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Effects of culture conditions on IVF outcome
European Journal of Obstetrics & Gynecology and Reproductive Biology 115S (2004) S72–S76
Effects of culture conditions on IVF outcome B. Behr*, H. Wang Department of Obstetrics and Gynecology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305, USA
Abstract Although in vitro fertilization (IVF) success rates have improved over the past decade, multiple pregnancies have become a formidable problem. The solution to this problem seems simple by mandating the reduction in numbers of embryos transferred. However, this is typically not accomplished without a compromise in the pregnancy rate. There have been a number of approaches designed to address high order multiple pregnancies from multi factorial analysis of early cleavage stage embryos to the development of extended culture systems, both of which require manipulations in the culture environment. Manipulations in embryo culture environment may not be benign. Several studies have demonstrated that adverse culture conditions have effects on gene expression and imprinting. Studies have also demonstrated that singleton human IVF babies have lower birth weight and higher incidence of congenital anomalies than natural conception babies. All of these factors need to be considered in relation to long term viability of IVF babies and the Barker hypothesis. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Embryo culture; Growth factors; Multiple pregnancy; Barker hypothesis; Blastocyst transfer; Imprinting
1. Introduction Despite numerous advances in the field of in vitro fertilization (IVF), many of the widely applied embryo culture techniques and resulting implantation rates have relatively unchanged since the first treatment was performed in the mid 1970’s [1]. For the past two decades the majority of embryos conceived through IVF have been transferred between days 1 and 3 at either the pronucleate or cleavage stage. The reason for this stems primarily from the inability of past culture system to support the development of viable blastocysts at acceptable rates. Recently, with the advent of newer culture systems it is now more consistent that day 5 or day 6 blastocysts be available for transfer. Impressive blastocyst formation and pregnancy results have been reported using in vitro blastocyst culture by a number of clinics and investigators [2–7]. The primary rational for blastocyst culture is to provide a mechanism for self-selection of viable embryos and improve the synchronicity of uterine and embryonic development. The commercialization of sequential culture media has led to the worldwide introduction of the technique despite much debate about its effect on IVF outcomes. Because culture in vitro can affect gene expression and embryo metabolism in model systems, concerns have arisen abut the effects of extended culture on these
* Corresponding author. Tel.: þ1-650-723-0951; fax: þ1-650-723-7737. E-mail address: [email protected] (B. Behr).
human embryos that may in part be responsible for the increasing risk of monozygotic twinning [8,9], or a propensity to skew the sex ratio after blastocyst transfer [10]. Therefore, clinics today are not only faced with what culture system and what media to choose from, but also face which stage and which embryo to select for transfer.
2. Preimplantation development and energy sources In the human, preimplantation development of the embryo from zygote to blastocyst occurs as it travels through the fallopian tube towards the uterus from day 1 to 5 after ovulation. As the embryo reaches the blastocyst stage on day 5, within 48 h of reaching the uterus, the blastocyst begins to hatch from the zona pellucida, and then implant. Many studies have been undertaken to understand the energy requirements of the preimplantation embryo. At the stage of the zygote, the embryo exhibits relatively low levels of biosynthesis prior to embryonic genome activation and expression. It is commonly accepted that pyruvate and lactate are the preferred energy sources for this early stage of development. Glucose consumption is limited by the early embryo. As development proceeds, and energy demands increase with cell multiplication and an increase in protein synthesis, there is a concomitant increase in glucose utilization. By the blastocyst stage, the embryo exhibits high oxygen utilization and an ability to readily utilize glucose, along with other energy sources [11].
0301-2115/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2004.01.016
B. Behr, H. Wang / European Journal of Obstetrics & Gynecology and Reproductive Biology 115S (2004) S72–S76
3. Culture system 3.1. Culture medium selection Over the past decade there has been considerable interest in optimizing culture media for supporting human embryos after IVF and before embryo transfer. Some of the first culture media used for human IVF, such as Ham’s F10 and Earle’s Medium were constructed to support the development of somatic cells and cell lines. Others, such as Tyrode’s T6 and Whitten’s WM1 had been used for IVF and embryo culture in laboratory animals. Human tubal fluid (HTF) medium was one of the first media specifically designed for human IVF, its formulation based on the imitative principle using the then known chemical composition of human tubal fluid [12]. IVF culture media can generally be categorized on whether they are a complex mixture of inorganic salts, energy sources, amino acids, vitamins and other substances (e.g. Ham’s F10, Menezo’s B2 and B3 media, Eagle’s MEM) or whether they are simple balanced salt solutions containing energy sources (e.g., Earle’s, Tyrode’s T6, WM1) [13]. However, prior to the mid ‘90s, low production and accompanying low viability of blastocysts during human IVF due to the inability of any single culture medium to meet the changing nutritional requirements of preimplantation embryos resulted in blastocyst transfer (BT) being abandoned early. Subsequently, applying a sequential culture approach resulted in acceptable blastocyst development and pregnancy rates to warrant its application in routine IVF [25,14]. The blastocyst culture provides greater opportunities for selection of more viable and, in certain cases, genetically normal embryos. Blastocyst transfer also provides more physiologic synchronization of the embryo stage with the endometrium. Therefore, the transfer of human blastocysts in IVF should result in an increased implantation rate and enable the transfer of fewer, but higher quality embryos, without any negative impact on pregnancy rates while, at the same time, decreasing high order multiples. With the wide commercial availability of sequential media, extend culture has become a practical tool within reach of all IVF programs. There is still the question as to which sequential media would be the best to culture cleavage or blastocyst stage embryos. There have been many studies that have compared various culture media but they provide conflicting data. Also, as with many studies in clinical IVF, the sample sizes in these studies are relatively small and more prospective randomized studies are therefore needed.
