Comment on the article by Kawachiya et al.: Blastocyst culture is associated with an elevated incidence of monozygotic twinning after single embryo transfer.

21 03 2011

To the Editor:

We read with great interest the article by Kawachiya et al., “Blastocyst culture is associated with an elevated incidence of monozygotic twinning after single embryo transfer”(1). We want to congratulate the authors for their nice and interesting work. However, this study also raises a few questions.

Were all the cycles independent or were some patients included for a fresh and a following frozen-thawed embryo transfer (FTET)? Were some couples included more than once over the 8 years, which might influence multivariable analysis?

Could the authors confirm that embryo culture conditions remained stable throughout the long study period, as culture media and/or atmosphere have been suspected by some authors to be responsible for monozygotic twinning (2)?

The authors conclude that blastocyst transfer is associated with an increased incidence of MZT as compared to cleavage stage embryo transfer, which has been the subject of some debate (3, 4). However, we were surprised by the discrepancy in ratios between fresh and FT ET in the two groups. Indeed, of the 22,855 cleavage-stage SET, 20,453 (89.5%) were fresh ET and 2,402 (10.5%) were FTET. On the other hand, 1,540 (6.2%) of the 24,926 SBT were fresh ET whereas 23,386 (93.8%) were FTET. The authors explain that FTET was chosen because the endometrial effect of clomiphene citrate (CC) results in lower than expected implantation rates for blastocysts. Did CC have no effect on endometrial receptivity after cleavage stage ET or were the women undergoing cleavage stage SET stimulated by a different protocol, enabling fresh ET without reducing the probability of implantation (in table 1, group A and group B had comparable success rates)? Indeed, it has been discussed that ovulation induction might increase the number of oocytes with an inborn propensity to undergo splitting after fertilization (5). Could the authors provide a table or additional information showing that SBT and cleavage stage ET concerned comparable patients undergoing comparable stimulation protocols?

After 47,841 SET, there were 31.3% clinical pregnancies and 23.3% live births. Of the 14,956 clinical pregnancies, there were 151 (1.01%) MZT and also 2 triplet pregnancies. The study confirms that MZTP is associated with a high incidence of prematurity, low birth rate and perinatal mortality (6). The authors concluded that patients should be informed of a less than 1% twin delivery after SET. However, if we look at table 1, there were 8/302 (2.6%) and 85/7,031 (1.2%) twin deliveries after single fresh and thawed blastocyst transfer, respectively. Although a low risk, this is not a “less than 1%” risk. Moreover, this incidence is lower than what has been reported in the literature with an average of 3% calculated over clinical pregnancies (3, 4, 6).

In table 1, vitrification appeared to be associated with a trend to a lower risk of MZT after SBT (9/399 (2.3%) after FTET versus 111/9483 (1.2%) after fresh ET, p=0.052), which has not been reported in previous studies. How would the authors explain that trend, which was not discussed in the article?

Lionel Dessolle M.D., M.Sc.
Thomas Fréour, Pharm.D., M.Sc.
Service de gynécologie-obstétrique et médecine de la reproduction, service de biologie de la reproduction et du développement
Centre Hospitalier Universitaire de Nantes
Nantes Cedex 1, France

References
1. Kawachiya S, Bodri D, Shimada N, Kato K, Takehara Y, Kato O. Blastocyst culture is associated with an elevated incidence of monozygotic twinning after single embryo transfer. Fertil Steril 2011 Jan5 Epub ahead of Print.

2. Yanaihara A, Yorimitsu T, Motoyama H, Watanabe H, Kawamura T. 2007 Monozygotic multiple gestation following in vitro fertilization: analysis of seven cases from Japan. J Exp Clin Assist Reprod 2007; 4: 4.

3. Vitthala S, Gelbaya TA, Brison DR, Fitzgerald CT, Nardo LG. The risk of monozygotic twins after assisted reproductive technology. A systematic review and meta analysis. Hum Reprod Update 2009; 15: 45-55.

