Blastocyst versus cleavage stage transfer in in vitro fertilization: differences in neonatal outcome?

lle n, M.D., Ph.D.,a Orvar Finnstro m, M.D., Ph.D.,b Anna Lindam, M.Sc.,c Emma Nilsson, Ph.D.,c Bengt Ka d sta Nygren, M.D., Ph.D., and Petra Otterblad Olausson, Ph.D.c Karl-Go
a ping c Centre for Tornblad Institute, University of Lund, Lund b Department of Paediatrics, University Hospital, Linko Epidemiology, National Board of Health and Welfare, Stockholm and d IVF and Fertility Clinic, Sophiahemmet, Stockholm, Sweden

Objective: To compare neonatal outcome of blastocyst and cleavage stage embryo transfers after IVF. Design: Register study. Setting: Births recorded in the Swedish Medical Birth Register after IVF performed, 20022006. Patient(s): Treatments reported from all Swedish IVF clinics. Intervention(s): None. Main Outcome Measure(s): Some neonatal characteristics were compared in 1,311 infants born after blastocyststage transfer and 12,562 infants born after cleavage-stage transfer. Comparisons were also made with all births, 20022007 (n 598,687). Result(s): After adjusting for year of birth, maternal age, parity, smoking habits, and body mass index, the risk of preterm birth among singletons was signicantly greater after blastocyst-stage transfer than after cleavage-stage transfer. The risk of congenital malformations was also signicantly higher. When the analysis was restricted to clinics where blastocyst transfers were made, the risk estimates increased for preterm birth, low birth weight, low APGAR score, and respiratory diagnoses, but did not change for congenital malformations. Conclusion(s): The results indicate a small increase in risk associated with blastocyst transfer, perhaps owing to the longer period of in vitro culture. There is a possibility that this effect is due, at least in part, to a selection of women for blastocyst transfers. Further studies are needed either to verify or to refute the found associations. (Fertil Steril 2010;94:16803. 2010 by American Society for Reproductive Medicine.) Key Words: Blastocyst, preterm birth, stillbirth, congenital malformations

During the past decade, extension of embryo culture to 56 days has become part of the routine IVF procedures in some clinics. The embryo then develops into a blastocyst. A number of studies, summarized in a Cochrane review (1), demonstrated a higher pregnancy rate and live birth rate after blastocyst transfer than after cleavagestage transfer. When an equal number of embryos was transferred, the summarizing odds ratio (OR) for a live birth was 1.35 (95% condence interval [CI], 1.051.74). Another metaanalysis found a similar OR of 1.39 (95% CI, 1.101.76) (2). A recent study (3) veried the higher pregnancy rate after fresh blastocyst transfer than after cleavage-stage transfer, but for frozen embryos the opposite was seen; therefore, the total pregnancy rate was similar when both fresh and frozen embryo transfers were included. Few studies have investigated the neonatal outcomes after blastocyst transfer. An increased rate of monozygotic twinning and an increased sex ratio have been found (4, 5). One study described a normal malformation rate for 200 infants (n 5) born after blastocyst transfer (6). In this study, we have analyzed neonatal outcomes including a number of neonatal diagnoses and the presence of congenital malformations in neoReceived October 13, 2009; revised and accepted December 7, 2009; published online February 4, 2010. Supported by a grant from Evy and Gunnar Sandberg Foundation, Lund, Sweden, to B.K. B.K. has nothing to disclose. O.F. has nothing to disclose. A.L. has nothing to disclose. E.N. has nothing to disclose. K.-G.N. has nothing to disclose. O.P.O. has nothing to disclose. lle n, M.D., Ph.D., Tornblad Institute, BiskopsReprint requests: Bengt Ka gatan 7, SE-223 62 Lund, Sweden (TEL: 46-46-222-7536; FAX: 46-46-222-4223; E-mail: Bengt.Kallen@med.lu.se).

nates born after blastocyst transfer, and we compared them with neonates after cleavage-stage embryo transfer.

