the blastocyst stage (days 4 to 7 after ovulation). Corner rationalized that ‘if during this period some accident of development causes division of the inner cell mass, or starts the growth of two inner cell masses, then we have the beginning of twin embryos that will be enclosed in a single chorion’. He illustrates this possibility with Assheton's and Streeter's non-human specimens, and describes the findings from four own specimens (see Note Footnote 15; Corner, Reference Corner1955). He concluded that ‘obstetricians are familiar with this arrangement as seen at term, when careful examination of the afterbirth reveals the doubled amnion and permits the retrospective deduction that the two infants must have come from two inner cell masses in one blastocyst’ (see Note Footnote 16; Corner, Reference Corner1955). Third, the latest variety of twinning, the duplication of the embryonic rudiment of the germ disc, at about 15 days. This event can occur ‘if two embryonic nodes develop instead of one. Speaking in the technical language of general embryology, this process is one of double gastrulation. . .When such twinning occurs in man. . .two embryos will appear on the floor of the amniotic cavity, and will have a common yolk sac. This arrangement will persist until birth, when the obstetrician will find two infants in a single chorioamniotic chamber’. Corner could not find a satisfactory illustration of this kind of twinning with normal embryos in an early stage (only damaged specimens and some cases of conjoined twins).
The triumph of the model
In a few years, the model evolved from a morphological theory into a precise record of facts. The reception of the model by the scientific community was almost immediate and approving. Published at the end of 1955, the next year Corner's article was included in some bibliographical reviews (see Note Footnote 17; Anonymous, 1956; Author, 1956; Ebert, Reference Ebert1956; Greenhill, Reference Greenhill1956); in 1957 it was repeatedly cited in books, journal articles or short communications (see Note Footnote 18; Beck & Rosenthal, Reference Beck and Rosenthal1957; Craig, Reference Craig1957; Librach & Terrin, Reference Librach and Terrin1957; Morton, Reference Morton1957; Osborne & de George, Reference Osborne and de George1957). In successive years the number of citations grew steadily both in journals and books (see Note Footnote 19; Stern, Reference Stern1960; Benirschke & Driscoll, Reference Benirschke and Driscoll1967; Strong & Corney, Reference Strong and Corney1967; Bulmer, Reference Bulmer1970; Boyd & Hamilton, Reference Boyd and Hamilton1970; O'Rahilly, Reference O'Rahilly1973). Fifteen years after its publication, the model became the standard wisdom (see Note Footnote 20; Corner, Reference Corner1955; Benirschke & Driscoll, Reference Benirschke and Driscoll1967; Strong & Corney, Reference Strong and Corney1967; Dollander, Reference Dollander1970; Leroy, Reference Leroy, Edwards, Purdy and Steptoe1985; Nance, Reference Nance1990). Remarkably, despite the lack of direct embryological evidence, the model was accepted as proven. Contrasting views were few and, as time passed, weaker (see Note Footnote 21). For decades, no other alternative theory disputed its supremacy.
Various factors have contributed to the rapid diffusion and acceptance of the model. Undoubtedly, the indisputable scientific prestige of Corner was a determining factor for its success (see Note Footnote 22; Hertig et al., Reference Hertig, Rock and Adams1956; Corner Reference Corner1981). Corner's ideas were supported by his outstanding academic achievements in the fields of endocrinology, human reproduction, medical education and history, and his 15 years as Director of the Department of Embryology of the Carnegie Institution (see Note Footnote 23; Hartman, Reference Hartman1956; Lord Zuckerman, 1983; Ramsey, Reference Ramsey1994).
Another factor responsible for the triumph of the model was its internal, straightforward, logic: the model is quite reasonable. It connects mutually two variables: on one side, the morphology of the fetal membranes with its gradations of double/single (DC/DA, MC/DA, MC/MA); and, on the other, the ‘three critical stages’ (see Note Footnote 24; Corner, Reference Corner1955), i.e. the successive stages of embryonic development at which twinning was thought to occur.
