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warmed day-5 embryos predicts rates of implantation, pregnancy and live birth
RBMOnline - Vol 19. No 1. 2009 72-78 Reproductive BioMedicine Online; www.rbmonline.com/Article/3730 on web 15 May 2009
Article Morphology of vitrified/warmed day-5 embryos predicts rates of implantation, pregnancy and live birth Thomas Ebner, PhD, graduated with honours from the Paris Lodron University of Salzburg, Austria, in 1992. His doctorate (1994) on cancer research was taken at the Institute for Pathology, General Hospital, Salzburg. After 2 years’ research at Salzburg University, he started in IVF in Linz. Completing his post-doctoral thesis, he became a university lecturer in Salzburg. He has published more than 60 papers. Research interests include non-invasive IVF selection processes, laser application, apoptosis, cryopreservation and culture media. He was certified as a senior clinical embryologist in 2008. Currently he is scientific director of the European School of Assisted Reproductive Technologies in Linz.
Dr Thomas Ebner T Ebner1,7, P Vanderzwalmen2,4, O Shebl1, W Urdl3, M Moser1, NH Zech5,6, G Tews1 1 Landes-Frauen-und Kinderklinik, IVF Unit, Linz, Upper Austria; 2Institute for Reproductive Medicine and Endocrinology, Bregenz, Vorarlberg; 3University of Graz, Klinische Abteilung für Gynäkologische Endokrinologie und Fortpflanzungsmedizin, Styria, Austria; 4Centre Hospitalier Inter Regional Cavell (CHIREC), Braine l´Alleud, Brussels, Belgium; 5Reproductive Genetics Institute, Chicago, Illinois, USA; 6University Hospital Zürich, Department of Obstetrics, Zürich, Switzerland 7 Correspondence: e-mail: [email protected]
Abstract Although some post-thaw morphological predictors of pregnancy have been investigated in slow freezing of blastocysts, no such data have been published for vitrified and warmed blastocysts. Therefore, a prospective four-part score was applied to vitrified/warmed day-5 embryos to evaluate whether certain morphological parameters could serve as predictors of implantation, pregnancy and live birth. All morulae/blastocysts that were considered to be viable after warming were scored according to a previously unpublished grading system based on re-expansion, hatching (out of an artificial gap in the zona pellucida), extensive cytoplasmic granulation and presence of necrotic foci. Overall, 74% (202/273) of the vitrified concepti were found to be viable after warming. Early blastocysts showed better survival versus extended/hatching blastocysts (P < 0.01). Of the morphological parameters analysed, immediate re-expansion (P < 0.05) and hatching (P < 0.001) were positive predictors of the rates of implantation, pregnancy and live birth. The opposite holds for extensive cytoplasmic granulation (P < 0.05), which was negatively related. Accurate scoring of warmed blastocysts (within the first 2 h) allows for prediction of pregnancy outcome, and thus will help to further reduce the number of transferred embryos. Keywords: blastocyst, blastocyst quality, cytoplasm, hatching, re-expansion, vitrification
Introduction
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More detailed blastocyst scoring systems allow for better prediction of implantation, and. as a consequence, better scoring systems should lead to a reduction in the number of blastocysts considered for transfer (Gardner et al., 2000). This strategy, however, increases the number of supernumerary blastocysts in culture, and if they are scored as having a good prognosis for cryosurvival, these day-5 concepti should be stored in liquid nitrogen. Approximately 15 years after the successful cryopreservation of a human blastocyst (Cohen et al., 1985), two major approaches have been developed and are currently applicable in routine IVF work.
Slow freezing is a safe and feasible option in human blastocyst cryopreservation, and results in adequate survival and pregnancy rates (Gardner et al., 2003; Van den Abbeel et al., 2005). However, as vitrification offers some obvious benefits compared with slow freezing, reports favouring this rapid freezing technique have become more frequent in the literature (Cho et al., 2002; Mukaida, 2003; Son et al., 2003; Vanderzwalmen et al., 2003, 2006), indicating that this technique for blastocysts is equivalent to (Liebermann and Tucker, 2006) or even better than (Stehlik et, 2004; Youssry et al., 2008) slow freezing.
