Monozygotic twinning associated with day 5 embryo transfer in pregnancies conceived after IVF

Victoria Wright1,2, Laura A. Schieve1, Anjel Vahratian1 and Meredith A. Reynolds1

1 Division of Reproductive Health, National Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA

2 To whom correspondence should be addressed. Email: vwright{at}cdc.gov


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: This study examines the association between day of embryo transfer and monozygotic (MZ) twinning. METHODS: We used a population-based sample of 108 336 IVF/embryo transfer procedures in which the patients oocytes' were freshly fertilized (non-frozen; non-donor) and 39 198 resultant pregnancies from US clinics in 1999 and 2000. Cases were pregnancies for which the number of fetal hearts observed on ultrasound exceeded the number of embryos transferred. These pregnancies were considered to contain at least one set of MZ twins. A total of 226 MZ pregnancies were compared with two control groups: 23 880 singleton pregnancies (one fetal heart) and 15 092 other multiple-gestation pregnancies (≥2 fetal hearts but the number of fetal hearts on ultrasound was less than or equal to the number of embryos transferred). RESULTS: Cases of presumed MZ multiple-gestation pregnancies were more likely to have had a day 5 embryo transfer compared with day 3 embryo transfers than singleton pregnancies [adjusted odds ratio (AOR)=3.92, 95% confidence interval (CI)=2.97–5.17] or other multiple-gestation pregnancies (AOR=3.91, 95% CI=2.96–5.17) conceived with IVF/embryo transfer. CONCLUSIONS: Day 5 embryo transfer may be associated with increased MZ twinning.

Key words: blastocyst/extended culture/IVF/monozygotic/twinning


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The increased use of assisted reproductive technologies (ARTs) has been an important contributing factor to increasing multiple birth rates in the USA (Wright et al., 2003Go).

Multiple embryo transfer is still the norm in ART treatment because there is concern that limiting the number of embryos transferred might reduce the likelihood of achieving pregnancy. Many advocate developing strategies to improve selection of high-quality embryos, thus maximizing the potential for implantation while limiting the number of embryos transferred (Racowsky et al., 2000Go). With the development of commercially available culture media, an increasingly feasible and popular treatment option is to extend embryo culture and transfer embryos at the blastocyst stage. This method allows the evaluation of embryo quality and the selection of fewer, high-quality embryos for transfer which, in turn, should reduce the number of multiple births.

Several randomized studies have demonstrated high efficacy with blastocyst transfer in comparison wirh the transfer of cleavage stage embryos (Gardner et al., 1998Go; Van der Auwera et al., 2002Go; Frattarelli et al., 2003Go). However, a recent meta-analysis suggests no difference between blastocyst and cleavage stage transfer in implantation and pregnancy rates (Blake et al., 2004). Moreover there is an emerging concern that extending embryo culture and transfer to the blastocyst stage may increase the risk for embryo splitting and subsequent monozygotic (MZ) multiple-gestation pregnancies. Several case studies, case series and small comparative studies suggest an association between blastocyst transfer and MZ twinning (Rijnders et al., 1998Go; Peramo et al., 1999Go; Behr et al., 2000Go; Van Langendonckt et al., 2000Go; da Costa et al., 2001Go; Sheiner et al., 2001aGo,bGo; Milki et al., 2003Go). MZ multiple gestations are of particular concern because morbidity and mortality rates are even higher for MZ twins than for dizygotic (DZ) twins (Deromet al., 1991). We used a population-based data set of IVF/embryo transfer procedures to perform a case–control analysis of the association between day of embryo transfer and MZ twinning.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Study population
The Fertility Clinic Success Rates and Certification Act of 1992 (FCSRA) requires that each medical centre in the USA that performs IVF/embryo transfer or related ARTs report data for every ART cycle initiated to the Centers for Disease Control and Prevention (CDC) annually for the purpose of reporting clinic-specific pregnancy success rates (US Department of Health and Human Services, 1992Go). An ART cycle begins when a woman begins taking fertility drugs or starts ovarian monitoring with the intent of having embryos transferred. The ART clinic submits data obtained from clinic records for each cycle initiated for a given reporting year, January 1 to December 31, in a standardized format to the Society for Assisted Reproductive Technology (SART). SART annually creates a database of ART procedures and provides these data to CDC. The data file is organized with one record per cycle. Multiple cycles from a single patient are not linked. Some clinics (6–7%) did not report their data, despite the federal mandate; however, most of these clinics are believed to be smaller than average practices.

