Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, West Orange, New Jersey, USA
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Abstract |
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Key words: blastocyst/cleavage rate/fragmentation degree/fragmentation pattern/multinucleation
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Introduction |
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It has been proposed that prolonged culture allows development of `normal' embryos with implantation potential, while `abnormal' and non-viable embryos arrest before or shortly after the onset of genomic activation (Huisman et al., 1994; Dawson et al., 1995
; Janny and Ménézo, 1996
; Gardner and Lane, 1997
).
Indeed, a number of intrinsic factors have been shown to influence survival to the blastocyst stage in extended culture in vitro; among them are sperm quality (Janny and Ménézo, 1994; Jones et al., 1998
), the aetiology of infertility (Ménézo et al., 1995
), and maternal age (Janny and Ménézo, 1996
; Schoolcraft et al., 1999
).
Cleavage patterns have also been linked to blastocyst formation (Bolton et al., 1989; Wiemer et al., 1995
; Balakier and Cadesky, 1997
; Rijnders and Jansen, 1998
). This, together with the link between chromosomal abnormalities and aberrant early embryo morphology (Pellestor et al., 1994
; Munné et al., 1994
, 1995
; Sadowy et al., 1998
), as well as the observed viability of in-vitro grown blastocysts (Gardner et al., 1998a
,b
, 2000
; Schoolcraft et al., 1999
) lend support to the `survival of the fittest' proposal.
However, the relationship between morphology, chromosomal integrity, embryogenesis in vitro, and viability is clearly more complex. Embryos with normal morphology may be chromosomally abnormal, but can reach the blastocyst stage; on the other hand, many chromosomally normal embryos with atypical or normal morphology fail to undergo differentiation in extended culture (Sandalinas et al., 2000). There is also some clinical evidence that suggests a loss of development potential with extended culture of compromised embryos. It has been reported that patients with no 8-cell embryos on day 3 had a 33% pregnancy rate after day 3 transfers, but failed to achieve pregnancies after day 5 transfers (Racowsky et al. 1999
).
So, extrinsic factors such as prolonged culture itself may also contribute to the loss of in-vitro-generated embryos over time in culture. But, it is so far unclear which embryos may be affected and to what extent. In the present study, the impact of cleavage rate, fragmentation, and multinucleation on compaction, cavitation, along with ICM and TE formation after prolonged culture is assessed. Morphological abnormalities in the pattern of blastulation are described. The relationship between morphological profiles of blastocysts and implantation after day 5 transfer was also evaluated. The findings have important implications for the application of day 5 transfer after IVF.
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Materials and methods |
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Procedures during which embryos were pooled in groups (n = 11) were excluded from the study, as the embryos could not be individually tracked during culture.
A standard down-regulation protocol included 1 mg of Lupron (TAP Pharmaceuticals, Lake Forest, IL, USA) starting on day 21 of the cycle prior to the stimulation cycle. On day 3 of menses, suppression was verified by both ultrasound and blood oestradiol and progesterone concentrations. On the day of gonadotrophin start (recombinant FSH; Gonal-F; Serono Laboratories, Norwell, MA, USA), Lupron dose was decreased to 0.25 mg and continued until the day of human chorionic gonadotrophin (HCG) administration. The usual daily dose of gonadotrophin was 4 ampoules, given singly on the evening of the first day, and split between morning and evening thereafter. Follicular development was monitored by ultrasound and blood hormone concentrations. When the lead follicle reached 1617 mm in diameter and a minimum of four mature follicles was present, HCG was administered. Oocyte retrieval was scheduled to 36 h after HCG.
Gamete and embryo culture
Oocytes were collected in HEPES-buffered human tubal fluid medium (HTF) (Quinn et al., 1985). After cumulus dissection and wash, they were placed in 100200 µl droplets of protein-supplemented HTF under mineral oil (Squibb, Princeton, NJ, USA). Protein supplementation was provided either by 10% heat-inactivated maternal serum, 6% plasmanate (Miles Inc., Elkhart, IN, USA) or 6% synthetic serum substitute (SSS; Irvine Scientific, Irvine, CA, USA).
Normally fertilized oocytes with two pronuclei were cultured individually in 100 µl droplets of HTF supplemented with protein, or in droplets of G1.2 (courtesy of D.Gardner; nine patients only). They remained in these media until early afternoon of day 3, when they were placed in 100 µl drops of G2.2 (courtesy of D.Gardner or purchased from Scandinavian IVF Science, Gothenburg, Sweden) or S2 (Scandinavian IVF Science) following a 5-drop rinse in the appropriate medium. A second changeover to fresh G2.2 or S2 was done on the morning of day 5, after embryo evaluation and before embryo transfer. Embryo transfers were scheduled between noon and 14.00 h on day 5.