4. Growth factors and preimplantation development There is increasing evidence in a variety of mammalian species that growth factors play an important role in preimplantation development, regulation of cellular events and
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in maternal embryonic dialogue [15,16,47]. A wide range of growth factor ligands and their receptors are expressed in embryos during the preimplantation period [16]. In addition, supplementation of culture medium with growth factors has been shown to be beneficial for preimplantation development. For example, LIF, IGF-1, HBEGF and GM-CSF have all been shown to increase the proportion of embryo development to the blastocyst stage [17–22]. Furthermore, IGF-1 and GM-CSF increase blastocyst cell number specifically in inner cell mass [19,20]. Previous studies have demonstrated that GM-CSF may act as a regulator in murine embryo development. This may be attributed to decreasing cell apoptosis during the preimplantation stage, achieving apoptosis rates and blastocyst cell numbers significantly different from the control embryos with similar degrees of apoptosis and cell numbers to in vivo derived embryos [22]. In the human, IGF-1 was shown to significantly reduce the percentage of apoptotic nuclei by 50% in blastocyst. Thus the preimplantation embryo is able to respond in vitro to growth factors produced by the reproductive tract, which appear to be important in cell survival at the preimplantation stage [21]. More recently, Mitchell et al. (2002) assessed the effect of LIF addition at the time of blastocyst transfer to recipient females on vivo implantation and embryo–fetal development. They found that exposure of murine blastocysts to LIF at the time of transfer resulted in pronounced positive effects on implantation and pregnancy rates without affecting fetal development. On the imitative principle therefore, it would seem reasonable that the addition of growth factors to culture medium for embryos is warranted. However, it is important to assess the safety of exogenous growth factors before routine clinical application. It is also worth noting the development of the mammalian preimplantation embryo is influenced by many factors. Even embryo culture conditions themselves can have considerable effects on the expression of imprinted genes [23].
5. Culture system optimization It is important to consider the embryo culture system rather than embryo culture media in isolation, as all aspects involved in the culture system need to be optimized in order for culture media to achieve their best outcomes. Some aspects of culture technology that should be considered are types of incubation chambers, O2 levels, oil versus open culture, volume of medium, embryonic density, and the time of gamete coincubation during the insemination period. Other aspects of ART that go beyond the scope of the topic of ART media but need to be kept in mind when considering the final outcome of the whole procedure include assisted hatching, preimplantation genetic diagnosis, embryo transfer technique and ovarian stimulation protocols, to name a few. In other words, apart from the consideration of the actual media to be used in various ART procedures, it is important to remember that the way in which the media are
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B. Behr, H. Wang / European Journal of Obstetrics & Gynecology and Reproductive Biology 115S (2004) S72–S76
used and the culture strategies employed play critical interacting roles in the final outcome [24].
6. Cleavage or blastocyst stage transfer? Realizing the potential of blastocyst transfer to improve implantation rates, some IVF clinics throughout the world have chosen the approach to transfer blastocysts rather than earlier cleavage-stage embryos. The assumption is that extended culture permits selection of the one or two embryos with the highest developmental potential for transfer. Moreover, transfer of blastocysts into the uterus provides the appropriate temporal synchronization of the embryo and uterus. An increasing number of studies have confirmed that high implantation rates can be achieved after transfer of blastocysts [4,6,25]. This is not to say that high implantation rates cannot be achieved with cleavage stage transfer with appropriate multi-factorial embryo selection. However, extremely high pregnancy rates of 87% have been obtained using sequential media in patients who had a good response to gonadotropin stimulation and two top scoring blastocysts which have yet to be equalled with day 3 transfers [7]. Blastocyst transfer has been demonstrated to be useful even for patients with multiple failures of IVF [4]. For example, other studies have reported comparable pregnancy and implantation rates after days 3 and 5 embryo transfers [26,27]. Table 1 summarizes the reports of prospective randomized studies from different groups applying extend culture system and blastocyst transfer. More recently, several studies have shown either no difference between day 3 versus day 5 embryo transfers [28,29] or lower pregnancy rates for day 5 versus day 3 embryo transfer [48]. Extended culture may have a negative effect on in vitro development and implantation potential in cases where the culture conditions are suboptimal. This is a significant confounding factor in studies that compare day 2 or day 3 embryo transfers with day 5 embryo transfers. Because of the contradictory outcomes of extended culture, certain doubts Table 1 Comparison of outcomes of blastocyst transfer in prospective randomized studies Reference
IR (%) day 2–3/ day 5–6
PR (%) day 2–3/ day 5–6
Multiple PR (%) day 2–3/day 5–6
[49] [25]
13/23 I 37/55 I 15/26 I 21/24 18.9/24 13/26 I 29/46 I 38.7/20.2 d
26/40 I 66/71 25/28 39/39 41.7/38 26/29 35/60 I 45.5/18.6 d
15/20 N/A N/A 33/38 28/24 29/35 53/37.5 40/50
[26] [27] [50] [51] [48]
IR: implantation rate; PR: pregnancy rate; I: significant increase, IR or PR of day 5–6 compared to day 2–3; d: significant decrease, IR or PR of day 5–6 compared to day 2–3.
