Uses of embryo duplication in humans: Embryology and ethics

E.C. Wood1,3 and A. Trounson2

1 Department of Obstetrics and Gynaecology, Monash University, and 2 Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia


    Introduction
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
The use of artificial twinning in the cattle and sheep industry to improve the reproductive efficiency raises the possibility of applying this technique to infertile couples who at present have 10–28% success rates for single in-vitro fertilization (IVF) procedures. This raises ethical and embryological considerations in determining whether the technique should and could be applied to humans. The rejection of our application to the Victorian Government body controlling the use of new infertility procedures led us to raise the matter for wider discussion in an endeavour to help clarify the reasons for approval or disapproval. The Victorian committee's reasons for rejecting the application were that artificial twinning is a form of `cloning'.


    Natural and IVF monozygotic twinning
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
Placentation in monozygous twins depends on the temporal sequence of division of the inner cell mass. Cleavage up to the third day after fertilization results in a dichorionic diamniotic pregnancy, whereas with twinning occurring at later gestational ages, the fetuses will be firstly monochorionic, diamniotic (days 5–8), and then monochorionic, monoamniotic (days 8–10). The complete separation of the early embryo into a dichorionic diamniotic pregnancy is possible as blastulation does not occur until days 5 or 6. The blastocyst consists of the inner cell mass and trophoblast. The trophoblast forms a monochorionic (fused) placenta and two discrete inner cell masses form twins within or after escape from the zona pellucida. Later division of the embryonic lineage is limited to twin formation with a single amnion and chorion, which both have already been formed.

Monozygous twins occur in IVF programmes and are associated with consequences similar to naturally conceived monozygous twins. In two thirds of monozygous twins, monochorionic placentation occurs.

Vascular anastomosis occur between the two halves of the shared placenta. In most cases this is a normal event (Table IGo). Imbalance in the placental support between the twins may lead to clinical sequelae of intertwin transfusion, which may be acute, chronic or reversed (Table IIGo).


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Table I. Vascular connections in monochorionic twins
 

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Table II. Intertwin transfusions
 

    Acute intertwin transfusion
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
Acute intertwin transfusion results from the death of one twin after which the healthy twin transfuses blood into the dead fetus, itself becoming anaemic and hypovolaemic. As a result the healthy twin has a 25% chance of fetal death and a similar incidence of survival with necrotic neurological and/or renal lesions (Fusi and Gordon, 1990Go; Nijhuis et al., 1997Go).

The impending fetal death of one twin represents a complex management issue: (i) delivery of both twins which at 25–28 weeks exposes the healthy twin to the risks of prematurity; (ii) conservative management which allows a single fetal death but exposes the surviving fetus to the risks of acute intertwin transfusion. Fetal sampling and fetal blood transfusion may succeed if the diagnosis can be made at an appropriate time; and (iii) selective feticide occluding the circulation of the compromised fetus to avoid acute intertwin transfusion. This has been achieved by laparoscopy (Denbow and Fisk, 1998Go).


    Chronic intertwin transfusion
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
Of monochorionic twins, 4–35% are affected by feto–fetal transfusion syndrome (FFTS) (Robertson and Neer, 1983Go; Patten et al., 1989Go; Radestad and Thomassen, 1990Go) and this accounts for 15–17% of overall perinatal mortality in twins (Weir et al., 1979Go; Steinberg et al., 1990Go), and if untreated in the second trimester the loss rate approaches 100% (Weir et al., 1979Go; Chescheir and Seeds, 1988Go; Saunders et al., 1992).

The clinical changes are thought to be due to intertwin transfusion in a state of flux or free mixing of blood between the twins. The differences between the twins, a large amniotic sac (hydramnios) enlarged heart and oedema, and the growth restricted small amniotic volume of the other, are due to volume changes rather than haematological differences, the haemoglobin in utero being similar.

Treatment options are suboptimal and include, serial aggressive amniotic fluid reduction, laser ablation or ligation of placental vasculature, amniotic septostomy to join the two unequal sized sacs, or selective feticide.