A total of 1395 zygotes from the 102 patients was cultured, of which 112 were cryopreserved on or before day 3. An additional 69 embryos were discarded before reaching day 5, as they were judged to have completely arrested. Thus the total number of embryos cultured to day 5 was 1214. The number of embryos in the cleavage rate analyses differed slightly from this number, since the pertinent information was not available in the database for all the embryos in the study.
Embryo evaluation
Fertilization was confirmed ~14 hours post insemination. On days 2, 3, 4 and 5 of development, embryos were evaluated on an Olympus IX70 inverted microscope (Olympus America, Melville, New York, NY, USA), equipped with Hoffman Modulation Optics (Narishige, Tokyo, Japan). Total magnification was x600. Cell number, degree and pattern of fragmentation, and the presence of multinucleate blastomeres were recorded on days 2 and 3 of development.
Cell numbers of embryos on days 2 and 3 were adjusted according to individual time in culture after oocyte retrieval using the following formula:
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The average number of hours in culture from oocyte retrieval to day 2 and day 3 was 48.28 and 71.68 respectively.
The degree of fragmentation was expressed as a percentage and defined as the embryonic volume occupied by anucleate cytoplasmic fragments (Puissant et al., 1987). Fragmentation pattern was defined based on spatial distribution and relative size of the fragments, as described previously (Alikani and Cohen, 1995
; Warner et al., 1998
; Alikani et al., 1999
).
Development on days 4 and 5 was recorded in detail during the study but was not classified until data analysis. At that time, embryos were classified according to the criteria listed in Table I. As is the nature of all morphological classification systems, the one proposed here is subjective, but based on our own observations as well as those of others on morula and blastocyst morphology in the human (e.g. Cohen et al., 1985; Hardy et al., 1989; Dokras et al., 1991, 1993; Van Blerkom, 1993; Gardner and Schoolcraft, 1999; Ménézo et al., 1999). Our analyses do not include day 6 development since (i) the majority of the blastocysts in this study, as in other studies (Gardner et al., 1998a
,b
) formed on day 5, (ii) embryos that did not undergo differentiation by day 5 rarely formed normal blastocysts on day 6. This is not to say that day 6 blastocysts do not have an implantation potential, rather that in the context of this study, timing was an important factor in definition of normal development.
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A distinct ICM, organized as a compacted mass of numerous cells, was considered normal. If the inner cells formed a small mass, or if the mass comprised loosely packed cells, the formation was suboptimal but considered normal (Gardner and Schoolcraft, 1999) (Figure 1F and G
respectively). The absence of an ICM was abnormal (Hardy et al., 1989
) (Figure 1H
). A cohesive layer of numerous tightly packed cells in the TE was normal; large TE cells were suboptimal but again considered normal (Figure 1I and J
). Highly irregularly arranged ICM and/or TE cells were considered abnormal (Figure 1K and L
). A blastocyst was considered morphologically normal if it contained either a normal or suboptimal ICM in combination with either a normal or suboptimal TE on day 5 (see Table I
). The appearance of either an abnormal ICM or an abnormal TE, or a full day's delay in differentiation placed the day 5 embryo in the abnormal category. Ten representative blastocysts of normal appearance are shown in Figure 2
; six of these have become babies.
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After retrieval, oocytes are numbered and tracked individually with separate pages or tables for each day of development, just as in a daily journal. When embryos are replaced, separate records for each are again created and linked to the record of the replacement procedure. As pregnancy results become available, they are entered in a further table, always linked by procedure number. Data for the analyses in this study were extracted by creating selection lists or queries from relevant tables in the database. EggCyte has over 40 data tables but this study has used about 14 altogether. Over 300 queries of varying complexity with Structured Query Language (SQL) and Visual Basic programming functions (Microsoft Corp.) were used to generate the data presented here.
Differences in the number of embryos compacting, cavitating, and forming blastocysts in each fragmentation group as well as in each cleavage group were tested for significance using 2 analysis. Differences between mean cell numbers were analysed using analysis of variance. P < 0.05 was considered significant.