We limited our analysis to IVF/embryo transfer procedures performed in 1999 and 2000 in which the embryos were freshly fertilized with the patient's oocytes (i.e. non-frozen, non-donor). Of 110 381 such transfer procedures, we excluded small proportions of procedures for which the number of embryos transferred was either missing (n=134) or implausible (n=6); the data necessary to calculate the day of embryo transfer were missing (n=32) or resulted in implausible values [i.e. <2 (n=884) or >6 (n=257)]; and where the patient age was aged either <20 years (n=17) or >44 years (n=715). Among the remaining 108 336 transfer procedures, there were 41 534 clinical pregnancies. We excluded those pregnancies missing data on the number of fetal hearts on ultrasound (n=2336). Thus, our final sample consisted of 39 198 pregnancies.

Definitions of cases and controls
Cases were defined as pregnancies in which the number of fetal hearts on ultrasound exceeded the number of embryos transferred. We considered these pregnancies to contain at least one set of MZ twin fetuses. We acknowledge that such pregnancies might not entirely be MZ multiple gestations; our definition allows for the inclusion of pregnancies with a mix of MZ and DZ fetuses. We identified 226 cases.

We selected two control groups: (i) IVF/embryo transfer singleton pregnancies (one fetal heart); and (ii) other IVF/embryo transfer multiple-gestation pregnancies (≥2 fetal hearts on ultrasound, but the number of fetal hearts was less than or equal to the number of embryos transferred). These control groups included 15 092 and 23 880 pregnancies, respectively. Having two control groups was desirable because each group had a possible limitation. First, comparison of our cases of MZ pregnancies with the control group of other multiple-gestation pregnancies could be biased toward the null hypothesis of no association between presumed blastocyst culture and MZ twinning. The ‘other multiples’ control group could contain some MZ and DZ multiple pregnancies, a misclassification that would be independent of day of embryo transfer, i.e. a non-differential misclassification. On average, non-differential misclassification tends to attenuate associations between two variables (Schlesselmann, 1982Go). In contrast, comparison of MZ multiples with a control group of singleton pregnancies might produce a spurious association. Previous studies suggest that blastocyst culture may increase implantation and pregnancy rates; if true, such a comparison might overstate an association with blastocyst culture if the MZ case pregnancies were actually a mix of MZ and DZ fetuses. This is likely to be the case with many case pregnancies in this study.

Exposure of Interest
Day of embryo transfer was calculated using dates of oocyte retrieval and embryo transfer. All embryo transfers that occurred on day 5 were defined as ‘presumed’ blastocyst transfer. Data were not available to classify specifically whether blastocyst sequential culture medium was used, nor to determine the developmental stage of the embryos transferred.

Embryo transfers that occurred on day 3 (71%) were considered the current standard of care and were thus considered the reference category for our analyses. In addition to examining day 5 transfers, we also considered cycles in which embryo transfer occurred on days 2, 4 or 6.

Data analysis
We calculated the percentage of pregnancies that were multiple gestations and the percentage with at least one MZ multiple gestation for embryo transfers conducted on days 2, 3, 4, 5 and 6. We also calculated odds ratios (ORs) and 95% confidence intervals (CIs) for the association between MZ twinning and day of embryo transfer.

We then considered the possibility of confounding or effect modification by patient and treatment factors for pregnancies resulting from a day 5 or day 3 transfer. We performed stratified analyses and multivariable logistic regression to evaluate and adjust for maternal age, number of prior ART procedures and number of prior births. We calculated adjusted ORs and 95% CIs for comparisons of case pregnancies with pregnancies from each control group. To address concerns that an association between MZ twinning and extended embryo culture was attributable to various treatment subgroups, we repeated these analyses after restricting our sample to procedures with a minimum of five oocytes retrieved; procedures with a minimum of 10 oocytes retrieved; procedures with extra embryos available and cryopreserved; procedures in which ICSI or assisted hatching techniques were not used; and procedures in which three or more embryos were transferred in a cycle. To address concerns that MZ twinning might occur through a natural conception concurrent with ART, we restricted the analyses to women with tubal infertility, a subset least likely to experience such an effect. We used Statistical Analysis Software (SAS), Version 8.2, to perform all analyses.

This study was approved by the Institutional Review Board at the CDC.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The 226 cases identified are described in Table I. For 215 cases (95%), the number of fetuses exceeded the number of embryos transferred by one; for 10 cases (4%), the number of fetuses exceeded the number of embryos transferred by two; and for one case the number of fetuses exceeded the number of embryos transferred by three. Because of fetal loss, the number of infants born was often less than the number of fetal hearts reported early in pregnancy (data not shown).


