Monozygotic twinning: is it related to apoptosis in the embryo?

The advent of prolonged in-vitro preimplantation embryo culture using sequential culture media (Gardner et al., 1998) has permitted many IVF laboratories to successfully adopt blastocyst transfer. Unfortunately, an unexpected side effect of this success has seen a small but significant appearance of monozygotic twinning. This has now been described by several authors (Peramo et al. Curiously enough, no monozygotic twinning was reported during our 10 years of practice, including over 800 deliveries, when co-culture was used to obtain blastocysts. The phenomenon of monozygotic twinning is seemingly a result of an in-vitro artefact and leads us to presume that although culture techniques have been dramatically improved in recent years, we are still far from providing the best environment. A number of hypotheses have been proposed in relation to this phenomenon. An association with in-vitro related zona hardening has been implicated, whereby embryos are pinched into two at the time of assisted hatching (Alikani et al., 1994). However, this aspect would not always lead to monozygotic twinning, as a small percentage would presumably form a fetal sac and an empty sac containing no fetus, if only trophectodermal cells were pinched off. This may have been missed during diagnosis. In addition, the above reports linked to monozygotic twinning are not exclusively linked to the use of assisted hatching. The appearance of monozygotic twinning leads us to suspect a possible anomaly in the distribution of inner cell mass (ICM) cells within the embryo. The question therefore arises as to whether the ICM is more sensitive to culture media. Interestingly, the cells within the ICM appear to be more sensitive to apoptosis and less resistant to disruption than trophoblastic cells (Pampfer, 2000). It is possible that some human embryos are hypersensitive and that there could be an over-stimulation of apoptosis in media containing an excessive glucose level. Moley et al. have shown that hyper-glycaemia induces apoptosis in preimplantation mouse embryos (Moley et al., 1998). The pro-apoptotic effector Bax was found to be increased in murine blastocysts exposed to hyperglycaemia, through free radical formation (Pampfer, 2000). Pampfer et al. found that two intracellular effectors of apoptosis, caspase-3 and caspase-activated deoxyribonuclease (CAD), were involved in the embryotoxicity of high glucose (Pampfer et al., 2001). Subsequent data has suggested that Bcl-2 is involved in the protective response against the induction of chromatin degradation in blastocysts on their exposure 247 to high concentrations of D-glucose in vitro, whereas nuclear …