still remain, such as whether the culture media totally satisfy the needs of preimplantation embryos for nutrients, whether more blastocysts would develop in vivo rather than in vitro, whether prolonged culture has any effect on the developmental potential of the embryo, and whether 5 day cultivation is suitable and justifiable in all cases, even in cycles with a low number of oocytes [30]. Our clinic used a conservative approach to the introduction of blastocyst culture. Rather than transferring blastocysts immediately, we continued with day 3 transfers but cultured the supernumerary embryos for a further 2 or 3 days. If embryos made it to the blastocyst stage by day 6, they were cryopreserved. It ensured that the culture system employed was able to facilitate blastocyst development without compromising day 3 pregnancy rates, and that a suitable cryopreservation system was in place for when the move to fresh blastocyst transfer was accomplished [6,14].
7. Potential concerns of embryo culture IVF success has improved substantially since its inception. However, these successes have also generated increased multiple pregnancies. Concerns regarding the risk of multiple pregnancy and the consequences on the mother and children have been raised and are substantial [31]. Woman undergoing IVF treatment face a 20-fold increased risk of twins and 400-fold increased risk of higher order pregnancy [32]. This is related to the current practice of transferring multiple embryos into uterus. Therefore, the greatest challenge associated with an embryo transfer during an ART cycle is to optimize the chance of pregnancy while minimizing the chance of a multiple gestation. Blastocyst culture has been proposed as a tool that will allow us to limit the number of embryos we transfer, while overcoming the risks of multiple pregnancy and maintaining pregnancy rates. However, a concept that is rarely addressed is culture in vitro can affect gene expression and embryo metabolism in model systems, especially with extended culture. Concerns have arisen about the effects of extended culture on human embryos that may in part be responsible for the increased risk of monozygotic twinning [8] and increased proportion of male offspring after blastocyst transfer [10]. Recently, Hansen et al. (2002) reported that infants conceived with use of ART were more likely than naturally conceived infants to have multiple major defects and to have chromosomal and musculoskeletal defects. Furthermore, embryo culture conditions are known to affect several parameters including the kinetics of development, cell allocation to the inner cell mass and trophectoderm, embryo metabolism and genomic expression [33]. Several studies have shown that culture conditions can also effect the expression of imprinted genes [34–37]. Decreased expression of the IGF2 and the imprinting gene H19 were observed in many fetuses derived from blastocyst transfer after IVF in sheep [37]. It has been proposed that fetal
B. Behr, H. Wang / European Journal of Obstetrics & Gynecology and Reproductive Biology 115S (2004) S72–S76
abnormalities observed as a consequence of culture during preimplantation development could in large part be due to changes of the status of imprinted genes [36,38]. Moreover, several imprinted genes are known to play key roles in fetal growth and development [39–41]. In bovine and ovine embryos, culture to the blastocyst stage before embryo transfer can result in a higher incidence of fetal and perinatal loss. These abnormalities have been attributed to embryo culture conditions and in sheep may be linked to reduced expression of the imprinted Igf2r gene [37]. Doherty et al. [42] found that H19 expression was affected by different blastocyst culture media in the mouse model. This suggested that culture effects on imprinting genes are dependent on the culture medium used. Whether culture conditions used in human ART programs affect the expression of imprinted genes is unknown. In a recent human IVF study, children born after the application of ICSI were analyzed for their allelic methylation status at a key regulatory region in the imprinted Prader–Willi syndrome (PWS) and Angelman syndrome (AS) regions on chromosome 15q11-q13 [43]. No abnormal DNA patterns were detected in 92 children analyzed, and none showed clinical symptoms of the neurodevelopmental imprinting disorders of PWS or AS. However, a recent study by Schieve and his colleagues showed that the use of ART accounts for a disproportionate number of low-birth weight and very-low-birth weight in infants in the United States, in part because of absolute increases in multiple gestations but also in part because of higher rates of low-birth weight among singleton infants conceived with IVF [44]. This issue, therefore, arises a potentially worrisome concern of IVF and Barker hypothesis. Based on an exhaustive analysis of 80-year-old medical records, Barker discovered that low-birth weight is related to increased risk of cardiovascular disease and diabetes in the adulthood [45]. Many imprinted genes can be disrupted in the early embryo by environmental factors that are crucial for fetal growth and determine birthweight. The disruptions of imprinting genes could affect adult disease in a variety of direct and indirect means. So, Young [38] proposed that imprinting genes may be, at least somehow, related to Barker hypothesis. The question as to whether ART increases the incidence of congenital malformations or has subtle, longterm, and adverse effects on behavior and cognition remains contentious [46]. Although there are reports of an increase in the birth of abnormal babies, these findings could be specific to these patient populations and need to be confirmed, they nevertheless serve as an impetus to refine ART procedures to minimize multiple gestation and any other potential detrimental effects of ART. However, in the light of the observations made in animal models [33,40,41], the new technologies in ART such as embryo biopsy, extend culture, cytoplasmic transfer and culture media reformulations need to be monitored with extreme care and should ally with longterm programs of research on the responses of early embryos to these manipulations.