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Table I. Cases of MZ multiple pregnancies by number of embryos transferred and number of fetal hearts on ultrasound

 
Of the 108 336 transfer procedures included in this study, >70% occurred on day 3 (Table II). Approximately 23% of these transfer procedures were extended beyond day 3, the majority of which were transferred on day 5 (16%). The overall pregnancy rate was 38% and the highest pregnancy rate was among day 5 transfers (45%); pregnancy rates were slightly lower than average for procedures with embryos transferred on days 2, 4 and 6. Thirty-nine percent of pregnancies were multiple gestations. Day 5 transfer procedures had the highest multiple-gestation rate (41%), despite a lower than average number of embryos transferred (2.50 versus 3.15 embryos transferred, respectively). Procedures in which embryo transfer occurred on day 6 also had a relatively low average number of embryos transferred (2.39 versus 3.15 embryos transferred, respectively); however, the multiple-gestation rate was also reduced (32%).


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Table II. Relationship between day of transfer and pregnancy, multiple gestation and MZ multiple gestation

 
Overall, the percentage of pregnancies with at least one presumed MZ multiple gestation was 0.6%; however, the rate varied by the day of embryo transfer (Table II). In comparison with transfer procedures that occurred on day 2, the percentage of MZ multiples increased 2-fold among transfer procedures that occurred on days 3 and 4, and >7-fold to 1.5% for day 5 transfers.

Women who had MZ multiple-gestation pregnancies were significantly younger than women with a singleton gestation (Table III). When compared with pregnancies in either of the control groups, MZ case pregnancies were less likely to be the result of ART procedures that included assisted hatching and were more likely to be the result of ART procedures with ≥10 oocytes retrieved, only one or two embryos transferred, and extra embryos cryopreserved for future use. We were unable to determine whether the difference in having embryos cryopreserved was because cases were more likely to have had extra embryos available for future use or whether some other patient or treatment factor was different between the case and control groups. Cases of MZ pregnancies were not significantly different from either control group for prior ART, prior live birth, tubal infertility or use of ICSI.


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Table III. Percentage distribution of patient characteristics and treatment factors in case and control groups

 
Using day 3 as the reference category, we found that case pregnancies were ~4 times more likely to have had a day 5 embryo transfer and 2–3 times more likely to have had a day 6 embryo transfer than pregnancies in either control group (Table IV). Although case pregnancies were also less likely to have had a day 2 embryo transfer, this finding was not statistically significant for either control group.


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Table IV. Odds ratios and 95% confidence intervals for presumed MZ multiple gestation, by day of embryo transfer

 
After adjusting for potential confounders and restricting our analyses to various treatment subgroups, we found that statistically significant associations between day 5 embryo transfer procedures and MZ multiple gestations persisted (Table V). In fact, this association increased for case pregnancies compared with both control groups when we restricted our analyses to patients who had undergone IVF only, had extra embryos available and cryopreserved for future use, and had a diagnosis of tubal infertility.


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Table V. Odds ratios and 95% confidence intervals for the association between MZ multiple gestation and day 5 embryo transfer before and after adjustment for patient factors and restrictions to various ART treatment subsets

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Our finding that MZ twinning was significantly associated with extended embryo culture is consistent with several previous studies. Rijnders et al. (1998)Go reported 2.7% MZ multiple gestations among day 5 embryo transfers compared with 0.52 and 0.68% for day 2 and day 3, respectively. Milki et al. (2003)Go reported a 5.6% incidence of MZ twinning after blastocyst transfer compared with 2% with day 3 embryo transfer; their findings were not explained by use of ICSI or assisted hatching techniques. Da Costa et al. (2001)Go reported five MZ twin gestations among 129 (3.9%) ICSI pregnancies with blastocyst transfer compared with 0.7% among ICSI pregnancies with 4- to 8-cell embryo transfers.

Methodological limitations must be considered when interpreting our results. We did not have specific information on zygosity. We relied on early ultrasound data to classify cases of MZ pregnancies based on the number of fetuses relative to embryos transferred and, thus, may have missed some cases. However, we do not believe this limitation biased our comparisons. Our study compared presumed MZ case pregnancies with pregnancies in two different control groups: other multiple gestations and singleton gestations. Potential biases related to case ascertainment were expected to be in opposite directions for these control groups. Nonetheless, our results were quite comparable across the two control groups. Because we did not have specific zygosity data, the percentages of presumed MZ twinning reported in this study should not be used to assume absolute risk for MZ twining; the strength of this study is in the assessment of relative differences. Although some past investigators have assessed the absolute risk for MZ twinning based on data limited to single embryo transfer (SET), in the USA, <2% of the pregnancies reported were conceived with SET. In addition to the limitation this poses with respect to statistical power, pregnancies from SET in the USA are likely to be a very select subgroup and thus are not representative of the entire ART population.

