Neonates born after in vitro fertilization (IVF) are at higher risk of perinatal morbidity.1–10 The majority of adverse outcomes such as prematurity and low birth weight (LBW) are the result of multiple pregnancies.9,11,12 However, increased perinatal morbidity has also been demonstrated in singleton neonates. Investigations regarding the potentially modifiable, IVF-specific treatment modalities that may confer this risk are needed and are ongoing.
Embryo culture is a vital component of the IVF process. The optimal conditions for embryo culture and the optimal time of embryo replacement are a continued area of debate.13–17 In the last two decades, most centers adopted the practice of transferring cleavage-stage embryos 3 days after oocyte retrieval. In recent years, blastocyst-stage transfer after 5 or 6 days of extended embryo culture has been advocated as a result of higher implantation and pregnancy rates.14,18–22 This practice has been widely adopted either as the primary protocol for embryo transfer or limited to good prognosis patients to select a single embryo for transfer and thus minimize the risk of a multiple gestation. However, the effect of extended embryo culture on neonatal adverse outcomes has not been well studied.
The goal of this study was to isolate one aspect of IVF and estimate its association with perinatal morbidity. Specifically, this study assessed whether extended embryo culture is associated with an increased risk of preterm delivery, very preterm delivery, LBW, or a combination of these. The U.S. 2004–2006 Society for Assisted Reproductive Technologies database was used as the source of data. We assessed over 69,000 neonates born after non-donor IVF cycles, comparing the outcomes of neonates born after blastocyst-stage embryo transfer with neonates born after cleavage-stage embryo transfer. Given the size of the database, we were able to stratify by singleton and twin birth. In addition, we performed subanalyses in primiparous patients and donor oocyte recipients with singleton live birth after cleavage-stage compared with blastocyst transfer in an effort to explore the effect of patient selection.
MATERIALS AND METHODS
This was a retrospective cohort study using the Society for Assisted Reproductive Technologies database to identify liveborn neonates conceived with IVF between 2004 and 2006. Exposure was defined as live birth resulting from transfer of a fresh embryo after extended embryo culture (embryo transfer on day 5 or 6 after oocyte retrieval). The comparison group was a live birth resulting from transfer of a fresh embryo at the cleavage stage (embryo transfer on day 3 after oocyte retrieval). Outcomes of this study included: preterm delivery (liveborn neonate between 32 and 37 weeks of gestation), very preterm delivery (liveborn neonate of gestational age of less than 32 weeks), and LBW (liveborn neonate weighing less than 2,500 g). This study was approved by the institutional review board of the University of Pennsylvania.
All data management and analyses were performed using STATA MP 12. Patient characteristics were compared using generalized estimating equations, with appropriate distributional assumptions for the characteristic being compared. Associations between day of embryo transfer and preterm delivery or LBW were assessed using univariable and multivariable analysis, both of which were conducted using generalized estimating equations to adjust for the possible contribution of multiple cycles from the same patient. Analyses were stratified by singleton and twin live birth. In addition to the primary exposure of time in embryo culture, other factors that have been associated with possible adverse outcome were also investigated. These possible confounders included patient age, prior parity, infertility diagnosis, number of embryos transferred, number of prior assisted reproductive technology cycles, history of prior miscarriage, reduction in fetal heart on ultrasonography (vanishing twin), and implantation rate.
Subanalyses were performed to assess the effect of patient selection on the study findings. The first subanalysis was restricted to singleton live birth after blastocyst compared with cleavage-stage transfer in women who had never been pregnant before. Comparisons were performed using logistic regression analysis. The goal of restricting to primiparous women was to control for the potential of selection bias resulting from the possibility of increased uptake of elective single blastocyst embryo transfer in women with a history of previous adverse perinatal outcome. In addition, singleton birth outcomes resulting from cleavage compared with blastocyst-stage transfer in oocyte donor recipients were assessed through generalized estimating equations. Finally, sensitivity analyses were performed in two small subgroups. The first included paired analysis of the same woman who underwent two separate IVF cycles at different times resulting in a singleton live birth with one birth resulting from a cleavage stage and the other from a blastocyst transfer. This analysis used conditional logistic regression to compare the results of the cycles from the same woman. The second analysis assessed the association in singleton births in “best prognosis” patients, defined as patients younger than age 35 years undergoing their first IVF cycle without an underlying infertility diagnosis of diminished ovarian reserve, endometriosis, or both.