Finally, a third factor aiding acceptance of the model has been the convincing force of its graphic representation. Corner's original diagram and its numerous adaptations, as well as new versions produced by others over the years, made it readily comprehensible, and above all, gave a semblance of ‘reality’ to the model (see Note Footnote 25).
Surprisingly, Corner did not hold his theory in great esteem (see Note Footnote 26; Corner, Reference Corner1955; Ebert, Reference Ebert1956; Corner, Reference Corner1958; Corner, Reference Corner1981). It would be interesting to know the reasons behind such an attitude. Probably, he realized that the embryological data he used could not confirm the core of his theory, because in none of the specimens was he able to observe directly the central event of MZ twinning: the splitting of the embryo. The specimens he examined belonged to post-splitting stages, not to the original moment of twinning: his ‘observed embryology’ was compatible with his theory, but unable to prove it.
Despite that fact, Corner convinced himself that his model corresponded to what really happened. Some meaningful modifications in his 1955 article attest to his change of mind. It suffices to compare his 1922 and 1955 articles: The Morphological Theory of Monochorionic Twins was the title chosen by the young Assistant Professor of Anatomy of the Johns Hopkins University, a title revealing the tentative character of his proposal; while The Observed Embryology of Human Single-Ovum Twins suggests a matter-of-fact assertion by the prestigious Director of the Department of Embryology of the Carnegie Institution. Another example of the change of tone is the caption for Fig. 6 in his first article, which reads ‘Hypothetical Diagrams of Human Monochorionic Twins’. Thirty-three years later, the same figure bears this other legend: ‘Diagrams illustrating two types of single-ovum twinning in man’. The transition of theoretical proposal to factual description appears unmistakably clear.
Corner was not alone in ‘seeing the light’. Benirschke, the leading authority on human placenta and twinning for the last half of the 20th century, made a similar transition. In 1965, he acknowledged the lack of data to establish with a certain degree of certitude the timing of twinning (see Note Footnote 27; Benirschke, Reference Benirschke1965). Then, without any good reason in 1973, ignorance appears to be overcome and the old doubts cleared up (see Note Footnote 28; Benirschke & Kim, Reference Benirschke and Kim1973).
Now for more than a half century, the model dwells in the realm of facts. Now, its adherents are not only biologists and physicians: with the expansion of bioethics, its main supporters are philosophers, theologians, jurists, bioethicists and politicians. The model has played a decisive role in the disputes on the legal and ethical status of the human embryo.
The weaknesses of the model
In fact, the rationality and persuasive force of the model have sufficed to quench the few criticisms raised against it, so that it has remained practically unchanged and, what is worse, unchallenged. But, if closely examined, one can find some weak points in its apparent solidity. The difficulties to explain the genesis of twinning increase as development of the embryo goes forward: for example, the suggested mechanism of formation of conjoined twins is complex and elusive in comparison with the simple mechanism proposed for the genesis of DC DA twins. The following are some weaknesses are found in all three critical stages.
Part (a)
Let us begin with the simplest case. It is said that DC DA twins result from the first cleavage division. Thenceforth the two cells become reciprocally independent; each of them initiates its separate individual existence, and each one develops as a whole embryo. Despite inhabiting both the same pellucida, both make their own independent and autonomous passage from cleavage to hatching. Afterwards, they implant separately in the endometrium and separately develop their sets of membranes (see Note Footnote 29).
This short story is practically all the model indicates regarding the origin of DC DA twins. It conveys the comfortable idea that this type of twinning is due to a casual separation of the blastomeres, a random or fortuitous event. Some causes have been suggested for the partition of the embryo at this critical stage, as, for example, subtle genetic differences which translate into a repulsive force that leads to the splitting of the zygote (Hall, Reference Hall1996), alterations of the calcium levels in the maternal environment or inside the embryo (Steinman & Valderrama, Reference Steinman and Valderrama2001), or a supposed absence or scarcity of binding molecules between embryonic cells (Bamforth et al., Reference Bamforth, Brown, Senz and Huntsman2003).