© 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
Although some post-thaw predictive morphological parameters have been investigated in slow freezing of blastocysts (Desai and Goldfarb, 2005; Van den Abbeel et al., 2005; Shu et al., 2008), e.g. immediate re-expansion or 24-h survival, no such data have yet been published for vitrified blastocysts. This led to the introduction and prospective application of a four-part scoring system to vitrified/warmed blastocysts, in order to evaluate whether certain morphological parameters could serve as predictors of implantation, pregnancy, and live birth.
Materials and methods In the present study all consecutive transfers of the LandesFrauen und Kinderklinik Linz deriving from vitrified/warmed blastocysts (n = 129) were prospectively analysed (June 2006 to December 2007). As the routine strategy in the present laboratory is to cryopreserve only day-5 embryos, all patients involved in this study either showed an adequate response to ovarian stimulation, or had surplus embryos of good quality after transfer at cleavage stage. Institutional Review Board approval was not required.
Corresponding fresh cycles In their corresponding fresh cycles, patients (33.9 ± 4.2 years) were either stimulated with a long agonist (n = 40) or an antagonist (n = 89) protocol, both of which allowed collection of a comparable number of oocytes. The agonist protocol was performed with a combination of buserelin (Suprecur; Aventis Pharma, Vienna, Austria) and human menopausal gonadotrophin (Menogon; Ferring, Kiel, Germany); the antagonist approach used a gonadotrophin-releasing hormone antagonist (Cetrotide; Merck Serono, Vienna, Austria) and recombinant FSH (Puregon; Organon, Vienna, Austria). In 28% (n = 36) of the cycles conventional IVF was applied, whereas in the remaining patients intracytoplasmic injection of ejaculated spermatozoa was performed. Three patients had no embryo/blastocyst transfer procedure, due to high oestradiol concentration and the risk of ovarian hyperstimulation syndrome (OHSS). Of the remaining 126 cycles, almost half of the women had a blastocyst transfer (n = 60); the remainder had a cleavage stage transfer (day 3). Thus, depending on the outcome of the corresponding fresh cycle, embryos that were vitrified on day 5 were kept in liquid nitrogen for periods of time that ranged from 2 months (e.g. no fresh transfer due to OHSS) and 3 years (in case of pregnancy). The fresh cycles resulted in a clinical pregnancy rate of 43%. Although the time of storage differed slightly, all of the embryos that were vitrified were cultured under the same conditions, allowing for adequate comparison of their morphology. In more detail, embryos were initially incubated in groups in 80 μl drops of BlastAssist System Medium 1 (MediCult, Copenhagen, Denmark) for the first 2 days. On day 3, medium was changed to BlastAssist System Medium 2 (MediCult).