There were a total of 368,833 cycles in the Society for Assisted Reproductive Technologies database. The primary analysis assessed liveborn neonates resulting from non egg donor cycles. Cycles that failed to result in a live birth (n=240,533) and cycles with unknown (n=560) or missing (n=2,623) pregnancy outcomes were excluded. In the primary analysis, oocyte donation cycles (n=18,032) were excluded. Cycles were included only if transfer of embryos occurred on day 3 or on day 5 or 6, and an additional 7,402 cycles were excluded as a result of transfer of embryos before day 3, on day 4, or after day 5 to 6. Finally, cycles with frozen embryo transfer (n=30,644) were also excluded. A total of 69,039 liveborn neonates were identified for analysis; 46,288 followed transfer of day 3 embryos and 22,751 followed transfer of blastocyst-stage embryos. Table 1 provides a comparison of demographic and treatment characteristics between the two groups. Maternal age was, on average, 1 year younger in mothers of neonates born after blastocyst transfer. Despite the fact that fewer blastocysts were transferred, twin pregnancy was significantly more likely after blastocyst embryo transfer as a result of a significantly higher implantation rate.
Associations between extended culture and preterm delivery and LBW are presented in Table 2. Analyses were stratified by singleton (n=47,133) and twin (n=20,418) gestation. Unadjusted and adjusted analyses are presented. The variables included in final adjusted analyses included variables that were considered clinically important, had been previously reported to be associated with adverse outcome, or both, including reporting year, patient age, parity, infertility diagnosis, number of embryos transferred, number of prior assisted reproductive technology cycles, history of prior miscarriage, “vanishing” fetus on ultrasonography, and implantation rate. In the analysis of singleton births, preterm delivery was significantly more likely with extended embryo culture (18.6% compared with 14.4%, respectively; odds ratio [OR] 1.37, 95% confidence interval [CI] 1.30–1.44, P<.001; adjusted OR 1.39, 95% CI 1.29–1.50; P<.001); very preterm delivery was also significantly more likely (2.8% compared with 2.2%, respectively; OR 1.36, 95% CI 1.20–1.54; adjusted OR 1.35, 95% CI 1.13–1.61; P<.001). There was an association with extended culture and LBW in the crude analysis (10.3% compared with 9.1%, respectively; OR 1.13, 95% CI 1.06–1.20, P<.001), but this did not meet statistical significance in the adjusted analysis (adjusted OR 1.10, 95% CI 1.00–1.21, P=.06). Given the finding that there was a significant increase in preterm delivery that did not correspond, as expected, to a similar increase in LBW, we performed an analysis to estimate the likelihood of small for gestational age in neonates born after extended embryo culture compared with cleavage-stage transfer and found that small for gestational age was less likely in neonates born after extended culture (5.6% compared with 6.9%, respectively; OR 0.80, 95% CI 0.74–0.87, P<.001).
We performed a similar analysis in a population restricted to twin gestations. As expected, the absolute risk of adverse outcome was higher in twin gestations compared with singletons; preterm delivery was fourfold higher (relative risk [RR] 4.70, 95% CI 4.51–4.90, P<.001), very preterm delivery 13-fold higher (RR 13.10, 95% CI 11.60–14.70, P<.001), and LBW was sevenfold higher (RR 7.19, 95% CI 6.82–7.57, P<.001) in twins compared with singletons.
Twins conceived after extended culture as compared with cleavage-stage transfer were at a further increased risk of preterm delivery (67.3% compared with 60.5%, respectively; OR 1.63, 95% CI 1.52–1.75, P<.001; adjusted OR 1.81. 96% CI 1.63–2.00, P<.001) and very preterm delivery (14.0% compared with 12.0%, respectively; OR 1.70, 95% CI 1.53–1.88, P<.001; adjusted OR 1.75, 95% CI 1.50–2.04). Twins born after extended embryo culture also demonstrated a slight increase in LBW (71.1% compared with 68.6%, respectively; OR 1.13, 95% CI 1.05–1.20, P<.001; adjusted OR 1.19 95% CI 1.09–1.31).
Subanalyses to attempt to control for selection bias were performed (Table 3). The first subanalysis was restricted to singleton neonates born to primiparous women. The risk of preterm delivery was higher in neonates born after extended culture, 19.9% compared with 15.9%, respectively (OR 1.42, 95% CI 1.35–1.49, P<.001; adjusted OR 1.30, 95% CI 1.20–1.41, P<.001). There was an association of extended embryo culture with LBW in the crude analysis, 10.6% compared with 9.7% (OR 1.23, 95% CI 1.17–1.30, P=.02) but this failed to meet statistical significance in the adjusted analysis (adjusted OR 1.10, 95% CI 0.99–1.22, P=.08). The second subanalysis was restricted to singleton neonates born to donor recipients (Table 3). In this subpopulation, extended embryo culture was also associated with a higher risk of preterm delivery, 25.4% compared with 21.8%, respectively (OR 1.24, 95% CI 1.11–1.38, P<.001; adjusted OR 1.33, 95% CI 1.15–1.54, P=.001). No association was found between extended embryo culture and LBW (11.7% compared with 12.4%, OR 0.96, 95% CI 0.83–1.11, P=.60; adjusted OR 0.98, 95% CI 0.78–1.24, P=.87).