Also some objections have been raised against the plausibility of twinning at this critical stage. The co-existence of two embryos within the same zona has been considered unlikely by those convinced that early embryos tend to fuse together in the closed and progressively overcrowded space of the pellucida. They think, therefore, that twinning is by necessity a post-hatching phenomenon (see Note Footnote 30; Allen, Reference Allen1969; Leroy, Reference Leroy, Edwards, Purdy and Steptoe1985). As pre-compaction morulas tend to fuse, others suppose that the independence of MZ twins within the zona is only possible after the differentiation of the trophectoderm, so that twinning must be a post-compaction event (Tarkowski & Wojewodzka, Reference Tarkowski and Wojewodzka1982). To make more credible the separation of early blastomeres as a mechanism of twinning, a few authors have dispensed with the pellucida, at least in their graphic representations, as if twinning were easier in ‘naked’ zygotes (see Note Footnote 31; Salerno, Reference Salerno1959; Derom & Derom, Reference Derom, Derom, Blickstein and Keith2005).
One last weakness of the model on the origin of DC DA twins consists in its inability of finding its own confirmation in IVF. Despite the countless human embryos produced and examined in vitro, the problem remains unsolved: the splitting and growth of twins within the pellucida has been never observed or live-recorded (see Note Footnote 32; Verpoest et al., Reference Verpoest, Van Landuyt, Desmyttere, Cremers, Devroey and Liebaers2009; Knopman et al., Reference Knopman, Kray, Lee, Fino, Novetsky and Noyes2010).
Part (b)
The second critical stage (days 4 to 8 after fertilization) is, according to the model, the time when MC DA twins are generated. It is customary to attribute this type of twinning to the splitting of the ICM (see Note Footnote 33; Roberts & Fisher Reference Roberts and Fisher2011; Roode et al., Reference Roode, Blair, Snell, Elder, Marchant, Smith and Nichols2012) or, rarely, to a supposed spontaneous development of two original ICMs (see Note Footnote 34; Sills et al., Reference Sills, Tucker and Palermo2000). Blastocysts with two separate ICMs have been observed in some non-human species (see Note Footnote 35; Assheton, Reference Assheton1893; Corner, Reference Corner1955); in more recent times, they have been occasionally found in the course of human IVF (Meintjes et al., Reference Meintjes, Guerami, Rodriguez, Crider-Pirkle and Madden2001). To date and despite the use of time-lapse cinematography techniques, no-one has observed directly the instant at which an ICM splits into two, neither the simultaneous formation of two separate ICMs (see Note Footnote 36; Mio & Maeda, Reference Mio and Maeda2008). Occasionally the visualization of the hatching of two blastocysts, instead of one, from the same pellucida has been reported (Van Langendonckt et al., Reference Van Langendonckt, Wyns, Godin, Toussaint-Demylle and Donnez2000).
The increase of MZ twinning linked to the practice of IVF has provoked a great interest in identifying the etiologic factors, in particular those responsible for the more frequent MC DA placentation. Many potential causes have been suggested for the fission of the ICM in two: a fissure in a hard and rigid pellucida can provoke an atypical hatching, with the result of an split ICM within an trophectoderm (see Note Footnote 37), or of two separate complete blastocysts (see Note Footnote 38; Leroy, Reference Leroy, Edwards, Purdy and Steptoe1985; Edwards et al., Reference Edwards, Mettler and Walters1986; Behr & Milki, Reference Behr and Milki2003); a disruption of intercellular communication inside the ICM (see Note Footnote 39; Aston et al., Reference Aston, Peterson and Carrell2008); a confluence into a continuous dissecting plane of dispersed groups of apoptotic cells in the ICM (see Note Footnote 40; Corner, Reference Corner1955; Ménézo & Sakkas, Reference Ménézo and Sakkas2002); and, finally, the ectopic adhesion of a piece of the ICM to the inner surface of the abembryonic trophectoderm when the blastocyst collapses, with the result that, at re-expansion, the torn apart portion of the ICM can give origin to a twin (see Note Footnote 41; Payne et al., Reference Payne, Okuda, Wakatsuki, Takeshita, Iwata, Shimura, Yumoto, Ueno, Flaherty and Mio2007).