The long-term sequelae in FFTS are cerebral white matter lesions with neurological impairment, cardiac dysfunction and morbidity associated with prematurity (Denbow and Fisk, 1998Go).


    Twin reversed arterial perfusion sequence
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
Acardiac twinning occurs in 1% of monochorionic twins. It is characterized by a grossly abnormal perfused twin and its co-twin, denoted as the pump twin. The perfused twin receives blood from placental arterio–arterial communication from the other twin, deoxygenerated blood entering its umbilical artery. This results in absent or rudimentary structures, head, chest and upper trunk. The pump twin may develop cardiovascular disease similar to FFTS recipients, volume overload with cardiomegaly and hydrops fetalis. Of these pregnancies, 65% result in preterm labour, with perinatal mortality of 100% for the perfused twin and ~50% for the pump twin (Van-Allen et al., 1983Go).

Conservative treatment or separation of the twins cardiovascular systems are the two options in management. Separation techniques (laser, suture ligation, ultrasound-guided or laparoscopic) have been used but may cause premature labour.


    Monozygotic twins may not be identical in genotype and phenotype
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
Genetic differences may arise from natural or artificial homozygous twins (Machin, 1996Go; Edwards and Beard 1998Go). Machin has pointed out that the use of the adjective identical rather than monozygotic leads to a misunderstanding about the biology of the monozygotic twinning. Monozygotic twin pairs may not be identical; there may be major discordance for birth weight, genetic disease, and congenital anomalies. These differences indicate that post-zygotic events may lead to the formation of two or more cell clones in the inner cell mass of the early embryo that stimulate the monozygotic twinning event. There is also evidence that there may be unequal allocations of numbers of cells to the monozygotic twins. The genetic differences between monozygotic twins may arise from mosaicism, skewed X-chromosome inactivation in female twins and dominant post-zygotic gene mutation (Machin, 1996Go). This may have widespread implications for the developmental events during embryogenesis and formation and vascularization of the placenta. Discordant physical malformations also may arise from different placental vasculature of the twins (Machin, 1996Go; Machin et al., 1996Go).

While it is true that most monozygotic twins are phenotypically very similar, there are significant numbers of monozygotic pairs who are neither genotypically nor phenotypically identical (Machin, 1996Go). Cohorts of monozygotic twin pairs have been monitored to determine non-genetic influences on the risks of developing diseases and on behaviour, the assumption being that studies comparing genetically identical monozygotic twin pairs eliminate genetic variability when the twins are reared apart. Any differences that have been attributed to environmental factors in twins reared apart may overestimate the effect of the environment as genetic or placental influences may produce differences between the twins. The percentage of discordant genetic monozygotic twins is unknown (Edwards and Beard, 1998Go). Presumably there may also be genetic differences in artificial twins.


    Artificial twinning in cattle and humans
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
Everyone is familiar with identical twins which result from the natural splitting of human embryos. They are accepted by society and in many ways are comparable to non-identical twins. Scientists have been able to reproduce the process of twinning in the laboratory by splitting embryos during their early development (Williams et al., 1984Go; Kippax et al., 1991Go).

The immediate concern is whether artificial twinning could result in identical twins where complications occur. Artificial identical twins would represent the one third of naturally occurring monozygotic twins having their own placenta, which can still be separate or fused and their own amniotic sacs, and would result from separation of the inner cell mass and trophoblast. This avoids those complications which occur in natural monozygotic twins which include cord entanglement, locked twins during birth and cross circulation between twins leading to acute, chronic, or retrograde intertwin transfusion.

Artificial twinning has been used extensively in cattle (Lewis, 1994Go; Hygate, 1995). Many thousands of calves have been born worldwide and there have been no reports of the technique producing abnormalities in the offspring, including an extensive Australian experience (Lewis, 1994Go). The health of the calves resulting from embryo duplication is not different from normal calves. The establishment of a pregnancy depends upon an `all or nothing' effect. If there are enough viable cells in the demi-embryo to establish a pregnancy, a normal offspring results. If not, then no offspring result.