Finally, sensitivity analyses exploring the association in subgroups were performed to assess whether findings remained robust. In a paired analysis in the same woman (n=135) who conceived a singleton birth after both cleavage-stage and extended embryo culture, the direction and strength of the association went in the same direction but did not meet statistical significance (5.1% compared with 5.7%, OR 1.64, 95% CI 0.77–3.46, P=.20; adjusted OR 3.76, 95% CI 0.60–23.4, P=.16). A second analysis was limited to singletons born to “best prognosis patients” (n=13,895) and also demonstrated that those born after extended embryo culture compared with cleavage stage were at increased risk of preterm delivery (13.6% compared with 18.2%, respectively; OR 1.42, 95% CI 1.30–1.56, P<.001; adjusted OR 1.45, 95% CI 1.26–1.67, P<.001).
Neonates conceived with the assistance of IVF are at an increased risk of preterm delivery. In addition to multiple pregnancy, the factors contributing to this increased risk observed even in singletons are not clearly understood. The purpose of this study was to isolate one of the many complex factors of the IVF process, specifically extended embryo culture, and to independently assess its potential association with perinatal morbidity. Extended embryo culture has increased in frequency because it allows embryo development to the blastocyst stage, potentially enhancing embryo selection and pregnancy rates. Given that, any independent effect of extended culture on neonates conceived with the assistance of IVF would have important scientific and public health consequences.
This analysis consisted of approximately 47,000 liveborn singleton neonates born to women undergoing autologous IVF cycles registered in the 2004–2006 Society for Assisted Reproductive Technologies database and thus represents contemporary practice in the United States. The results suggest that extended embryo culture to the blastocyst stage increased the risk of preterm delivery by approximately 30–40% compared with culture of embryos up to the cleavage stage (day 3). This association remained consistent in singleton deliveries, twin deliveries, and in subanalyses performed to control for patient selection. The initial subanalysis of primiparous patients was performed to minimize bias that may have been introduced if patients with a history of prior adverse outcome were disproportionately chosen to have a blastocyst transfer. In addition, we evaluated outcomes of singleton neonates born to donor oocyte recipients to assess whether our findings were generalizable to this population of women who often undergo blastocyst transfer as well and to explore whether the association remained robust in the absence of ovarian stimulation and the resultant nonphysiological, high-estrogen peri-implantation endocrine milieu. The association of preterm delivery and extended culture also remained consistent when restricted to a cohort of “best prognosis” patients undergoing their first cycle of IVF. Finally, in a paired analysis of women who conceived a singleton neonates after both a cleavage stage and an extended embryo transfer, the direction of the association remained the same but was limited by a small sample size and thus low power. Of interest, LBW in singleton neonates was not associated with extended embryo culture, suggesting that the underlying etiology of LBW may be different than that of preterm delivery. It is noteworthy, however, that despite similar mean birth weights, neonates born after extended embryo culture were less likely to be small for gestational age.
The mechanism underlying the consistently demonstrated association between preterm delivery and extended embryo culture cannot be elicited with this study design. It is possible that extended culture could alter trophoblast function and placentation resulting in predisposition of an embryo to early initiation of labor and preterm delivery.23 It has been demonstrated that nonimprinted genes that are involved in placental growth and parturition are under epigenetic control and in vitro studies have demonstrated that activation of genes critical to prostaglandin biosynthesis can be modified by changes in DNA methylation status in human placental explants.24 Furthermore, inflammatory mediators such as interleukin-6 that are critical in the initiation of preterm and term labor are also under epigenetic control and could be altered by changes in regulatory mechanisms at the time of implantation.25,26 An alternative hypothesis is that the shift in time of implantation at the level of the endometrium or the intrauterine hormonal environment is the mechanism underlying the increase in preterm delivery.27–29 Further investigation is needed to elucidate the mechanism for the observed association.