All these suggestions are a display of the ingenuity of their authors to find an answer to the riddle. Only suggestions that point to the possibility that an altered zona could bisect the blastocyst and the ICM have received a limited, but questionable support, in the literature.
Supposedly, in vitro embryo culture could afford some opportunities to observe the alleged process of twinning, but until now graphic evidence has been elusive or unconvincing. Certainly, twin blastocysts inside the pellucida are not subjects easy to detect, and, because of their thickness, more difficult still to photomicrograph (see Note Footnote 42). Moreover, photographs provide an image frozen in time: the use of continuous time-lapse video could help to observe what happens from beginning to end, that is, the whole course of the event (see Note Footnote 43). For now, one can only conclude that twinning took place before hatching when the ICMs appear situated at opposite poles of the twin blastocysts (see Note Footnote 44; Behr & Milki, Reference Behr and Milki2003). Recently, there has been convincing documentation of the presence inside the pellucida of two independent blastocysts prior to hatching (See Note Footnote 45; Shibuya & Kyono, Reference Shibuya and Kyono2012).
Part (c)
The difficulties in explaining the mechanism of MZ twinning grow as embryo development progresses and its structure becomes more complex. According to the model, MC MA twins result from the splitting of the embryonic disc on days 8 to 12. By this time, implantation has begun, the amnion and the exocoelomic cavity have formed, and the ICM is deeply changed by hypoblast delamination and epiblast epithelialization. Such evolved embryonic structure represents an important challenge to the model.
The model affirms that with the splitting of the germ disc (now, the floor of the amniotic cavity) into two portions, two embryonic anlagen result that develop to MC MA twins. Although much attention has been devoted to the obstetrical consequences of this type of placentation, practically nothing new has been added to the understanding of its morphogenesis. Hertig suggested that MC MA twinning was possible on the condition that the cells of the germ disc were sufficiently undifferentiated to form two equally potential halves, a circumstance only possible before the appearance of embryonic axial arrangement (see Note Footnote 46; Coulton et al., Reference Coulton, Hertig and Long1947).
The vague explanations offered to justify the low frequency of MC MA twinning (weakening of the twinning impetus, resistance of the embryo to splitting) have not contributed to the clarification of its genesis. There are no acceptable descriptions on how the disc can split into two halves, nor on how the resulting parts distance one from the other. This issue is important, as the type of separation (complete or incomplete) decides if the twins become separate or conjoined. Many questions (such as which forces are behind the splitting of the disc and the separation of its halves; how the amniotic epithelium can insinuate between those halves; and, if the splitting follows the longitudinal axis of the disc, how each half rebuilds its complementary missing parts, left and right) remain unanswered.
Part (d)
The deficiencies of the model culminate when the splitting theory tries to elucidate the twinning of the trilaminar embryo at 14 days or later. The majority of followers of the model limits their commentaries to state that the late splitting of the embryonic shield gives origin either to MC MA twins, or, if incomplete separation results, to conjoined twins. Other authors put forward the alternative hypothesis of a double gastrulation (see Note Footnote 47; Arey, Reference Arey1922; Corner, Reference Corner1955; McLaren, Reference McLaren, Austin and Short1982). Only very few authors mention the hypothesis of embryonic fusion, but apparently no serious critical evaluation on the plausibility of Spencer theory has been published (Spencer, Reference Spencer2000).
It must be asked: how and when a second primitive streak can be inserted in a germ disc? Until 40 years ago, drawing two parallel streaks on the same embryonal disc could be a simple mental experiment because, on the wake of Spemann–Mangold organizer's transplant experimentation, the disc was considered then a clean slate, apt to receive new determinations at any moment, at any place or in any direction. Now, however, we know that, at least in the mouse, the embryonic shield is a highly organized structure, its cellular population show gradients of specific gene activation and signalling activity, and that these gradients flow in the proximal–distal direction as well in a centripetal direction from the ring of extraembryonic tissues to the centre of the disc (Tam & Gad, Reference Tam, Gad and Stern2004). As regards the theory of double gastrulation it must be considered very unlikely (see Note Footnote 48; McGeady et al., Reference McGeady, Quinn, Fitzpatrick and Ryan2006).