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8. Condensation Differences exist between in vivo and in vitro derived embryos. Some of these differences may be attributed to culture induced stress.
References [1] Templeton A. Infertility and the establishment of pregnancy— overview. Br Med Bull 2000;56:577–87. [2] Barnes FL, Crombie A, Gardner DK, Kausche A, Lacham-Kaplan O, Suikkari AM, et al. Hum Reprod 1995;10(12):3243–7. [3] Jones GM, Trounson AO. Blastocyst stage transfer: pitfalls and benefits. The benefits of extended culture. Hum Reprod 1999;14:1405–8. [4] Cruz JR, Dubey AK, Patel J, Peak D, Hartog B, Gindoff PR. Is blastocyst transfer useful as an alternative treatment for patients with multiple in vitro fertilization failures? Fertil Steril 1999;72(2): 218–20. [5] Behr B, Pool TB, Milki AA, Moore D, Gebhardt J, Dasig D. Preliminary clinical experience with human blastocyst development in vitro without co-culture. Hum Reprod 1999;14:454–7. [6] Milki AA, Hinckley MD, Fisch JD, Dasig D, Behr B. Comparison of blastocyst transfer with day 3 embryo transfer in similar patient populations. Fertil Steril 2000;73:126–9. [7] Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155–8. [8] Behr B, Fisch JD, Racowsky C, Miller K, Pool TB, Milki AA. Blastocyst-ET and monozygotic twinning. J Assist Reprod Genet 2000;17:349–51. [9] Menezo YJ, Sakkas D. Monozygotic twinning: is it related to apoptosis in the embryo? Hum Reprod 2002;17(1):247–8. [10] Menezo YJ, Chouteau J, Torello J, Girard A, Veiga A. Birth weight and sex ratio after transfer at the blastocyst stage in humans. Fertil Steril 1999;72(2):221–4. [11] Gardner DK, Lane M, Stevens J, Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril 2001;76(6):1175–80. [12] Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44(4):493–8. [13] Loutradis D, Drakakis P, Kallianidis K, Sofikitis N, Kallipolitis G, Milingos S, et al. Biological factors in culture media affecting in vitro fertilization, preimplantation embryo development, and implantation. Ann NY Acad Sci 2000;900:325–35. [14] Behr B. Blastocyst culture and transfer. Hum Reprod 1999;14:5–6. [15] Diaz-Cueto L, Gerton GL. The influence of growth factors on the development of preimplantation mammalian embryos. Arch Med Res 2001;32:619–26. [16] Hardy K, Wright C, Rice S, Tachataki M, Roberts R, Morgan D, et al. Future developments in assisted reproduction in humans. Reproduction 2002;123:171–83. [17] Dunglison GF, Barlow DH, Sargent IL. Leukaemia inhibitory factor significantly enhances the blastocyst formation rates of human embryos cultured in serum-free medium. Hum Reprod 1996;11: 191–6. [18] Martin KL, Barlow DH, Sargent IL. Heparin-binding epidermal growth factor significantly improves human blastocyst development and hatching in serum-free medium. Hum Reprod 1998;13: 1645–52. [19] Lighten AD, Moore GE, Winston RM, Hardy K. Routine addition of human insulin-like growth factor-I ligand could benefit clinical invitro fertilization culture. Hum Reprod 1998;13:3144–50.