We also lacked specific data on embryo stage at transfer and culture media. In our study, day 5 embryo transfer procedures were presumed to have been blastocyst. We cannot completely discount the possibility that some embryo transfer dates were reported incorrectly; however, misclassification was likely to have been random, and this would have biased our results toward the null hypothesis. Nonetheless, the lack of specific laboratory data on the embryo and the culture media precluded us from evaluating specific media components that may have been related to MZ twinning.

Our study was based on pregnancies rather than women, so it is possible that the women in our sample with more than one pregnancy during the 2 year period of interest were represented more than once. However, when we limited our sample to a subset of women undergoing their first ART cycle, our results remained the same.

Finally, our study subjects were not randomized. Treatment options, such as the day of embryo transfer, were based on the patient's choice and the provider's assessment. Because blastocyst transfer may be differentially performed depending on the presumed prognosis for the treatment cycle, we performed various subset analyses to determine if our results persisted within more homogeneous treatment groups; we found similar results for all. We also considered that the association documented in this study may have been attributable to high MZ twinning in only a few ART centres. We thus examined the distribution of MZ cases across clinics. MZ cases were identified from 114 ART centres. There was no difference between MZ cases identified from day 5 and day 3 embryo transfer procedures (110 cases from day 5 embryo transfers were from 66 clinics and 98 cases from day 3 transfers were from 58 clinics). Moreover, no clinic contributed an excessive number of cases.

Despite limitations, our study had many strengths. We used a population-based data set containing a large patient population, which allowed for a more thorough analysis of various patient and treatment factors than in previous studies from clinical populations. We reported 226 cases of presumed MZ twinning. The number of MZ twins reported from previous clinical studies ranged from three to eleven.

The underlying mechanism for the association between blastocyst culture and MZ twinning is unclear. One hypothesis raised is that in vitro culture conditions are related to increased hardening of the zona pellucida (Alikani et al., 1994Go); this could become even more problematic with extended time in culture. It is possible that spontaneous or assisted hatching of the blastocyst through a less flexible zona may increase the risk for its division. Alternatively, Cassuto and colleagues suggested that embryo culture conditions (i.e. culture media) rather than time in culture increase the risk of MZ twinning (Cassuto et al., 2003Go). Other ART treatment factors that have been potentially related to MZ twinning are ovulation induction (Derom et al., 1987Go) and zona manipulation such as those that occur during assisted hatching techniques (Hershlag et al., 1999Go; Schieve et al., 2000Go). These factors, however, would not explain the increased risk with extended culture. In our study population, nearly all case and control pregnancies included ovulation induction. Additionally, our results persisted when we excluded pregnancies from embryos that had been exposed to either assisted hatching or ICSI. Clearly, further work is needed to disentangle the mechanism by which extending culture to blastocyst creates a risk for MZ twinning.

Although our study found the highest pregnancy success rates (45%) among women who had undergone embryo transfer on day 5, it is important to note that we found a large percentage of multiple gestations (41%) among these procedures, despite the fact that they involved the transfer of fewer embryos on average (2.5 embryos). Several other studies have reported similar findings. Gorrill et al. (2001)Go documented a high multiple-gestation rate (40%) when only two blastocysts were transferred. Levron et al. (2002)Go reported a multiple-gestation pregnancy rate of 50% for day 5 embryo transfers with an average of 2.3 embryos transferred. Finally, in a randomized study, Coskun et al. (2000)Go reported a multiple-gestation rate of 38% multiples when only 2.2 embryos were transferred on day 5. Thus, our findings suggest that extended embryo culture may be associated with increased DZ multiple gestations in addition to MZ multiple gestations. Although there is great potential to reduce dizygotic multiples by limiting the number of embryos transferred, even two embryos may pose a substantial risk.

Taken altogether, the comparative studies to date and the numerous case reports of MZ twinning in blastocyst transfer pregnancies lend support to the hypothesis of an association between blastocyst transfer and MZ twinning (Rijnders et al., 1998Go; Peramo et al., 1999Go; Behr et al., 2000Go; Van Langendonckt et al., 2000Go; da Costa et al., 2001Go; Sheiner et al., 2001aGo,bGo; Milki et al., 2003Go). While this and other studies also suggest that DZ multiple gestation remains a substantial problem with blastocyst culture, it is important not to minimize the risk for MZ twinning. Prevention of adverse health outcomes among ART pregnancies should not be a sequential approach. While extending embryo culture beyond day 3 holds promise of improved success with fewer embryos transferred, more research is needed to better understand the mechanisms underlying the MZ twin effect.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The data used for this study were collected by SART. The SART system is jointly supported by the CDC, Atlanta, Georgia; SART, Birmingham, Alabama; and ASRM, Birmingham, Alabama. The authors thank SART and ASRM, without whose contributions this work would not have been possible.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on February 24, 2004; accepted on May 5, 2004.