When the complex organization of the embryonic disc at the molecular and cellular levels is taken into account, the splitting theory of the model does not seem a reasonable explanation for the origin of both late MC MA and conjoined twins. Notwithstanding, some authors insist on the capacity of the embryonic axes to divide into two parallel ones capable of inducing two new primitive streaks (see Note Footnote 49; Kaufman, Reference Kaufman2004), or on the unlikely possibility that ‘if the streak splits along its length prior to completion of formation, conjoined twins result’ (see Note Footnote 50; Downs, Reference Downs, Kleiman, Cloud-Hausen, Matta and Handelsmaneds2008).
Conclusion
From this critical review it can be concluded that the current model of the timing of MZ twinning is not based on facts but only on apparently reasonable conjectures. When the model is analyzed in detail, it reveals itself as fragile and untenable. Consequently, from the scientific and bioethical perspectives, the model ought to be presented not as a reliable record of observed facts, but as a hypothetical construct. It lacks the required strength to adjudicate on biological or bioethical issues related to the early embryo. Its acceptance by many is grounded on an uncritical acceptance of these conjectures and the passage of time (see Note Footnote 51; Boklage, Reference Boklage, Blickstein and Keith2005).
Although this critical assessment goes against the grain, it has, however, some support in recent bibliography (see Note Footnote 52; Matias et al., Reference Matias, Montenegro, Blickstein, Kurjak and Chervenak2011). The model is inadequate and unable to explain some instances of twinning associated with assisted reproduction practices (Klein et al., Reference Klein, Grunfeld, Duke, Mukherjee, Sandler and Copperman2005). To overcome the present situation it seems necessary to ‘disenchant’ the model and invite the proposal of new assessable theories on the mechanism and timing of human twinning.
Addendum
After so much criticism on the model, the author is uncertain about the convenience of adding here a short note on a theory of himself on the origin of MZ twinning. After due consideration, he feels obliged to presented it for discussion and critical evaluation. A very similar theory on the origin of MZ twins based on a process of long fertilization that gives origin through an intermediate stage to two zygotes has been recently presented by López-Moratalla and Cerezo (see Note Footnote 53; López-Moratalla & Cerezo, Reference López-Moratalla, Cerezo, Terzis and Arp2011). My theory is based in two premises:
1. All MZ twinning is the result of the first zygotic division. In other words, in the case of MZ twinning, the first cleavage division of the fertilized egg, instead of giving origin to two blastomeres, generates twin zygotes.
2. The structure of the fetal membranes does not depend on the splitting of an embryo, but on different modes of fusion of the membranes of the twin embryos within the pellucida (or, in the case conjoined twins, of the two embryonic bodies).
With respect to the first premise: this theory puts forward that there is a unique timing for human twinning: the division of the zygote (see Note Footnote 54). That proposal presupposes that the zygote can adopt two basically different types of cleavage: (1) the common type, that gives origin to two blastomeres; and (2) the twinning type, that generates twin zygotes. The twin zygotes (if there is no further MZ twinning to triplets or quadruplets) initiate their own embryonic development when each of them, shifting immediately to the standard developmental mode, cleaves to form its first two blastomeres.
As the cleavage of the zygote to form the first two blastomeres can be legitimately considered as the last event of the process of fertilization (see Note Footnote 55; Hertwig, Reference Hertwig and Hertwig1906; Jones Jr & Schrader, Reference Jones and Schrader1987; Silver, Reference Silver1987; Lejeune, Reference Lejeune1992; Bompiani, Reference Bompiani2006), it could be easily understood that twinning belongs to the fertilization process and, therefore, is not a post-fertilization event. An immediate consequence of this theory is a radical change in the chronology of MZ twinning: it shifts from the 14 days customarily assigned by the model based on embryo splitting, to around 24 hours after sperm penetration, when the process of transition from zygote to blastomeres takes place. It could be the result of certain molecular and cellular decisions taken in that transition. Therefore the clarification of their nature and mechanisms acquires the utmost importance.