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[20] Sjoblom C, Wikland M, Robertson SA. Granulocyte-macrophage colony-stimulating factor promotes human blastocyst development in vitro. Hum Reprod 1999;14:3069–76. [21] Spanos S, Becker DL, Winston RM, Hardy K. Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development. Bio Reprod 2000;63(5):1413–20. [22] Wang H, Dasig D, Gebhardt J, Polan ML, Behr B. Granulocytemacrophage colony-stimulating: a regulator in preimplantation embryo development and apoptosis? Fertil Steril 2002;77(Suppl): 3:S7. [23] Young LE, Fairburn HR. Improving the safety of embryo technologies: possible role of genomic imprinting. Theriogenology 2000; 53:627–48. [24] Quinn P. Review of media used in ART laboratories. J Androl 2000;21:610–5. [25] 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. [26] Coskun S, Hollanders J, Al-Hassan S, Al-Sufyan H, Al-Mayman H, Jaroudi K. Day 5 versus day 3 embryo transfer: a controlled randomized trial. Hum Reprod 2000;15:1947–52. [27] Plachot M, Belaisch-Allart J, Mayenga JM, Chouraqui A, Serkine AM, Tesquier L. Blastocyst stage transfer: the real benefits compared with early embryo transfer. Hum Reprod 2000;15(Suppl 6):24–30. [28] Rienzi L, Ubaldi F, Iacobelli M, Ferrero S, Minasi MG, Martinez F, et al. Day 3 embryo transfer with combined evaluation at the pronuclear and cleavage stages compares favourably with day 5 blastocyst transfer. Hum Reprod 2002;17:1852–5. [29] Utsunomiya T, Naitou T, Nagaki M. A prospective trial of blastocyst culture and transfer. Hum Reprod 2002;17(7):1846–51. [30] Kovacic B, Vlaisavljevic V, Reljic M, Gavric Lovrec V. Clinical outcome of day 2 versus day 5 transfer in cycles with one or two developed embryos. Fertil Steril 2002;77:529–36. [31] Bergh T, Ericson A, Hillensjo T, Nygren KG, Wennerholm UB. Deliveries and children born after in-vitro fertilization in Sweden 1982–1995: a retrospective cohort study. Lancet 1999;354:1579–85. [32] Martin PM, Welch HG. Probabilities for singleton and multiple pregnancies after in vitro fertilization. Fertil Steril 1998;70:478–81. [33] Leese HJ, Donnay I, Thompson JG. Human assisted conception: a cautionary tale. Lessons from domestic animals. Hum Reprod 1998;13(Suppl 4):184–202. [34] Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation of completely cell culture-derived mice from earlypassage embryonic stem cells. Proc Natl Acad Sci USA 1993;90:8424–8. [35] Moore T, Reik W. Genetic conflict in early development: parental imprinting in normal and abnormal growth. Rev Reprod 1996;1: 73–7.
[36] Dean W, Bowden L, Aitchison A, Klose J, Moore T, Meneses JJ, et al. Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes. Development 1998;125:2273–82. [37] Young LE, Fernandes K, McEvoy TG, Butterwith SC, Gutierrez CG, Carolan C, et al. Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture. Nat Genet 2001;27:153–4. [38] Young LE. Imprinting of genes and the barker hypothesis. Twin Res 2001;4:307–17. [39] Beechey CV. Peg1/Mest locates distal to the currently defined imprinting region on mouse proximal chromosome 6 and identifies a new imprinting region affecting growth. Cytogenet Cell Genet 2000;90:309–14. [40] Khosla S, Dean W, Reik W, Feil R. Culture of preimplantation embryos and its long-term effects on gene expression and phenotype. Hum Reprod Update 2001;7:419–27. [41] Khosla S, Dean W, Brown D, Reik W, Feil R. Culture of preimplantation mouse embryos affects fetal development and the expression of imprinted genes. Biol Reprod 2001;64:918–26. [42] Doherty AS, Mann MR, Tremblay KD, Bartolomei MS, Schultz RM. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Bio Reprod 2000;62(6):1526–35. [43] Manning M, Lissens W, Bonduelle M, Camus M, De Rijcke M, Liebaers I, et al. Study of DNA-methylation patterns at chromosome 15q11-q13 in children born after ICSI reveals no imprinting defects. Mol Hum Reprod 2000;6:1049–53. [44] 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(10):731–7. [45] Barker DJ, Fall CH. Fetal and infant origins of cardiovascular disease. Arch Dis Child 1993;68:797–9. [46] Schultz RM, Williams CJ. The science of ART. Science 2002;296(5576):2188–90. Review. [47] Hardy K, Spanos S. Growth factor expression and function in the human and mouse preimplantation embryo. J Endocrinology 2002;172:221–36. [48] Levron J, Shulman A, Bider D, Seidman D, Levin T. A prospective randomized study comparing day 3 with blastocyst-stage embryo transfer. Dor J Fertil Steril 2002 Jun;77(6):1300–1. [49] Scholtes NC, Zeilmaker GH. A prospective, randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertil Steril 1996;65(6):1245–8. [50] Karaki Raja Z, Samarraie Sadoon S, Younis Nagham A, Lahloub Tarek M, Ibrahim Mohammed H. Blastocyst culture and transfer: a step toward improved in vitro fertilization outcome. Fertil Steril 2002;77(1):114–8. [51] Van der Auwera I, Debrock S, Spiessens C, Afschrift H, Bakelants E, Meuleman C, et al. A prospective randomized study: day 2 versus day 5 embryo transfer. Hum Reprod 2002;17(6):1507–12.