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Vitrification Prior to vitrification, all blastocysts were scored according to the guidelines of Gardner and Schoolcraft (1999), focusing on expansion, inner cell mass and trophectoderm. Only adequate morulae/blastocysts were considered for cryostorage. According to in-house definitions, an adequate quality was reached if morulae or early blastocysts had only minor fragmentation and fully expanded blastocysts showed either a perfect inner cell mass (ICM) and/or trophectoderm (TE) (with none of these cell types of poorest quality ‘C’ according to the Gardner score). Artificial reduction of the blastocoelic cavity prior to vitrification (Vanderzwalmen et al., 2002) was not performed in expanded blastocysts. In addition, images of the blastocysts considered for vitrification were prospectively stored (Octax EyeWare; MTG, Altdorf, Germany) in order to facilitate morphological comparison after warming. In all cases, vitrification was carried out utilizing a commercially available kit (VitriFreeze; FertiPro, Beernem, Belgium). All morulae/blastocysts were pre-incubated at room temperature in a medium containing phosphate-buffered saline (PBS) and human serum albumin (HSA) for 1 min. This step was followed by two incubations in vitrification media with different compositions. The duration of time that blastocysts were kept in the first medium [PBS, HSA, ethylene glycol and dimethyl sulphoxide (DMSO)] depended on the individual degree of expansion. In detail, morulae and early blastocysts were incubated for 1 min, full blastocysts for 2 min and expanded or hatching blastocysts for 3 min. As compared with vitrification medium 1, medium 2 had higher concentrations of cryoprotectant (PBS, HSA, ethylene glycol, DMSO and sucrose), and the exposure time was therefore kept to a minimum (30 s). Within this last crucial period, shrunken morulae/blastocysts had to be placed on the tip of a semi-straw (VitroPlug; Astro Med Tec, Salzburg, Austria) in very small volumes of vitrification medium 2 (<0.5 μl), followed by direct plunging into liquid nitrogen. Before storage in a special container (Arpege 170; Air Liquid, Vienna, Austria) all semi-straws were sealed with high security straws (Cryo Bio System, L’Aigle, France). It should be noted that blastocysts from fully expanded stage onwards were individually vitrified, whereas morulae and early blastocysts were placed as a pair on the semi-straw.
Warming Special care was taken to ensure very high warming rates, which required rapid separation of the semi- and the protective high security straw followed by immediate plunging into the first warming solution. Using the VitriThaw kit (FertiPro, Beernem, Belgium) at room temperature all concepti involved in this study were warmed in four thawing media (based on PBS and HSA) of descending concentrations of sucrose (0.5, 0.25, 0.125, 0 mol/l). This was followed by direct transfer into pre-warmed medium (BlastAssist; MediCult) for intrauterine blastocyst transfer.
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Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
All warmed blastocysts (except those that had started to leave the zona before vitrification) had their zonae opened by means of a laser in order to minimize any negative impact of cryopreservation on zona pellucida constitution (Vanderzwalmen et al., 2003; Liebermann and Tucker, 2006). In order to allow for an exact analysis of viability (e.g. showing distinct cell membranes, no discolouration) after vitrification (Figures 1, 2a,b), warmed blastocysts were kept in culture for 2 h prior to scoring and transfer. All blastocysts considered to be viable after warming were scored according to a previously unpublished grading system based on re-expansion (Figures 1, 2a), hatching out of the artificial gap in the zona pellucida (Figure 1), cytoplasmic granulation (Figure 2a) and presence of necrotic foci (Figure 1). It should be noted that morulae or early blastocysts were considered to be re-expanded when they reached their original size prior to vitrification. Cytoplasmic granulation should be homogeneous and never localized, whereas necrotic foci comprised a maximum of two or three cells.
Statistics Subsequently, these parameters were related to rates of implantation and pregnancy focusing exclusively on single blastocyst transfers and double transfers homogeneous for the prevailing morphology. Thus, predictive values of certain morphological attributes could be accurately estimated. Comparison was performed using chi-squared and Mann−Whitney U hypothesis tests. Statistical significance was defined as P < 0.05.
Results After fresh transfer, or in case of OHSS, a total of 400 surplus morulae/blastocysts were vitrified in 129 study patients (3.1 ± 2.0; range: 1–10). Of these cryopreserved day-5 embryos, 273 morulae/blastocysts were considered for warming during the study period. Although all of them (100%) could be retrieved, only 202 were found
Figure 1. Re-expanded blastocyst hatching out of an artificial gap created by laser pulses. One trophectodermal cell did not survive vitrification (9 o’clock position).