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Results |
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Implantation potential of embryos with cleavage anomalies after short-term culture
The EggCyte database was searched for homogeneous day 3 transfers involving embryos with either no abnormality or one or more of the anomalies described here. Homogeneous transfers were those where all the embryos transferred belonged in the same morphological category. The resulting implantation rates were compared to blastocyst formation rate in extended culture and the theoretical implantation rate after day 5 transfer (Table V). A similar concept was previously suggested by Edwards and Beard (1999).
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The theoretical implantation rate of embryos in prolonged culture was calculated based on the following formula:
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For embryos with slow development and/or fragmentation, the theoretical implantation rate after day 5 transfer appeared to be lower than their actual implantation rate after day 3 transfer, since many were lost during extended culture. Embryos with multinucleation, on the other hand, appeared to perform similarly after short- or long-term culture (Table V).
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Discussion |
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The culture system used during this study included culture in HTF on days 0 to 3, then in G2.2 or S2 on days 3.5 to 6. This is a variation on the currently advocated sequential systems, hence the possibility that the observed effects were partly a result of deviation from the established sequence. Although this possibility cannot be excluded, the culture system reported here produced an overall blastocyst formation rate similar to that reported by others (e.g. Gardner et al., 1998a,b). Moreover, a 49% implantation rate reflects the ability of this system to support development of viable blastocysts (Table II). Nevertheless, we are currently evaluating the use of G1.2/G2.2 (commercial preparation) in the blastocyst transfer programme.
The reduced ability to form blastocysts of normal appearance was in many instances already obvious at compaction, which occurs on day 4 of development in the human (Nikas et al., 1996). Regional compaction with exclusion of a number of cells and fragments from the morula occurred among fragmented embryos (Figure 1
). Blastocyst formation rate among normally compacted day 4 embryos was 46.8%, but this rate was reduced to 28.6% among regionally compacted embryos and 10.3% among embryos that did not show compaction on day 4 (data not shown). These data suggest that (i) the absence of compaction on day 4 is highly prognostic for normal blastocyst formation, and (ii) while regional compaction by itself does not preclude blastocyst formation, the extent to which exclusion occurs may determine development potential; we did not note the number of excluded cells when regional compaction occurred. Exclusion of cells in the blastocoelic cavity was also observed. This anomaly is associated with reduced embryo viability in sheep and cattle (Steen Willadsen, personal communication), but its impact on human embryo viability is uncertain.
Other abnormal features in human blastocysts described in this and other studies have been associated with reduced total cell count, low or absent HCG secretion in vitro (Dokras et al., 1993), and reduced implantation after intrauterine transfer (Jones et al., 1998
; Gardner et al., 2000
). Furthermore, total blastocyst cell count has been associated with hatching ability in vitro (Van Blerkom, 1993
).
The appearance of a single cleavage anomaly on day 2 and/or 3 of development led to a significant reduction in normal blastocyst formation on day 5. Only 17% of embryos with >15% fragmentation formed a morphologically normal blastocyst. Such embryos have been shown to develop into fetuses at a higher rate after assisted hatching, fragment removal, and uterine transfer on day 3 (Alikani et al., 1999). A further analysis for this study of 118 homogeneous transfers (all embryos transferred in one morphological category) involving embryos with >15% fragmentation revealed that ~18% implanted; on the other hand, the loss of ~80% of such embryos between day 3 and day 5 in vitro yields a theoretical day 5 implantation rate of ~8%. This suggests that the developmental potential of some of these embryos may be reduced in extended culture.
The 8% value seems to be in agreement with the ~5% implantation rate for fragmented embryos reported by a number of investigators (Staessen et al., 1993; Giorgetti et al., 1995
; Ziebe et al., 1997
). However, this low rate should be surprising, in view of the prevalence of fragmentation among in-vitro-generated embryos. Eighty per cent of all day 3 embryos in our database (40 438/49 801) had some degree of fragmentation. Also, fluorescence in-situ hybridization (FISH) analysis of embryos with
35% fragmentation shows that about one-half are normal for the number of chromosomes tested (S.Munné, personal communication), and aneuploidy is not a major abnormality among such embryos (Munné et al., 1995
; Marquez et al., 2000
). Instead, these embryos display different forms of mosaicism, some of which are compatible with normal development (reviewed by Munné and Cohen, 1998). Together, these findings suggest that fragmentation per se is not an abnormality. Moreover, with the exception of extreme cases where 40100% of the embryonic volume has been lost and chromosomal abnormality is almost certain, it has been shown that the potential of moderately fragmented embryos for implantation may be determined by the distribution and size of the fragments (Alikani and Cohen, 1995
; Alikani et al., 1999
; Antczak and Van Blerkom, 1999
). Here, reduced rates of normal compaction, cavitation, and blastocyst formation were observed in embryos with type IV fragments (large scattered fragments associated with one or more uneven cells) but not types I, II or III. If fragmentation results in the depletion of cortically positioned regulatory proteins essential to the embryo, as suggested by Antczak and Van Blerkom (1999), then type IV fragmented embryos may be specially affected, since the fragments are much larger than those in other types. These embryos had the lowest implantation rate after transfer on day 3 (Alikani et al., 1999
) but extended culture may reduce their potential even further as less than a quarter may survive to the blastocyst stage (Table V
).