Effects of culture conditions on IVF outcome B. Behr*, H. Wang Department of Obstetrics and Gynecology, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305, USA
Abstract Although in vitro fertilization (IVF) success rates have improved over the past decade, multiple pregnancies have become a formidable problem. The solution to this problem seems simple by mandating the reduction in numbers of embryos transferred. However, this is typically not accomplished without a compromise in the pregnancy rate. There have been a number of approaches designed to address high order multiple pregnancies from multi factorial analysis of early cleavage stage embryos to the development of extended culture systems, both of which require manipulations in the culture environment. Manipulations in embryo culture environment may not be benign. Several studies have demonstrated that adverse culture conditions have effects on gene expression and imprinting. Studies have also demonstrated that singleton human IVF babies have lower birth weight and higher incidence of congenital anomalies than natural conception babies. All of these factors need to be considered in relation to long term viability of IVF babies and the Barker hypothesis. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Embryo culture; Growth factors; Multiple pregnancy; Barker hypothesis; Blastocyst transfer; Imprinting
1. Introduction Despite numerous advances in the field of in vitro fertilization (IVF), many of the widely applied embryo culture techniques and resulting implantation rates have relatively unchanged since the first treatment was performed in the mid 1970’s [1]. For the past two decades the majority of embryos conceived through IVF have been transferred between days 1 and 3 at either the pronucleate or cleavage stage. The reason for this stems primarily from the inability of past culture system to support the development of viable blastocysts at acceptable rates. Recently, with the advent of newer culture systems it is now more consistent that day 5 or day 6 blastocysts be available for transfer. Impressive blastocyst formation and pregnancy results have been reported using in vitro blastocyst culture by a number of clinics and investigators [2–7]. The primary rational for blastocyst culture is to provide a mechanism for self-selection of viable embryos and improve the synchronicity of uterine and embryonic development. The commercialization of sequential culture media has led to the worldwide introduction of the technique despite much debate about its effect on IVF outcomes. Because culture in vitro can affect gene expression and embryo metabolism in model systems, concerns have arisen abut the effects of extended culture on these
* Corresponding author. Tel.: þ1-650-723-0951; fax: þ1-650-723-7737. E-mail address: [email protected] (B. Behr).
human embryos that may in part be responsible for the increasing risk of monozygotic twinning [8,9], or a propensity to skew the sex ratio after blastocyst transfer [10]. Therefore, clinics today are not only faced with what culture system and what media to choose from, but also face which stage and which embryo to select for transfer.
2. Preimplantation development and energy sources In the human, preimplantation development of the embryo from zygote to blastocyst occurs as it travels through the fallopian tube towards the uterus from day 1 to 5 after ovulation. As the embryo reaches the blastocyst stage on day 5, within 48 h of reaching the uterus, the blastocyst begins to hatch from the zona pellucida, and then implant. Many studies have been undertaken to understand the energy requirements of the preimplantation embryo. At the stage of the zygote, the embryo exhibits relatively low levels of biosynthesis prior to embryonic genome activation and expression. It is commonly accepted that pyruvate and lactate are the preferred energy sources for this early stage of development. Glucose consumption is limited by the early embryo. As development proceeds, and energy demands increase with cell multiplication and an increase in protein synthesis, there is a concomitant increase in glucose utilization. By the blastocyst stage, the embryo exhibits high oxygen utilization and an ability to readily utilize glucose, along with other energy sources [11].
0301-2115/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ejogrb.2004.01.016
B. Behr, H. Wang / European Journal of Obstetrics & Gynecology and Reproductive Biology 115S (2004) S72–S76
3. Culture system 3.1. Culture medium selection Over the past decade there has been considerable interest in optimizing culture media for supporting human embryos after IVF and before embryo transfer. Some of the first culture media used for human IVF, such as Ham’s F10 and Earle’s Medium were constructed to support the development of somatic cells and cell lines. Others, such as Tyrode’s T6 and Whitten’s WM1 had been used for IVF and embryo culture in laboratory animals. Human tubal fluid (HTF) medium was one of the first media specifically designed for human IVF, its formulation based on the imitative principle using the then known chemical composition of human tubal fluid [12]. IVF culture media can generally be categorized on whether they are a complex mixture of inorganic salts, energy sources, amino acids, vitamins and other substances (e.g. Ham’s F10, Menezo’s B2 and B3 media, Eagle’s MEM) or whether they are simple balanced salt solutions containing energy sources (e.g., Earle’s, Tyrode’s T6, WM1) [13]. However, prior to the mid ‘90s, low production and accompanying low viability of blastocysts during human IVF due to the inability of any single culture medium to meet the changing nutritional requirements of preimplantation embryos resulted in blastocyst transfer (BT) being abandoned early. Subsequently, applying a sequential culture approach resulted in acceptable blastocyst development and pregnancy rates to warrant its application in routine IVF [25,14]. The blastocyst culture provides greater opportunities for selection of more viable and, in certain cases, genetically normal embryos. Blastocyst transfer also provides more physiologic synchronization of the embryo stage with the endometrium. Therefore, the transfer of human blastocysts in IVF should result in an increased implantation rate and enable the transfer of fewer, but higher quality embryos, without any negative impact on pregnancy rates while, at the same time, decreasing high order multiples. With the wide commercial availability of sequential media, extend culture has become a practical tool within reach of all IVF programs. There is still the question as to which sequential media would be the best to culture cleavage or blastocyst stage embryos. There have been many studies that have compared various culture media but they provide conflicting data. Also, as with many studies in clinical IVF, the sample sizes in these studies are relatively small and more prospective randomized studies are therefore needed.