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Figure 2. Blastocyst showing suspicious cytoplasmic granulation after vitrification and warming. (a) Surviving blastocyst with cell membranes still identifiable (arrows). (b) Degenerating blastocyst with no functional cell membranes present. RBMOnline®
Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
to be viable immediately after warming (74%). As indicated in Table 1, a significant association between blastocyst size (before vitrification) and survival after warming was observed. In detail, morulae/early blastocysts (ca. 160 μm) showed a better survival rate versus expanded (ca. 180 μm) or hatched blastocysts (P < 0.01). Of all morulae/blastocysts found to be viable after warming, 147 (72.8%) showed re-expansion within 2 h in culture, and 75 (37.1%) hatched from the artificial gap. Less than 20% were found to be positive for cytoplasmic granulation (n = 38) and necrotic foci (n = 40). Table 1 indicates that hatching of embryos was related to the degree of blastocyst expansion (P < 0.001). In addition, morulae or early blastocysts revealed granular cytoplasm more frequently than expanded ones (P < 0.01). Overall, 57 pregnancies were achieved (44.2%). However, one ectopic pregnancy, three biochemical pregnancies, and five
delayed miscarriages reduced the live birth rate to 37.2%. The corresponding implantation rate was 33.7% (68/202). Focusing on single blastocyst transfers and on transfers of two blastocysts of equivalent blastocoele expansion (68 elective single and 45 double blastocyst transfers) it was demonstrated (Table 2) that there was no relationship between the size of the blastocyst and the rates of pregnancy and implantation. It was also found (Table 3) that re-expansion (P < 0.05) and spontaneous hatching after warming (P < 0.01) can be considered as positive predictors of outcome. When both positive prognostic markers, re-expansion and hatching, were combined, every second blastocyst (52.2%) implanted. In contrast, abnormal dense granulation after warming (P < 0.05) was a negative predictor. The presence of necrotic foci in either ICM or TE did not affect implantation behaviour.
Table 1. Survival and morphology of vitrified day-5 embryos of different expansion.
No. warmed Immediate survival Re-expansion Hatching Granulation Necrotic foci
Morula or early blastocyst
Full blastocyst
Expanded or hatching blastocyst
86 72 (83.7)a 52 (72.2) 6 (8.3)b 21 (29.2)d 14 (19.4)
53 39 (73.6) 25 (64.1) 8 (20.5)c 8 (20.5) 7 (17.9)
134 91 (67.9)a 70 (76.9) 61 (67.0)b,c 9 (9.9)d 19 (20.9)
Values in parentheses are percentages. Values with the same superscript letter are significantly different: aP < 0.01; bP < 0.001; c
P < 0.001; dP < 0.01.
Table 2. Rates of pregnancy, implantation and live birth in vitrified and warmed transfers homogeneous for blastocyst size (68 elective single and 45 double blastocyst transfers).
Patients Pregnancy rate Clinical pregnancy Live birth rate Implantation rate
Morula or early blastocyst
Full blastocyst
Expanded or hatching blastocyst
42 20 (47.6) 19 (45.2) 17 (40.5) 22/60 (36.7)
23 7 (30.4) 7 (30.4) 6 (26.1) 10/33 (30.3)
48 25 (52.1) 23 (47.9) 23 (47.9) 29/65 (44.6)
Values in parentheses are percentages. There were no statistically significant differences.
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Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
Table 3. Morphological aspects of vitrified blastocysts after thawing and 2-h culture. Only single blastocyst transfers and homogeneous double blastocyst transfers were considered. n Re-expansion Alla Nonea Precocious hatching All None Granulation Allf Nonef Necrotic foci All None
β-HCG
Clinical PR Live birth
Implantation rate
81 21
45 (55.6) 6 (28.6)
39 (48.2) 5 (23.8)
38 (46.9) 4 (19.0)
53/121 (43.8) 6/29 (20.7)
30 77
21 (70.0)b 27 (35.1)b
19 (63.3)c 22 (28.6)c
19 (63.3)d 22 (28.6)d
23/39 (59.0)e 31/125 (24.8)e
18 102
4 (22.2) 50 (49.0)
3 (16.7) 43 (42.2)
3 (16.7) 42 (41.2)
4/26 (15.4) 59/156 (37.8)
19 93
8 (42.1) 42 (45.2)
7 (36.8) 35 (37.6)
7 (36.8) 35 (37.6)
8/25 (32.0) 47/143 (32.9)
Values in parentheses are percentages. Values with the same superscript letter are significantly different: a,fP < 0.05; bP < 0.01; cP < 0.001; d,eP < 0.0001. All = patients with transfer of day-5 embryos that were exclusively positive for the given phenomenon. None = patients with transfer of day-5 embryos that were exclusively negative for the given phenomenon. HCG = human chorionic gonadotrophin; PR = pregnancy rate.