With respect to the second premise: in normal circumstances, the embryos, twinned or not, develop for the first 5 days within the pellucida. In the case of twinning, the eventual fusion of their membranes can in principle follow one of two basic paths.
In the first one, no fusion is produced between the two closely apposed trophectoderms. Both twins follow their own independent course through the stages of cleavage, morula and blastocyst. They remain tightly packed together within the zona until the moment of hatching, when two independent blastocysts emerge and expand, to implant and develop as DC DA twins. This is the same and well known history of the DC DA twins of the model, with only one difference: the starting point of DC DA twins would not be, as the model proposes, the splitting of blastomeres during the first four days after fertilization, but the production of two independent zygotes at the fertilization process.
The second path concerns monochorionicity. This type of placentation in its two forms of amnionicity (DA or MA) requires, according to this the new proposed theory, the fusion of the trophectoderms of the two twins, an event that in all probability happens within the zona or during hatching. There are divergent opinions on the ability of the trophectoderm of two embryos for mutual adhesion and fusion. Some maintain that fusion of embryos before trophoblast differentiation results in the formation of aggregate chimeras, never in the production of twins (see Note Footnote 56; Boklage, Reference Boklage2010). Others think that there are distinct possibilities of trophectoderm fusion that depend upon the developmental stage (early or late) of the blastocyst. Redline has suggested that there could be a short period, immediately preceding the blastocyst stage, when trophoblast fusion is possible and compatible with the independent growth of the twins (see Note Footnote 57; Redline, Reference Redline2003). Another circumstance that would favour fusion of trophoblasts is the phenomenon of repeated cycles of collapse and expansion of blastocysts. Its mechanism and meaning have not been clarified until now, but it can be suspected that the collapse of the blastocyst, with the emptying of the blastocoelic cavity and the draining of the fluid into the perivitelline space, cannot be done without some disruption of the trophectoderm (see Note Footnote 58; Niimura, Reference Niimura2003). It can be speculated that welding the edges of such discontinuities could facilitate the fusion of blastocysts and bring about monochorionicity. Mio and Maeda have suggested the possible relationship between cycles of collapse-expansion and MC twinning (Mio & Maeda, Reference Mio and Maeda2008).
Lastly, the type of amnionicity could be determined, as Redline observes, by the distance separating the two ICMs within the fused twin blastocyst: ‘If the inner cells were sufficiently far apart, dizygotic twins with separate amnions could develop’ (see Note Footnote 59; Redline, Reference Redline2003). The concept that proximity-remoteness of ICMs within the blastocyst can decide amnionicity is not new; it has a long history. Although controversial, it was widely accepted at the beginning of the twentieth century (see Note Footnote 60; Bumm, Reference Bumm1902).
For the mechanism of origin of conjoined twins, the fusion theory of Spencer appears to offer a more reasoned and acceptable explanation that the simplistic theory of very late and incomplete splitting.
A few years ago, Blickstein devised two main requirements for future new theories on MZ twinning (see Note Footnote 61; Blickstein, Reference Blickstein2006). The first was the capability of give some reason of the consistent increase of MZ twinning with every method of assisted conception. The new theory that considers twinning as a fertilization event is in the best position to explain that IVF and its technical variants can affect directly the process of fertilization and be responsible for the observed increased incidence of twins.
The second provision required by Blickstein is that any new theory on MZ twinning must be able to explain why embryologists do not observe any physical splitting of the embryo in in vitro fertilization. If, according to the present theory, there is no embryo splitting, such a condition could be excused. Certainly, the visualization of two separate embryos within the confined space a single pellucida is technically a very difficult task, due to cellular overcrowding and the limited room for blastocyst expansion. Perhaps, continuous time-lapse video micrographic analysis and confocal microscopy could help to overcome this difficulty. In any case, we need a more complete knowledge of the molecular markers of the zygote–blastomere transition. Only then, the riddle of MZ twinning could start to be solved.
