I'd like to present some questions and observations to the group, to gain a greater understanding of the mechanisms driving monozygotic twinning (MZT) in IVF. Most papers suggest a relationship between MZT and time in culture (day 3 vs. day 5/6), and/or use of sequential media, and/or use of zona breaching techniques (ICSI, AH, other?). A recent paper (Jain et al., 2004 JARG 21:103-107) also suggests the possibility of a mechanism involving signaling by closely implanted embryos.
When do the MZT actually form? Pre-hatching, at hatching, or post-hatching/implantation?
I've observed one pre-hatching, expanded blastocyst with two distinct inner cell masses (ICM), and several that appear to have cell masses closely opposed but 'bridged' by a cell or cells - and there are a few papers where others have observed these phenomenon as well - demonstrating that at least some of these MZT are formed pre-implantation, pre-hatching. In discussions with a former professor, he observed blastocysts retreived from superovulated cattle with two ICM's present - is this then a function, in part, of the ovarian stimulation? If so, what is the incidence of MZT in COH intrauterine insemination cycles?
I've also observed embryos hatching directly across the inner cell mass, shaping the ICM into a classic dumbell configuration, suggesting also that hatching (breach from ICSI, AH, or hardened zona) may influence ICM integrity. What is the incidence of MZT from programs that remove the zona entirely before replacement? What is the incidence of MZT in programs that perform ICSI on all patients, or assisted hatching on all patients, or PGD for that matter? And as cryopreservation appears to have an effect on the zona, are there more or less MZT in FET cycles, compared to fresh embryo replacement cycles?
As to the concept proposed by Jain et al., it appears that in my data sets at least - not statistically analyzed - there are more MZT present with multiple embryos implanted - that is, two sacs with three FHT total, as compared to single sac with two FHT.
What about group culture vs. single embryo culture - are there any data that suggest an embryo/volume ratio effect? I group up to six embryos in approximately 50ul (overall MZT of about 1.5%) however I recall that in the very early use of extended embryo culture in his program, Barry Behr mentioned culturing entire cohorts of embyros in a single microdrop (not sure if this is the case now), but Behr et al. reported a MZT rate of approximately 5% in a later paper (2000 JARG 17:349-351) with authors from several programs, however there were no details regarding how the embryos were cultured, respective to groups or single embryos.
Last, what about the details of the culture conditions themselves - oxygen tension, pHi, microdrops vs. tubes (oil overlay), co-culture, sequential (two or more stage-specific media) vs. single non-stage-specific media, and so on?
Mike [Respond to this question]