Discussion
difficult to assess and score a vitrified blastocyst after warming than a fresh one (Shu et al., 2008).
Several factors (unrelated to the method of cryopreservation) are known directly to influence the fate of a cryopreserved blastocyst after warming/thawing and transfer. It is important to realize that survival rates in the literature may not be comparable, due to the fact that some embryologists focus on immediate survival while others suggest an additional waiting period of 24 h to monitor survival and growth (Vanderzwalmen et al., 2003). Differences in implantation rates may be attributed to the fact that not all groups apply assisted hatching to the thawed blastocysts (whilst collapsed), although this was found to improve outcome (Vanderzwalmen et al., 2003).
Re-expansion of the blastocoele (and consequently the blastocyst) after warming is expected within 24 h after thawing (Vanderzwalmen et al., 2003; Van den Abbeel et al., 2005). However, immediate re-expansion, e.g. within the first 2 h after warming, has not been used for prognostic purposes in vitrified blastocysts. In slow freezing, however, approximately half of the frozen blastocysts (197/402) did re-expand after 2–4 h in culture (Shu et al., 2008). Present results indicate that a higher rate of re-expansion might be observed using vitrification.
Most importantly, the morphology of the blastocyst will have a significant impact on survival; therefore, only blastocysts with good to moderate cell lineages will usually be considered for cryostorage. Nevertheless, cryosurvival of morphologically inconspicuous blastocysts may also fail if they derive from a cohort of poor quality day-3 embryos (Vanderzwalmen et al., 2003). There is some evidence in the literature that the efficiency of the vitrification method may depend on the expansion of the blastocyst, with better survival in morulae or early blastocyst stages compared with full or expanded blastocysts (Vanderzwalmen et al., 1999; 2002; Son et al., 2003; Zech et al., 2005). This phenomenon could, theoretically, be related to the size of the blastocoele, which in turn is related to the volume of watery fluid within the blastocyst. Present data are in line with this hypothesis, and routinely bringing forward the day of vitrification to compaction stage (exclusively vitrifying adequate concepti) could be considered (Ebner et al., 2008).
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As a result of the presence and size of the blastocoelic cavity, vitrified−warmed blastocysts experience several morphological changes and collapse during cryopreservation. Thus, it is more
Table 3 indicates that failure of the re-expansion process in vitrified/warmed blastocysts is associated with significantly reduced rates of implantation (P = 0.022) and clinical pregnancy (P = 0.021), suggesting that rapid blastocoelic re-expansion may also be an indicative marker of viability after vitrification. Even if it might be assumed that all viable blastocysts will re-expand after several more hours, any delay in this process could be a manifestation of altered osmotic and/or metabolic conditions. These events are comparable with the situation found during blastocoele development, when water enters the blastocoelic cavity via tight junctions, either diffusing passively or being pumped actively (Veeck and Zaninoviv, 2003). It may be postulated that in blastocysts with delayed reexpansion, vitrification may have influenced their permeability to water. This could either be due to a cryoartefact per se, eventually causing a delay in active and efficient pumping, or to laser manipulation during assisted hatching, although the latter assumption is less likely, since zona drilling is usually performed immediately after warming, when the blastocyst is completely shrunk. One morphological feature of warmed blastocysts that often goes with re-expansion is precocious hatching of the blastocyst RBMOnline®
Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
