1 Division of Reproductive Medicine, Department of Obstetrics and Gynaecology and 2 Department of Public Health, Centre for Clinical Decision Sciences, Erasmus Medical Centre, Rotterdam, The Netherlands
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Abstract |
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Key words: blastocyst/culture medium/IVF/pregnancy rates/randomized controlled trial
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Introduction |
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Early reports of human blastocysts grown in culture employed culture medium alone (Steptoe et al., 1971; Edwards et al., 1981
). Using simple defined media supplemented with serum, other workers obtained high blastocyst formation rates (Hardy et al., 1989a
,b
; Fitzgerald and di Mattina, 1992). However, implantation rates remained poor (Bolton et al., 1991
) until co-culture systems in which trophoblast cells were included in the culture environment were developed and implemented (Ménézo et al., 1990
, 1992
; Ménézo and Ben Khalifa, 1995
; Fong et al., 1997
). At the same time, culture media capable of sustaining embryo development to the blastocyst stage without the use of co-culture systems were developed (Huisman et al., 1994
, 2000
). Increasing understanding of the dynamic nature of early embryo metabolism (Leese et al., 1986
; Hardy et al., 1989b
; Gott et al., 1990
) and the changing environment encountered by the preimplantation embryo in vivo (Gardner et al., 1996
; Gardner, 1998
) led to the formulation of sequential culture media. These were designed to support the growth of human pronuclear embryos to the blastocyst stage (Barnes et al., 1995
; Gardner et al., 1996
; Gardner and Lane, 1997
; Ménézo et al., 1998
; Behr et al., 1999
; Milki et al., 1999
).
Central to the design of sequential media is the switch from pyruvate (the preferred nutrient of the early cleavage stage when low levels of oxygen are consumed) to glucose to meet the increased energy demands as development to the blastocyst stage proceeds (Hardy et al., 1989b). Moreover, the complex structure of the blastocyst is considered to merit recognition in the design of culture media since the inner cell mass requires the essential group of amino acids, whereas the trophectoderm utilizes the non-essential amino acids and glutamine (Lane and Gardner, 1997a
). Consistent with this notion is the supposition that conditions supporting optimal development of the early cleavage stage embryo are unlikely to support optimal blastocyst development and differentiation (Gardner and Lane, 1997
) and vice versa (Lane and Gardner, 1997b
).
Addressing these changes in the requirements for both carbohydrates and proteins during early embryonic development, two commercially available culture media were formulated to support the growth of human pronuclear embryos to the blastocyst stage (Gardner et al., 1996, 1998a
,Gardner et al., b
). The first medium (G1.1) is based on the levels of carbohydrate present in the human Fallopian tube at the time when the cleavage stage embryo is present. It also contains amino acids, which have been shown to stimulate development of the cleavage stage embryo (non-essential amino acids and glutamine). The chelator EDTA is also present to sequester any toxic divalent cations present in the system. It also helps to minimize glycolytic activity of the embryo thus minimizing the metabolic perturbations (Lane and Gardner, 1997b
). The second medium is based on the levels of carbohydrate present in the human uterus and contains both essential and non-essential amino acids to facilitate both blastocyst formation and differentiation. However, EDTA is not present as it may selectively impair inner cell mass development and function (Hewitson and Leese, 1993
).
Initial experience with sequential culture systems designed to address the complexities and dynamic nature of embryo metabolism was very encouraging and high rates of blastulation and implantation were reported (Gardner and Lane, 1997b; Gardner et al., 1998a; Ménézo et al., 1998
). A subsequent prospective study comparing outcomes from blastocysts produced by sequential culture systems with those from day 3 embryos produced from monoculture systems confirmed the potential of this approach (Gardner et al., 1998b
). The compelling theoretical advantages of sequential culture systems and number of retrospective studies suggesting their superiority over monoculture and co-culture techniques (Jones et al., 1998a
), have led to the widespread use of sequential culture systems which are now commercially available. However, most studies of blastocyst culture published so far are uncontrolled, based on small numbers and with highly selected patients, making comparisons of results between different culture systems difficult. Moreover, the superiority of sequential culture of embryos over monoculture systems for producing blastocysts for transfer in IVF treatment awaits confirmation in prospective studies. We set out to address this by prospectively comparing blastulation, implantation and viable pregnancy rates from embryos randomized to culture in sequential and non-sequential culture systems using our own complex medium or the commercially available sequential G1/G2 medium.