MATERIALS AND METHODS

Data were collected from all IVF clinics in Sweden regarding IVF treatments with an embryo transfer during the period 20022006. Information was requested on the identication number of the woman, the date of embryo transfer, IVF method, and embryo age at transfer. All reported IVF infants were included in the study. To study delivery outcomes, we used information from the Swedish Medical Birth Register (7), which supplied information on parity, smoking habits in early pregnancy, prepregnancy weight and height (which made it possible to calculate body mass index [BMI]), years of unwanted childlessness, birth weight, and gestational duration. Small-for-gestational age (SGA) was calculated using normal graphs for singletons, based on data in the Medical Birth Register (8). Information on congenital malformations was obtained from three sources: the Medical Birth Register (International Classication of Diseases [ICD] codes given at the pediatric examination of the newborn), the Register of Birth Defects (former Register of Congenital Malformations, primarily based on reports from neonatologists), and the National Patient Register (formerly the Hospital Discharge Register) (9). Respiratory diagnoses were dened as ICD-10 codes P22P28. Comparisons were made between infants born after blastocyst transfer and those born after cleavage-stage transfer. Comparisons were also made between infants born after blastocyst transfer or after cleavage-stage transfer and all infants born during 20022007 and registered in the Medical Birth Register (n 598,687). All diagnoses with an ICD-10 code beginning with Q (i.e., Chapter 17, congenital malformations, deformations, and chromosomal anomalies) were primarily included to dene malformations. To reduce variability, some common and relatively frequent minor anomalies with little clinical

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Fertility and Sterility Vol. 94, No. 5, October 2010 Copyright 2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

0015-0282/$36.00 doi:10.1016/j.fertnstert.2009.12.027

importance were excluded: preauricular appendix, tongue tie, patent ductus arteriosus in preterm infants, undescended testicle, hip luxation, hip subluxation, clicking hip, single umbilical artery, and nevus. The remaining malformations were called relatively severe malformations, although some mild conditions could be included. An analysis of cardiovascular anomalies (except patent ductus in preterm infants and single umbilical artery) was also made. Identication of vanishing twins was made from the observation of two emryonic sacs in early pregnancy followed by a singleton birth. ORs were estimated using the Mantel-Haenszel procedure and the 95% CIs were estimated with Miettinens method. Adjustments were made for year of birth, maternal age, parity, smoking habits, and prepregnancy BMI. When the expected number was less than 10, risk ratios as observed over expected numbers were calculated instead, and the 95% CIs were based on exact Poisson distributions. This study was performed within the responsibilities of the National Board of Health and Welfare; therefore, no ethical approval from outside ethical committees was needed.

RESULTS
This study is based on 1,311 infants born to 1,190 women after blastocyst transfer and 12,562 infants born to 11,548 women after cleavage-stage transfer. Births occurred during 20022007. There were 115 twin pairs (228 recorded infants, two missing twins) and four triplet sets (12 recorded infants) after blastocyst transfer and 1,022 twin pairs (2,022 recorded infants, 22 missing twins) and seven triplet sets (21 recorded infants) after cleavage-stage transfer. The rate of multiple births was thus 10% and 8.9%, respectively, and the two rates do not differ signicantly (c2 1.56; P0.21). Infant sex was known for both twins in 113 pairs after blastocyst transfer and in 1,000 pairs after cleavage-stage transfer (c2 0.01; P0.95). The percentage of like-sexed twins was similar: 52 and 53%, respectively. Among singletons born after blastocyst transfer,

there were signs of vanishing twins in 1% after cleavage stage transfer in 1.8 % (c2 3.2; P0.07). There were slightly fewer intracytoplasmic sperm injection procedures associated with blastocyst transfer (43%) than with cleavage-stage transfer (46%), but this difference may be random (c2 3.4; P0.07). Table 1 describes some maternal characteristics in the two groups. They differ in regard to maternal age, parity, and BMI, whereas no certain difference is seen between the groups in regard to smoking and years of unwanted childlessness when stated. Table 2 shows data on preterm birth, low birth weight, and SGA for singleton infants. Infants born after blastocyst transfer are more often born preterm than are infants born after cleavage-stage transfer. Both groups show a higher risk of preterm birth than does the population (OR, 1.66; 95% CI, 1.352.05; and OR, 1.24; 95% CI, 1.151.34, respectively). No statistically signicant differences exist in regard to low birth weight or SGA, but for low birth weight the risk estimate is higher after blastocyst than after cleavage-stage transfer. Table 2 also shows neonatal data. These refer to all infants born and thus include multiples. Although all ORs are higher after blastocyst than after cleavage-stage transfer, none of the differences reaches statistical signicance. The four variables tabulated are also strongly related. Data on congenital malformations are also presented in Table 2. The ORs for any congenital malformation vs. population gures was higher after blastocyst transfer (OR, 1.53; 95% CI, 1.231.90) than after cleavage-stage transfer (OR, 1.11; 95% CI, 1.021.21), and a direct comparison between the two types of embryo transfer showed statistically signicant higher risk for the former group. When the analysis was restricted to relatively severe

TABLE 1
Comparison of some maternal characteristics after blastocyst and cleavage stage transfer: embryo transfers 20022006. Blastocyst transfer Characteristic Maternal age, y <30 3034 R35 Parity 1 2 R3 Smoking in early pregnancy Unknown No Yes Body mass index Unknown <19.8 19.825.9 R26 Years of unwanted childlessness Not stated 12 3 R4
a

Cleavage-stage transfer No. % c2 P value

No.