We previously suggested that fragment removal may, at least in part, explain the high implantation rates we have reported for 635%, non-type IV fragmented embryos (Cohen et al., 1994; Alikani et al., 1999
). Whether fragment removal on day 2 or 3 can lead to better blastocyst formation in-vitro remains to be clarified, but there is some firm indication that both cleavage between days 2 and 3 (Zaninovic et al., 1999
) and compaction between days 3 and 4 (M.A., unpublished observation) are promoted if fragments are microsurgically removed.
Embryos that successfully underwent normal compaction, cavitation and ICM formation had significantly higher mean cell numbers on days 2 and 3 compared to those that failed to complete these developmental stages (Figure 4). Somewhat surprisingly, fast-cleaving embryos, particularly those with 910 cells on day 3, showed a reduced capacity to form normal blastocysts. In a study by Ziebe et al. (1997), the transfer of 4-cell embryos on day 2 resulted in a significantly higher pregnancy rate than the transfer of embryos beyond the 4-cell stage. Unusually fast-developing embryos may exhibit high levels of chromosomal aberration (Magli et al., 1998
), some of which are due to polyspermic fertilization (Harper et al., 1994
). These observations conflict with actual transfer results obtained in our Institute. In homogeneous transfers on day 3, the implantation rate of embryos with >9 cells was 32.4% (35/108) a high rate, albeit slightly lower than that of embryos with 79 cells 35.5% (1019/2874).
In a large study by Huisman et al. (1994), slow embryos showed lower implantation potential after 2, 3 or 4 days in culture. In the present study, prolonged culture of such embryos often led to abnormal patterns of compaction, specifically `fusion-like' compaction, and abnormal cavitation (Figure 1). By contrast, homogeneous transfer of slow embryos on day 3 (between 4 and 5 cells) produced an implantation rate of 22%, suggesting either a normalization of development rate after intrauterine transfer of some slow embryos, or the receptivity of the uterus to late blastocysts developed in-vivo.
The reduced capacity of embryos with MNB to form blastocysts was demonstrated by Balakier and Cadesky (1997). The majority of the embryos in that study arrested at 215 cells and only 14% formed morphologically normal blastocysts. It has also been shown that multinucleation leads to low implantation rates after day 3 transfer (Jackson et al., 1998; Pelinck et al., 1998
), as was observed in this study. Interestingly, multinucleation was the only cleavage anomaly that had a marked negative association with the development potential of embryos regardless of the duration of culture (Table V
).
Many of the embryos with multinucleate blastomeres failed to show signs of compaction. When they did compact, their attempt at cavitation often ended with the persistence of what should be only transitory structures (e.g. intracellular vacuoles) involved in the formation of the blastocoel (Gualtieri et al., 1992). Embryos with MNB had a lower cell number (on average 6.5) on day 3, and formed blastocysts at a rate of 15.9%; this figure is not different for the rate at which all embryos with <7 cells formed blastocysts (13.8%). So, it is possibly both chromosomal anomalies associated with multinucleation (Kligman et al., 1996
; Laverge et al., 1997
; Staessen and Van Steirteghem, 1998
) as well as their lower cell number that contribute to reduced blastocyst formation and reduced implantation in this group.
Under the conditions of this study, extended culture led to a reduction in viability of embryos with cleavage abnormalities. On this basis, and until further refinement of culture media, we suggest that extended culture should be limited to those embryos with optimal development during the first 3 days in culture. In the future, embryo selection based on oocyte and embryo polarity (Edwards and Beard, 1997), zygote morphology (Wright et al., 1990
; Sadowy et al., 1998
; Scott and Smith, 1998; Tesarik and Greco, 1999
) and expression of developmental genes linked to implantation (Steuerwald et al., 1999
) may offer an alternative to extended culture and the associated loss of potentially viable embryos.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 21, 2000; accepted on September 15, 2000.