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Materials and methods |
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Earlier studies have indicated that following sequential culture with G1/G2, implantation rates per blastocyst of 4060% may be achieved (Gardner et al., 2000a). The mean implantation rate per blastocyst derived from monoculture systems in our centre is
20% (Huisman et al., 2000
). In order to show with 80% power at P < 0.05 that G1/G2 sequential culture as opposed to monoculture with the Rotterdam medium could increase implantation rates per blastocyst from 20 to 40%, 100 transferred embryos were required in each arm. Since these two culture systems were also to be compared with the Rotterdam medium in a serial culture system, and a maximum of two embryos were transferred per cycle, a total of 150 completed cycles was required.
Pituitary down-regulation was achieved by administering the GnRH agonist triptorelin (Decapeptyl®; Ferring, Hoofddorp, The Netherlands) 0.1 mg/day s.c. starting in the mid-luteal phase of the preceding cycle (long protocol). Following pituitary down-regulation, multiple follicle development was induced with 150300 IU/day recombinant FSH (Puregon®; NV Organon, Oss, The Netherlands) administered s.c. Follicular growth was monitored by ultrasonography until three or more dominant follicles measuring 17 mm in diameter were observed. A single i.m. injection of 10 000 IU of hCG (Pregnyl®; NV Organon) was administered to induce final stages of oocyte maturation. Transvaginal ultrasound-guided follicular aspiration was performed 3436 h later. Following oocyte retrieval, patients were randomized by sealed envelopes to one of three groups for embryo culture. In group A, embryos underwent non-sequential culture in our own complex medium. This culture system was termed the Rotterdam monoculture system. In group B, embryos were cultured in the same medium but were transferred to a fresh drop of this medium on day 3. This was referred to as the Rotterdam serial culture system. In group C the commercially available G1.2/G2.2 sequential medium (Vitrolife, Gothenburg, Sweden) referred to as the G1/G2 sequential system was employed (Table I).
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Embryo morphology scoring was performed on days 3 and 5 for all study groups. Scoring of day 3 embryos was performed according to previously published criteria including cell number, regularity of the blastomeres, fragmentation and morphological aspects such as granulation (Veeck, 1998; Huisman et al., 2000
). Day 3 embryos were thus scored on a scale of 1 (high grade) to 4 (low grade). On the day of embryo transfer all embryos were scored again according to strict day 5 criteria including embryonic stage, cavitation, inner cell mass and cell morphology (Veeck, 1998
; Huisman et al., 2000
). When no fragmentation was evident and the developmental stage was appropriate for their age, embryos were described as high grade (score 1). Embryos showing developmental delay and >50% fragmentation were described as low grade (score 4). Embryos of better and worse intermediate quality were scored as grade intermediate high and intermediate low respectively (score 2 and 3).
Blastulation rate was calculated from the number of blastocysts available for embryo transfer and cryopreservation divided by the total number of oocytes fertilized. Pregnancy was identified by a positive urinary hCG test result (sensitivity of >25 IU/l) (Clearview; Unipath Ltd, Bedfordshire, UK) performed 17 days after ovum retrieval. Pregnancy rates per embryo transfer procedure and implantation rates per transferred blastocyst were based on the detection of fetal heart beats by ultrasound carried out 5 weeks after embryo transfer.
Data analysis
Differences in blastulation, pregnancy and implantation rates per blastocyst between the three groups were analysed using the 2-test. Differences in embryo quality for the studied protocols on day 3 and day 5 were also evaluated using the
2-test. Data related to the cryopreservation of supernumerary embryos derived from study treatment cycles were not included in the analysis.
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Results |
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Discussion |
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The viability of the blastocysts obtained is an important measure of quality since a morphologically normal blastocyst is not necessarily viable. This concept may explain why previous attempts to culture the human blastocyst have resulted in a low implantation rate after transfer (Bolton et al., 1991). Indeed, Behr et al. concluded from their study that the sequential media approach may not necessarily result in a higher number of blastocysts being obtained but in an increase in the number of viable blastocysts as evidenced by high implantation rates (Behr et al., 1999
). We therefore used implantation rates per blastocyst (as represented by the presence on ultrasound examination of intrauterine gestational sacs and fetal poles with visible heart beats) as a means of comparing viability of the blastocysts formed by the different media and systems studied.