Most demi-embryos that have been split are transferred individually to separate cows and birth and rearing is normal. When the two demi-embryos are transferred to the same cow the only problems are the same as natural twins with possibly difficulty at birth or mothering, but no refusion of embryos to form Siamese twins.

The success of the embryo bisection is greatest at the blastocyst to expanded blastocyst stage (Lewis, 1994Go; Hygate et al., 1995Go). This is probably due to the fact that there are more cells in the inner cell mass of a blastocyst, and after bisection there are more cells left in the demi-embryo to establish a pregnancy (I.M.Lewis, personal communication). This hypothesis agrees with the finding that the technique does not work well in poor quality embryos, which have fewer cells and possibly other cell defects. Poor quality embryos are transferred whole as bisection results in a lower overall chance of pregnancy.

Transfer of good quality whole cattle embryos result in a pregnancy rate of ~70% per transfer. Where good quality embryos are bisected the pregnancy rate of the resulting demi-embryos are ~50–55%, resulting in a 100–110% pregnancy rate for the original whole embryo. This gives a 30–40% greater chance of conception. Poor quality embryos result in a pregnancy rate of around 40% and after bisection only 20% (I.M.Lewis, personal communication).

Demi-embryos may be cryopreserved as blastocysts and are viable after thawing and transfer (I.M.Lewis, personal communication). New cryopreservation methods enable the high survival of in-vitro produced, demi-and cloned embryos devoid of their zona (I.M.Lewis, personal communication).


    Prevention of monochorionic twins
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
It is hypothetical that unborn embryos are grown in culture for IVF that a bilobed inner cell mass will occur at the blastocyst stage (days 6–8 after fertilization) and gives rise to monozygotic twinning. When this occurs in the blastocyst, monochorionic twins may result with its associated hazards. Splitting the bilobed blastocyst at this stage may prevent monochorionic monozygotic twin pregnancy with its associated markedly increased risk of perinatal death or survival with brain damage.

It has been observed that monozygotic pregnancy is higher than expected with new embryo culture methods. Following the transfer of human blastocysts, the incidence of monozygotic twins is 2.7% of pregnancies (Rijnders et al., 1998Go). It could be argued that separation of the trophoblast of embryos with two distinct inner cell masses by microsurgery would benefit the probability of survival of both embryos formed and this would be the preferred ethical position. This is an argument for splitting embryos with naturally occurring twin inner cell masses.


    Artificial twinning may help infertile couples
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
Other arguments can be put forward to produce completely separated monozygotic dichorionic diamniotic twins by microsurgically separating the blastocyst into two equal halves cutting through the axis that includes the inner cell mass (Figure 1Go) (Trounson and Pera, 1998Go). This will result in the formation of two smaller but complete blastocysts with outer trophoblast and an inner cell mass. The benefits of this procedure may be for patients where it is possible to only obtain one or a few genetically normal embryos by IVF techniques. For example, women who are older (>37 years) or who have had severe pelvic diseases such as endometriosis that damage the ovary, frequently produce few eggs when the ovaries are stimulated on an IVF programme (Lancaster et al., 1995Go). The possibility of conceiving each cycle of IVF treatment with a single embryo transfer in a women aged >40 years is only 3–8% (Lancaster et al., 1995Go). Increasing duration of infertility and increasing number of previous failed treatments also are associated with lower IVF success rates (Templeton et al., 1996Go). Live birth rates per treatment declined from 15.3 to 8.6% when the duration of infertility was extended from 1–3 years to >12 years. A decline in live birth rates per treatment from 14% in women having their first IVF treatment to 8.9% in women having IVF after three previous failed treatments (Templeton et al., 1996Go).



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Figure 1. Blastocyst containing a duplicated inner cell mass with the plane of dissection indicated for twin embryos that are monochorionic and monoamniotic monozygotic twins.