4. Papanikolaou EG, Fatemi H, Venetis C, Donoso P, Kolibianakis E, Tournaye H et al. Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer. Fertil Steril 2010; 93(2): 592-7.

5. Blickstein I, Keith LG. On the possible cause of monozygotic twinning: lessons from the 9-banded armadillo and from assisted reproduction. Twin Res Hum Genet 2007; 10: 394–9.

6. Dessolle L, Allaoua D, Fréour T, Le Vaillant C, Philippe HJ, Jean M, Barrière P. Monozygotic triplet pregnancies after single blastocyst transfer: two cases and literature review. RBM Online 2010; 21: 283-9.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.03.055

The Authors Respond:

We thank Drs. Dessolle and Fréour for their interest and the thorough review of our recently published paper on the incidence of monozygotic twinning (MZT) after single embryo transfer. As follows here are the answers to the points raised by them, which we hope could clarify further the findings of our study.

1) In our large cohort, individual patients could have been included with more than one cycle. Although this might be considered as a limitation and also could have influenced the results of the multivariate analysis, this was chosen because only first-cycle inclusion would have reduced considerably the sample size. As mentioned in our paper, only sufficiently large cohorts are able to detect any significant difference in MZT rates because of its overall low incidence. In this respect, our study was similar to most of the other published MZT cohorts, and to our knowledge, to date only one study respected only first-cycle inclusion (1).

2) Although during a seven-year period some changes inevitably might have occurred in laboratory conditions, but the type of incubators used and gas concentrations were not changed, and the embryo culture medium used was the same during the most of the study period. Furthermore, no secular trends were observed in MZT rates, which remained stable during the whole study period.

3) As discussed in our paper, blastocyst implantation rates were significantly higher following cryopreserved embryo transfer cycles, which might be related to potential deleterious endometrial effects of clomiphene citrate in fresh cycles. This advantage in favor of cryopreserved cycles was less pronounced for cleavage-stage embryos, therefore other variables not evaluated in our study (such as embryo selection or specific patient characteristics) might have influenced pregnancy rates between groups. Any potential effect of ovarian stimulation was already evaluated in our paper by a multivariate analysis (including 6 variables), and it showed no association between the type of stimulation protocol used (unstimulated versus minimal stimulation with clomiphene-citrate) and MZT (OR: 1.74 95CI% 0.6-5.03; p=0.31).

4) In our paper, the overall risk of delivering an MZT twin was expressed as the ratio of MZT twins per obtained clinical pregnancy and not per delivery, which resulted in 0.79% (119/14.956). If calculated per delivery the MZT risk would be somewhat higher 1.07% (119/11,129). It must be also emphasized that in a different clinical setting where more than one embryo/blastocyst is transferred, the subsequent MZT risk probably would be even higher.

5) Although in our series there seemed to be a trend toward diminished MZT risk after embryo/blastocyst vitrification, this was not significant in multivariate analysis (OR: 0.64 95%CI: 0.17-2.37; p=0.51). A similar trend of limited statistical significance was also observed in the study of Knopman et al. (2), where frozen-thawed embryos had a lower MZT rate compared to fresh ones (0.8% versus 2.1%, p=0.05). Hence, currently it is not possible to hypothesize about the association of embryo cryopreservation and MZT risk because data are very scarce, especially in relation to the recently developed vitrification methods.

Satoshi Kawachiya, M.D.
Daniel Bodri M.D., M.Sc., Ph.D.
Osamu Kato, M.D.
Kato Ladies Clinic
Tokyo, Japan

References
1. Papanikolaou EG, Fatemi H, Venetis C, Donoso P, Kolibianakis E, Tournaye H et al. Monozygotic twinning is not increased after single blastocyst transfer compared with single cleavage-stage embryo transfer. Fertility and sterility;93:592-7.

2. Knopman J, Krey LC, Lee J, Fino ME, Novetsky AP, Noyes N. Monozygotic twinning: an eight-year experience at a large IVF center. Fertility and sterility;94:502-10.

Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.03.054

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