Previous studies have reported implantation rates for transferred blastocysts of between 25 and 70% (Jones et al., 1998b; Gardner et al., 1998b
, 2000b
; Milki et al., 2000
;). The implantation potential observed of transferred blastocysts in all three groups of the present study was lower than those previously published. In addition to a possible reduced performance in our laboratory, this difference may also be explained by the lack in our study of any selection of patients or embryos for culture to the blastocyst stage, as was also the case in our previous report on blastocyst transfer (Huisman et al., 2000
). Moreover, the blastulation rate was based on the entire cohort of the patients fertilized oocytes and not on the spare embryos as in other studies (Bolton et al., 1989
; Desai et al., 1996
). The present study therefore represents a comparison of the two media and culture systems as may be used in everyday clinical practice. When the analysis was restricted to those women in whom at least one top quality embryo was transferred, implantation rates of up to 30% of blastocysts transferred and ongoing pregnancy rates of up to 40% per embryo transfer procedure were observed in all groups. These high rates are similar to those reported by other groups analysing selected patients (Gardner et al., 2000b
; Milki et al., 2000
).
The success of our monoculture system in producing viable blastocysts may be due in part to fact that the mixture of Earles balanced salt solution and Hams F-10 medium used under our conditions contains all three energy substrates (pyruvate, lactate and glucose) required. The latter is not the case in many routinely used culture media (Gardner and Lane, 2001). Furthermore, none of the nutrients appears to be supplied in a concentration likely to lead to developmental arrest or retardation at any stage from the zygote to the blastocyst stage. In the present study, embryos were cultured in groups (with a maximum of four embryos) in microdrops of medium. Both human studies (Moessner and Dodson, 1995
; Amalgor et al., 1996), and studies in sheep and mice have indicated that culturing embryos in groups has beneficial effects on development, in part by improving cell allocation to the inner cell mass (Gardner et al., 1994
, 1997
). This effect is considered to be due to a relative decrease in the volume of culture medium (Paria and Dey, 1990
; Amalgor et al., 1996) and a relative increase in the concentration of paracrine and autocrine factors stimulating embryo development (Gardner et al., 1997
).
The serial culture protocol involving transfer to a new drop of medium on day 3 revealed slightly favourable results over the monoculture system when comparing biochemical and ongoing pregnancy rates. Although these differences did not reach statistical significance, they would appear to be consistent with improved blastocyst development and viability following the elimination of waste products from embryonic metabolism. Refreshment itself may also have a beneficial effect, simply due to renewal of the metabolic nutrients in a new medium drop. However, the corollary to this is the possible negative impact on embryo development of the additional handling required.
The benefits of continued improvements to blastocyst culture conditions would appear to be limited by genetic factors, since the presence of oocyte and sperm genetic abnormalities will prevent a proportion of the pronuclear embryos from reaching the blastocyst stage, independent of culture conditions. The majority of these abnormalities are chromosomal, as 25% of oocytes are aneuploid (Kola et al., 1993
; van Blerkom, 1994
). Since the number of blastocysts that develop in culture reflect the quality of the gametes from which they were derived (Behr et al., 1998
), further improvements in media used for blastocyst culture may only allow limited improvements in blastulation rate. In a recent prospective study comparing sequential blastocyst culture with co-culture with Vero cells, no significant difference in take-home baby rate was observed (Giscard dEstaing et al., 2001
). Moreover, in a comparison of two commercially available sequential culture systems, no difference in outcomes in the blastocyst programme was observed (Van Langendonckt et al., 2001
). Further development towards optimal culture conditions for viable blastocyst development may therefore require a new approach.
At present however, the design of blastocyst culture media continues to focus primarily on the successful production of viable embryos. In this, the first randomized prospective study comparing sequential culture with monoculture of human blastocysts, no significant differences in blastulation and implantation rates were observed between the Rotterdam medium used in monoculture and serial culture or the sequential G1.2 and G2.2 media system. It may be concluded that the Rotterdam medium contains the nutrients required by the preimplantation embryos at the different stages of development. The applicability of our results to other laboratory settings is inevitably limited by other local factors which may have affected the study outcome. However, the results of this study indicate that human embryos do not necessarily have to be cultured in specially designed sequential media to fully develop into viable blastocysts capable of resulting in ongoing pregnancy.
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Acknowledgements |
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Notes |
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References |
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Submitted on October 29, 2001; resubmitted on April 19, 2002; accepted on June 21, 2002.