Although this study strongly supports that extended embryo culture independently increases the risk of preterm delivery, the fact that the multiple birth remains the major contributor to adverse perinatal outcome in IVF neonates cannot be overlooked. High-order multiple birth, live birth of more than three neonates, was an infrequent outcome in both groups, comprising 2.4% of births after cleavage-stage transfer and 1.7% of births after blastocyst-stage transfer. It is well known that high-order multiple births carry the very highest risk of maternal, perinatal, and long-term morbidity. As such, an important advantage of blastocyst transfer in the current study was the 25% reduction in high-order multiple pregnancy. The paradox of this advantage, however, was that the advantage gained by reduction in high-order multiple pregnancy was offset by a 30% increase in twin births, suggesting that an additional mechanism whereby extended culture contributes to perinatal risk through an increase in twins. Consistent with previous reports, we demonstrated that twin births were associated with an approximately fourfold increase in preterm delivery and an approximately and sevenfold increase in LBW as compared with singleton births.
The major advantage of extended embryo culture is the ability to select the single best embryo for elective single embryo transfer and thereby reduce multiple pregnancy rates. The most recent data from the Society for Assisted Reproductive Technologies confirm, however, that health care practitioners still routinely transfer more than one blastocyst. Even in “best prognosis” patients—women younger than age 35 years with embryos available for cryopreservation—only 10% underwent elective single embryo transfer, demonstrating that the major advantage of extended culture has yet to be realized in the United States.30 Until the uptake of elective single embryo transfer increases, extended culture will not decrease morbidity and given the current findings may contribute further risk. It is important to note, however, that suggesting we shift to earlier transfer may be premature in certain populations. At present, it is common to transfer three cleavage-stage embryos in women older than age 35 years. Given that, cleavage-stage transfer may carry the inadvertent consequence of a further increase in morbidity through an increase in twin and high-order multiple pregnancy rates in those women. Before such a shift, reliable techniques to select fewer, viable cleavage-stage embryos need to be developed.
In summary, extended embryo culture, with transfer of blastocyst-stage embryos, is associated with an increased risk of preterm delivery in both singleton and twin gestations. Of importance is that blastocyst transfer is associated with increased perinatal morbidly in two ways, independently and by increasing the rate of twin gestation. To date, the potential of extended embryo culture to select and transfer the single best embryo to achieve high pregnancy rates and low multiple birth rates has not been realized.22,31,32 Our findings suggest that future research should focus on earlier selection and uptake of the most suitable single embryo for transfer and, in certain cases, transfer should be considered at the cleavage stage to avoid any additional independent detrimental effect of extended embryo culture.
1. Schieve LA, Meikle SF, Ferre C, Peterson HB, Jeng G, Wilcox LS. Low and very low birth weight in infants conceived with use of assisted reproductive technology. N Engl J Med 2002;346:731–7.
2. Klemetti R, Gissler M, Hemminki E. Comparison of perinatal health of children born from IVF in Finland in the early and late 1990s. Hum Reprod 2002;17:2192–8.
3. Hansen M, Kurinczuk JJ, Bower C, Webb S. The risk of major birth defects after intracytoplasmic sperm injection and in vitro fertilization. N Engl J Med 2002;346:725–30.
4. Ericson A, Kallen B. Congenital malformations in infants born after IVF: a population-based study. Hum Reprod 2001;16:504–9.
5. Jackson RA, Gibson KA, Wu YW, Croughan MS. Perinatal outcomes in singletons following in vitro fertilization: a meta-analysis. Obstet Gynecol 2004;103:551–63.
6. Schieve LA, Ferre C, Peterson HB, Macaluso M, Reynolds MA, Wright VC. Perinatal outcome among singleton infants conceived through assisted reproductive technology in the United States. Obstet Gynecol 2004;103:1144–53.
7. Shevell T, Malone FD, Vidaver J, Porter TF, Luthy DA, Comstock CH, et al.. Assisted reproductive technology and pregnancy outcome. Obstet Gynecol 2005;106:1039–45.
8. Chung K, Coutifaris C, Chalian R, Lin K, Ratcliffe SJ, Castelbaum AJ, et al.. Factors influencing adverse perinatal outcomes in pregnancies achieved through use of in vitro fertilization. Fertil Steril 2006;86:1634–41.
9. Kalra SK, Molinaro TA. The association of in vitro fertilization and perinatal morbidity. Semin Reprod Med 2008;26:423–35.
10. Wang YA, Sullivan EA, Black D, Dean J, Bryant J, Chapman M. Preterm birth and low birth weight after assisted reproductive technology-related pregnancy in Australia between 1996 and 2000. Fertil Steril 2005;83:1650–8.
11. Olivennes F, Fanchin R, Lédée N, Righini C, Kadoch IJ, Frydman R. Perinatal outcome and developmental studies on children born after IVF. Hum Reprod Update 2002;8:117–28.