 
Numerous other examples can be encountered in clinical reproductive medicine where small numbers of embryos are obtained but limit the opportunity for pregnancy. Duplication of embryos by microsurgical separation may improve their chance of pregnancy and since monozygotic twins occur in natural conception, their occasional birth by intention is unlikely to disturb the general community if the overall pregnancy and birth rates are substantially increased for patients where this may be indicated.


    Ethics
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
A variety of ethical objections have been raised to cloning human embryos in news reports ranging from; the possible sale of embryos to the infertile; the production of organs or tissue for children needing transplants; cloning being contrary to human values; an opportunity for mischief (Jacoby, 1993Go; Waldholz, 1993Go); a call for government and societal debate; a tunnel of madness (New York Times, 1993Go); banning cloning in federally financed institutions (New York Times, 1993Go); and the response of 58% of Americans to oppose cloning one week after the announcement of the birth of `Dolly' the sheep (Robertson, 1995Go). John Robertson writing in the Monash Bioethics Review listed concerns related to the cloning of embryos (Robertson, 1995Go). This involved the artificial method of reproduction; the manipulation and possible destruction of embryos; the effect on the resulting offspring; violation of inherent uniqueness of an individual; belief that identical embryos or massed produced embryos may be sold for their genetic desirability; embryos may be created to produce organs and tissues for children who need transplants; the procedure is a result of an uncritical approach resulting from scientific zeal; profit and desire of the infertile has encouraged such a procedure; eugenic possibilities in cloning selected embryos and adults and uncertain legal issues.

IVF has already been generally accepted and involves an artificial method of reproduction and manipulation and possible destruction of embryos if these are abnormal. Monozygotic twins do occur in natural reproduction, the effect on each twin being accepted. The violation of an inherent uniqueness of an individual already exists in natural reproduction with the occurrence of monozygotic twins. The use of artificial twinning may be controlled by the government as it has been in the use of donor eggs, donor spermatozoa and surrogacy in Australia. For example, a restriction could be placed on couples wishing to create embryos to produce organs and tissues for children who need transplants.

Twins are associated with some disadvantages, a four-fold increase in perinatal death rate, three per 100, and if severe prematurity occurs an increased risk of mental deficiency in offspring. The mother has double the chance of requiring Caesarean section (42%, compared to 19%) (Lancaster et al., 1995Go). The risk to artificial twins may be less than to natural monozygotic twins. Separate placenta and amnion are formed, so that cord entanglement, locking of twins during birth and cross circulation of placental vasculature, which may cause fetal death or growth retardation, would be avoided.

Artificial twinning has the second advantage that the twinning is planned and the parents informed of risks, which does not occur in natural twinning. Bryan emphasized the psychological effects of being born as a monozygotic twin (Bryan, 1998Go). Problems exist because of the unique situation of two similar individuals growing up together but neither behaviour problems nor long-term psychological disturbance are significantly greater than singletons (Rutter and Redshaw, 1991Go; Kendler et al., 1996Go).

Artificial twinning may provide donor embryos for infertile couples who cannot conceive naturally or with IVF. The shortage of adopted children and donor embryos may be overcome by the production of artificial twins. The offspring of the original embryo could be checked before using the frozen stored copy. Adoption or unwanted donor embryos can give little or no information relevant to the future health of the child. The donated embryo would enable the infertile woman to experience pregnancy, birth and early rearing. A donor embryo would also be advantageous for a couple with increased genetic risks to offspring from their own spermatozoa or oocytes.


    Notes
 
3 To whom correspondence should be addressed at: 284 High Street, Ashburton, Victoria 3147, Australia Back

This debate was previously published on Webtrack 99, December 13, 1999


    References
 Top
 Introduction
 Natural and IVF monozygotic...
 Acute intertwin transfusion
 Chronic intertwin transfusion
 Twin reversed arterial perfusion...
 Monozygotic twins may not...
 Artificial twinning in cattle...
 Prevention of monochorionic...
 Artificial twinning may help...
 Ethics
 References
 
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