4. Growth factors and preimplantation development There is increasing evidence in a variety of mammalian species that growth factors play an important role in preimplantation development, regulation of cellular events and
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in maternal embryonic dialogue [15,16,47]. A wide range of growth factor ligands and their receptors are expressed in embryos during the preimplantation period [16]. In addition, supplementation of culture medium with growth factors has been shown to be beneficial for preimplantation development. For example, LIF, IGF-1, HBEGF and GM-CSF have all been shown to increase the proportion of embryo development to the blastocyst stage [17–22]. Furthermore, IGF-1 and GM-CSF increase blastocyst cell number specifically in inner cell mass [19,20]. Previous studies have demonstrated that GM-CSF may act as a regulator in murine embryo development. This may be attributed to decreasing cell apoptosis during the preimplantation stage, achieving apoptosis rates and blastocyst cell numbers significantly different from the control embryos with similar degrees of apoptosis and cell numbers to in vivo derived embryos [22]. In the human, IGF-1 was shown to significantly reduce the percentage of apoptotic nuclei by 50% in blastocyst. Thus the preimplantation embryo is able to respond in vitro to growth factors produced by the reproductive tract, which appear to be important in cell survival at the preimplantation stage [21]. More recently, Mitchell et al. (2002) assessed the effect of LIF addition at the time of blastocyst transfer to recipient females on vivo implantation and embryo–fetal development. They found that exposure of murine blastocysts to LIF at the time of transfer resulted in pronounced positive effects on implantation and pregnancy rates without affecting fetal development. On the imitative principle therefore, it would seem reasonable that the addition of growth factors to culture medium for embryos is warranted. However, it is important to assess the safety of exogenous growth factors before routine clinical application. It is also worth noting the development of the mammalian preimplantation embryo is influenced by many factors. Even embryo culture conditions themselves can have considerable effects on the expression of imprinted genes [23].
5. Culture system optimization It is important to consider the embryo culture system rather than embryo culture media in isolation, as all aspects involved in the culture system need to be optimized in order for culture media to achieve their best outcomes. Some aspects of culture technology that should be considered are types of incubation chambers, O2 levels, oil versus open culture, volume of medium, embryonic density, and the time of gamete coincubation during the insemination period. Other aspects of ART that go beyond the scope of the topic of ART media but need to be kept in mind when considering the final outcome of the whole procedure include assisted hatching, preimplantation genetic diagnosis, embryo transfer technique and ovarian stimulation protocols, to name a few. In other words, apart from the consideration of the actual media to be used in various ART procedures, it is important to remember that the way in which the media are
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used and the culture strategies employed play critical interacting roles in the final outcome [24].
6. Cleavage or blastocyst stage transfer? Realizing the potential of blastocyst transfer to improve implantation rates, some IVF clinics throughout the world have chosen the approach to transfer blastocysts rather than earlier cleavage-stage embryos. The assumption is that extended culture permits selection of the one or two embryos with the highest developmental potential for transfer. Moreover, transfer of blastocysts into the uterus provides the appropriate temporal synchronization of the embryo and uterus. An increasing number of studies have confirmed that high implantation rates can be achieved after transfer of blastocysts [4,6,25]. This is not to say that high implantation rates cannot be achieved with cleavage stage transfer with appropriate multi-factorial embryo selection. However, extremely high pregnancy rates of 87% have been obtained using sequential media in patients who had a good response to gonadotropin stimulation and two top scoring blastocysts which have yet to be equalled with day 3 transfers [7]. Blastocyst transfer has been demonstrated to be useful even for patients with multiple failures of IVF [4]. For example, other studies have reported comparable pregnancy and implantation rates after days 3 and 5 embryo transfers [26,27]. Table 1 summarizes the reports of prospective randomized studies from different groups applying extend culture system and blastocyst transfer. More recently, several studies have shown either no difference between day 3 versus day 5 embryo transfers [28,29] or lower pregnancy rates for day 5 versus day 3 embryo transfer [48]. Extended culture may have a negative effect on in vitro development and implantation potential in cases where the culture conditions are suboptimal. This is a significant confounding factor in studies that compare day 2 or day 3 embryo transfers with day 5 embryo transfers. Because of the contradictory outcomes of extended culture, certain doubts Table 1 Comparison of outcomes of blastocyst transfer in prospective randomized studies Reference
IR (%) day 2–3/ day 5–6
PR (%) day 2–3/ day 5–6
Multiple PR (%) day 2–3/day 5–6
[49] [25]
13/23 I 37/55 I 15/26 I 21/24 18.9/24 13/26 I 29/46 I 38.7/20.2 d
26/40 I 66/71 25/28 39/39 41.7/38 26/29 35/60 I 45.5/18.6 d
15/20 N/A N/A 33/38 28/24 29/35 53/37.5 40/50
[26] [27] [50] [51] [48]
IR: implantation rate; PR: pregnancy rate; I: significant increase, IR or PR of day 5–6 compared to day 2–3; d: significant decrease, IR or PR of day 5–6 compared to day 2–3.