12. Pinborg A, Loft A, Nyboe Andersen A. Neonatal outcome in a Danish national cohort of 8602 children born after in vitro fertilization or intracytoplasmic sperm injection: the role of twin pregnancy. Acta Obstet Gynecol Scand 2004;83:1071–8.
13. Oatway C, Gunby J, Daya S. Day three versus day two embryo transfer following in vitro fertilization or intracytoplasmic sperm injection. The Cochrane Database of Systematic Reviews 2004, Issue 2. Art. No.: CD004378. DOI: 10.1002/14651858.CD004378.pub2.
14. Blake D, Proctor M, Johnson N, Olive D. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. The Cochrane Database of Systematic Reviews 2005, Issue 4. Art. No.: CD002118. DOI: 10.1002/14651858.CD002118.pub3.
15. van Os HC, Alberda AT, Janssen-Caspers HA, Leerentveld RA, Scholtes MC, Zeilmaker GH. The influence of the interval between in vitro fertilization and embryo transfer and some other variables on treatment outcome. Fertil Steril 1989;51:360–2.
16. Goto Y, Kanzaki H, Nakayama T, Takabatake K, Himeno T, Mori T, et al.. Relationship between the day of embryo transfer and the outcome in human in vitro fertilization and embryo transfer. J Assist Reprod Genet 1994;11:401–4.
17. Dawson KJ, Conaghan J, Ostera GR, Winstom RM, Hardy K. Delaying transfer to the third day post-insemination, to select non-arrested embryos, increases development to the fetal heart stage. Hum Reprod 1995;10:177–82.
18. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434–40.
19. Papanikolaou EG, D'haeseleer E, Verheyen G, Van de Helde H, Camus M, Van Steirteghem A, et al.. Live birth rate is significantly higher after blastocyst transfer than after cleavage-stage embryo transfer when at least four embryos are available on day 3 of embryo culture. A randomized prospective study. Hum Reprod 2005;20:3198–203.
20. Papanikolaou EG, Camus M, Kolibianakis EM, Van Landuyt L, Van Steirteghem A, Devroey P. In vitro fertilization with single blastocyst-stage versus single cleavage-stage embryos. N Engl J Med 2006;354:1139–46.
21. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil Steril 1998;69:84–8.
22. Blake DA, Farquhar CM, Johnson N, Proctor M. Cleavage stage versus blastocyst stage embryo transfer in assisted conception. The Cochrane Database of Systematic Reviews 2007, Issue 4. Art. No.: CD002118. DOI: 10.1002/14651858.CD002118.pub3.
23. Cutfield WS, Hofman PL, Mitchell M, Morison IM. Could epigenetics play a role in the developmental origins of health and disease? Pediatr Res 2007;61:68–75R.
24. Mitchell MD. Unique suppression of prostaglandin H synthase-2 expression by inhibition of histone deacetylation, specifically in human amnion but not adjacent choriodecidua. Mol Biol Cell 2006;17:549–53.
25. Rahnama F, Shafiei F, Gluckman PD, Mitchell MD, Lobie PE. Epigenetic regulation of human trophoblastic cell migration and invasion. Endocrinology 2006;147:5275–83.
26. Sato TA, Mitchell MD. Contact paracrine inhibitory networks within human gestational tissues. Am J Obstet Gynecol 2006;195:1396–7.
27. Athanassiades A, Hamilton GS, Lala PK. Vascular endothelial growth factor stimulates proliferation but not migration or invasiveness in human extravillous trophoblast. Biol Reprod 1998;59:643–54.
28. Kalra SK, Ratcliffe SJ, Coutifaris C, Molinaro T, Barnhart KT. Ovarian stimulation and low birth weight in newborns conceived through in vitro fertilization. Obstet Gynecol 2011;118:863–71.
29. Norwitz ER, Schust DJ, Fisher SJ. Implantation and the survival of early pregnancy. N Engl J Med 2001;345:1400–8.
30. Centers for Disease Control and Prevention. Figure 58: percentages of fresh nondonor cycles that involved the transfer of one, two, three, or four or more embryos, 1999–2008. Available at: www.cdc.gov/art/ART2008/sect5_fig51–63.htm#f57
. Accessed May 8, 2012.
31. Gelbaya TA, Tsoumpou I, Nardo LG. The likelihood of live birth and multiple birth after single versus double embryo transfer at the cleavage stage: a systematic review and meta-analysis. Fertil Steril 2010;94:936–45.
32. Criniti A, Thyer A, Chow G, Lin P, Klein N, Soules M. Elective single blastocyst transfer reduces twin rates without compromising pregnancy rates. Fertil Steril 2005;84:1613–9.