%a

221 478 491 760 334 96 91 1054 45 132 37 676 345 338 251 202 399

18.6 40.2 41.3 63.9 28.1 8.1 95.9 4.1 3.5 63.9 32.6 29.5 23.7 46.8

1682 4777 5089 8067 2915 566 1248 9937 363 1780 650 6209 2829 3254 2589 2144 3561

14.6 41.4 44.1 69.9 25.2 4.9 95.5 3.5 6.7 64.1 29.2 31.2 25.9 42.9

13.9

<0.001

29.8

<0.001

0.94

0.33

19.1

<0.001

4.86

0.09

Percentages counted on the total number of women with information on the variable in question

lle n. Blastocyst transfer after IVF. Fertil Steril 2010. Ka

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TABLE 2
Comparison of neonatal characteristics of infants born after blastocyst or cleavage-stage transfer: embryo transfers 20022006. Blastocyst transfer Neonatal characteristic <32 wa <37 wa <1500 g birth weight a <2500 g birth weight a SGAa Low APGAR score Intracranial hemorrhage Respiratory diagnoses CPAP treatment Any congenital malformation Relatively severe malformationb Cardiovascular malformation No. with outcome 18 97 16 55 25 25 6 94 23 90 61 20 Total no. 1071 1071 1071 1071 1071 1303 1311 1311 1311 1311 1311 1311 Cleavage-stage transfer No. with outcome 142 757 126 532 338 240 36 774 203 645 509 177 Total no. 10513 10513 10493 10493 10488 12469 12562 12562 12562 12562 12562 12562 OR 1.44 1.35 1.41 1.13 0.82 1.06 1.46 1.15 1.15 1.43 1.33 1.18 95% CI 0.872.40 1.071.71 0.832.41 0.841.54 0.541.26 0.711.60 0.643.34 0.901.47 0.711.85 1.141.81 1.011.75 0.941.90

Note: Adjustment was made for year of birth, maternal age, parity, smoking, and BMI. For pregnancy duration, birth weight, SGA, and neonatal diagnoses adjustment was also made for IVF clinic. ORs refer to blastocyst stage vs. cleavage-stage transfers. CPAP continuous positive airway pressure. a Singleton infants only. Information on gestational duration or birth weight is missing in a few cases. b The following common and variably registered conditions were excluded (see Material and Methods). lle n. Blastocyst transfer after IVF. Fertil Steril 2010. Ka

malformations, the ORs changed only marginally, and the difference between the two groups remained signicant. When only cardiovascular defects were studied, no signicant difference between the groups was seen. Among the 61 relatively severe malformations, there were four infants with CNS malformations (three of them with spina bida), 18 with cardiovascular defects, three with cleft palate, ve with ureter malformations, four with hypospadias, four with pes equinovarus, two with hand malformations, and three with Down

syndrome. Other malformations occurred in only one case each among them was one infant with Goldenhar syndrome and one with sirenomelia. Compared with the risk of spina bida after cleavage stage transfer (11 cases), the risk after blastocyst transfer is nonsignicantly increased (P0.10). IVF followed by blastocyst transfer was reported from only half of the IVF clinics, and a large majority (n 986) came from one clinic. Analyses were therefore made adjusting for IVF clinic (the three largest ones with 986, 87, and 74 cases, respectively, and all

TABLE 3
Repeated analyses of neonatal outcome, restricted to the IVF clinic that used blastocyst transfers. Cleavage-stage transfer Neonatal characteristic <32 sk gestation <37 wk gestationa <1500 g birth weighta <2500 g birth weighta SGAa Low APGAR score Intracranial hemorrhage Respiratory diagnoses CPAP Any congenital malformation Relatively severe malformation Cardiac defect
a