still remain, such as whether the culture media totally satisfy the needs of preimplantation embryos for nutrients, whether more blastocysts would develop in vivo rather than in vitro, whether prolonged culture has any effect on the developmental potential of the embryo, and whether 5 day cultivation is suitable and justifiable in all cases, even in cycles with a low number of oocytes [30]. Our clinic used a conservative approach to the introduction of blastocyst culture. Rather than transferring blastocysts immediately, we continued with day 3 transfers but cultured the supernumerary embryos for a further 2 or 3 days. If embryos made it to the blastocyst stage by day 6, they were cryopreserved. It ensured that the culture system employed was able to facilitate blastocyst development without compromising day 3 pregnancy rates, and that a suitable cryopreservation system was in place for when the move to fresh blastocyst transfer was accomplished [6,14].
7. Potential concerns of embryo culture IVF success has improved substantially since its inception. However, these successes have also generated increased multiple pregnancies. Concerns regarding the risk of multiple pregnancy and the consequences on the mother and children have been raised and are substantial [31]. Woman undergoing IVF treatment face a 20-fold increased risk of twins and 400-fold increased risk of higher order pregnancy [32]. This is related to the current practice of transferring multiple embryos into uterus. Therefore, the greatest challenge associated with an embryo transfer during an ART cycle is to optimize the chance of pregnancy while minimizing the chance of a multiple gestation. Blastocyst culture has been proposed as a tool that will allow us to limit the number of embryos we transfer, while overcoming the risks of multiple pregnancy and maintaining pregnancy rates. However, a concept that is rarely addressed is culture in vitro can affect gene expression and embryo metabolism in model systems, especially with extended culture. Concerns have arisen about the effects of extended culture on human embryos that may in part be responsible for the increased risk of monozygotic twinning [8] and increased proportion of male offspring after blastocyst transfer [10]. Recently, Hansen et al. (2002) reported that infants conceived with use of ART were more likely than naturally conceived infants to have multiple major defects and to have chromosomal and musculoskeletal defects. Furthermore, embryo culture conditions are known to affect several parameters including the kinetics of development, cell allocation to the inner cell mass and trophectoderm, embryo metabolism and genomic expression [33]. Several studies have shown that culture conditions can also effect the expression of imprinted genes [34–37]. Decreased expression of the IGF2 and the imprinting gene H19 were observed in many fetuses derived from blastocyst transfer after IVF in sheep [37]. It has been proposed that fetal
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abnormalities observed as a consequence of culture during preimplantation development could in large part be due to changes of the status of imprinted genes [36,38]. Moreover, several imprinted genes are known to play key roles in fetal growth and development [39–41]. In bovine and ovine embryos, culture to the blastocyst stage before embryo transfer can result in a higher incidence of fetal and perinatal loss. These abnormalities have been attributed to embryo culture conditions and in sheep may be linked to reduced expression of the imprinted Igf2r gene [37]. Doherty et al. [42] found that H19 expression was affected by different blastocyst culture media in the mouse model. This suggested that culture effects on imprinting genes are dependent on the culture medium used. Whether culture conditions used in human ART programs affect the expression of imprinted genes is unknown. In a recent human IVF study, children born after the application of ICSI were analyzed for their allelic methylation status at a key regulatory region in the imprinted Prader–Willi syndrome (PWS) and Angelman syndrome (AS) regions on chromosome 15q11-q13 [43]. No abnormal DNA patterns were detected in 92 children analyzed, and none showed clinical symptoms of the neurodevelopmental imprinting disorders of PWS or AS. However, a recent study by Schieve and his colleagues showed that the use of ART accounts for a disproportionate number of low-birth weight and very-low-birth weight in infants in the United States, in part because of absolute increases in multiple gestations but also in part because of higher rates of low-birth weight among singleton infants conceived with IVF [44]. This issue, therefore, arises a potentially worrisome concern of IVF and Barker hypothesis. Based on an exhaustive analysis of 80-year-old medical records, Barker discovered that low-birth weight is related to increased risk of cardiovascular disease and diabetes in the adulthood [45]. Many imprinted genes can be disrupted in the early embryo by environmental factors that are crucial for fetal growth and determine birthweight. The disruptions of imprinting genes could affect adult disease in a variety of direct and indirect means. So, Young [38] proposed that imprinting genes may be, at least somehow, related to Barker hypothesis. The question as to whether ART increases the incidence of congenital malformations or has subtle, longterm, and adverse effects on behavior and cognition remains contentious [46]. Although there are reports of an increase in the birth of abnormal babies, these findings could be specific to these patient populations and need to be confirmed, they nevertheless serve as an impetus to refine ART procedures to minimize multiple gestation and any other potential detrimental effects of ART. However, in the light of the observations made in animal models [33,40,41], the new technologies in ART such as embryo biopsy, extend culture, cytoplasmic transfer and culture media reformulations need to be monitored with extreme care and should ally with longterm programs of research on the responses of early embryos to these manipulations.
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8. Condensation Differences exist between in vivo and in vitro derived embryos. Some of these differences may be attributed to culture induced stress.
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