No. of infants with outcome 72 369 66 259 159 110 23 365 111 320 227 86

Total 5,221 5,221 5,206 5,206 5,206 6,135 6,178 6,178 6,178 6,178 6,178 6,178

OR 2.22 2.31 2.20 2.38 1.16 2.95 1.65 1.52 1.37 1.47 1.39 1.19

95% CI 0.657.61 1.423.74 0.411.90 1.344.18 0.542.52 1.436.08 0.387.37 1.042.23 0.692.72 1.151.88 1.041.87 0.721.97

Note: For data on blastocyst transfers, see Tables 2 and 3. Adjustment for year of birth, maternal age, parity, and BMI. For pregnancy duration, birth weight, SGA, and neonatal diagnoses adjustment was also made for IVF clinic. ORs refer to blastocyst stage vs. cleavage-stage transfers. CPAP continuous positive airway pressure. a The analyses of gestational duration, birth weight, and SGA are based on singletons with information on respective variable. lle n. Blastocyst transfer after IVF. Fertil Steril 2010. Ka

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other clinics with at least one blastocyst transfer together). The sample of cleavage-stage transfer was then reduced to approximately half (6,178 infants among a total of 12,562). Table 3 presents the results. All estimates that did not refer to congenital malformations increased and some (<2,500 g, low American Pediatric Gross Assessment Record [APGAR] score, respiratory diagnoses) became statistically signicant. The estimates for congenital malformations remained basically unchanged.

DISCUSSION
In our sample, women who had IVF with a blastocyst transfer differed in some aspects from women who had IVF with a cleavage-stage transfer. They were younger, more often of high parity, and showed an excess of high BMI. In the analysis, we adjusted for these variables. There was no difference in smoking habits, but both groups of women smoked less than other women who gave birth; therefore, adjustment was made also for smoking in the comparisons with the population. Previous studies have shown blastocyst transfer to be more effective than transfer of cleavage stage embryos. However, some drawbacks have been described concerning blastocyst transfer (e.g., problems at cryopreservation and an increased risk for monozygotic twinning) (1, 4, 5). Little interest has been paid to other aspects of neonatal outcomes such as neonatal complications or congenital malformations. In one study (10), a higher rate of preterm births was found after blastocyst transfer than after cleavage-stage transfer, but this was due to a higher twin rate. In this study, data are presented that might indicate an increased risk for neonatal problems after blastocyst transfer; they are not explainable by differences in multiple births or in the occurrence of vanishing twins. These differences in risk are not very strong, and the number of infants born after blastocyst transfer in the study is only 1,311, which is a relatively small number to evaluate rare outcomes. The most clearly indicated risk increase compared with cleavage-stage transfer refers to preterm birth and the presence of congenital malformations. For some other variables, increased risks are indicated but not statistically signicant. Larger data sets than the present one are needed to explore whether these effects are true or random. Blastocyst transfers were reported only from some IVF clinics. To adjust for IVF clinic and its catchment area, the analyses of outcomes were repeated after adjusting for IVF clinic. The idea behind

this analysis is that if there is an unidentied difference between women (putative high-risk women, perhaps owing to underlying cause of infertility) who get blastocyst transfers and women (putative low-risk women) who get cleavage-stage transfers, one would expect the difference between the two methods to increase when the analysis is restricted to clinics where both methods were used. The repeated analyses did increase the recorded differences between blastocyst and cleavage-stage transfer groups in regard to preterm birth, low birth weight, low APGAR score, and respiratory diagnoses, but not in regard to congenital malformations. The differences between outcomes after blastocyst and cleavagestage transfers could therefore be due to a selection of women undergoing blastocyst transfer, except for the characteristics for which we adjusted. If these women were selected for characteristics that increased the risk for preterm birth, this would explain higher risk estimates when comparisons were restricted to clinics where blastocyst transfers were done in contrast to comparisons with all infants after cleavage-stage transfer. The subgroup of women with blastocyst transferrelated characteristics will exist also among the patients of IVF clinics that did not perform blastocyst transfers, resulting in a higher risk for preterm birth. The difference between the blastocyst and the cleavage-stage transfer groups would be less marked than compared with the IVF clinics that performed blastocyst transfers. No major change in the risk estimates for congenital malformations was seen when infants born after blastocyst transfer were compared with infants born after cleavage-stage transfer, after restricting to clinics where blastocyst transfers were made. In this analysis, no adjustment for IVF clinic was made, but comparisons were restricted to cleavage-stage transfers made in clinics where blastocyst transfers were also made. The possible selection mechanism discussed previously does not seem to operate on congenital malformations. The differences between neonatal outcome after blastocyst and cleavage-stage transfer are not large, but they may suggest a problem that needs further exploration. It is possible that the extended period in culture increases the risk for monozygotic twinning (1, 4, 5) and for other developmental disturbances, including malformations. Our sample size is too small to study the question of imprinting errors after blastocyst transfer, but no such case was identied.
Acknowledgments: We express our thanks for the assistance of all IVF clinics in Sweden in collecting data.

REFERENCES
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