through the artificial gap created by laser pulses. Quite logically, the probability that a blastocyst will hatch is likely to be increased if the conceptus is already re-expanded. However, even shrunken blastocysts tend to leave their outer shell if the position within the zona is close to the opening. Present data suggest that double clinical pregnancy rates can be achieved (63%) if transferred blastocysts have already started to hatch as compared with blastocysts without (29%) this morphological attribute (Table 3). Blebbing out of the artificial gap (2 h after thawing) obviously characterizes a subgroup of blastocysts that can hatch completely without being trapped within the zona. In contrast to previous positive predictors, necrotic foci in the blastocyst are considered to have a negative impact on further development. Similar to cryopreservation at earlier cleavage stages, areas of necrosis in warmed blastocysts mark cells that did not survive vitrification. There is general agreement that at earlier stages at least 50% of the blastomeres have to survive in order to assess a thawed embryo as viable (Rienzi et al., 2002; Archer et al., 2003). Recently, it was shown that blastomere loss after thawing in day-2 embryos has a detrimental effect on blastocyst formation and cell number (Archer et al., 2003). Partially damaged day-3 embryos can be rescued if the necrotic blastomeres are removed prior to transfer (Rienzi et al., 2002, 2005). However, prior to compaction, cell−cell adhesion can be neglected and does not reflect cell fusion in blastocysts. In other words, removal of necrotic cells is impossible on day 5 and would possibly harm the affected cell lineages. It has to be stressed that in the present data set, only minor injuries were observed (one to three cells affected by necrosis), thus removal of necrotic cells would not bring any benefit. This is the first evidence demonstrating that partial damage of the blastocyst caused by vitrification does not reduce rates of implantation and pregnancy. This is made all the more interesting by the fact that whether ICM or TE was affected played no role. Obviously, the blastocyst can compensate for minor injuries as a result of vitrification. This seems not to be the case if the whole cytoplasm is harmed by vitrification. Occasionally, blastocysts show extensive granular cytoplasm immediately after thawing instead of an expected homogeneous appearance. Although these blastocysts could be considered for transfer once they have recovered, their implantation potential seems to be limited. This conspicuous morphological feature is different from previous cytoplasmic irregularities such as cytoplasmic pitting, the manifestation of which is probably culture dependent (Ebner et al., 2005). The granular cytoplasm sometimes observed after vitrification is characterized by a halo-like structure in the periphery of the cells (which is most likely to consist of water) and a dominant accumulation of some cytoskeletal components (Figure 2a). Warmed blastocysts showing this phenomenon have a reduced capacity to survive vitrification. In the presence of this cytoplasmic anomaly, blastocysts with a good prognosis for subsequent survival may be distinguished from those with a bad prognosis (Figure 2b) by a healthy appearance of the cell membranes (Zhu et al., 2001), which should appear as a small intact line (Figure 2a).
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It should be clearly stated that the present morphological data are exclusively based on vitrification of day-5 embryos (asthis is the routine technique in the authors’ laboratories), and theoretically, morphology of thawed day-5 embryos after slow freezing might differ slightly. However, this is the first evidence that accurate scoring of vitrified and warmed day-5 embryos allows adequate prediction of pregnancy outcome. Subsequent scoring, based on the suggested morphological parameters, could improve cumulative pregnancy rate and help to further reduce the number of transferred embryos, thus limiting multiple pregnancy rates.
References Archer J, Gook DA, Edgar DH 2003 Blastocyst formation and cell numbers in human frozen−thawed embryos following extended culture. Human Reproduction 18, 1669–1673. Cho HJ, Son WY, Yoon SH et al. 2002 An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Human Reproduction 17, 2419–2422. Cohen J, Simons R, Fehilly C et al. 1985 Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage. Lancet 1, 647. Desai N, Goldfarb J 2005 Examination of frozen cycles with replacement of a single thawed blastocyst. Reproductive BioMedicine Online 11, 349–354. Ebner T, Moser M, Shebl O et al. 2008 Morphological analysis at compacting stage is a valuable prognostic tool in ICSI patients. Reproductive BioMedicine Online 18, 61–66. Ebner T, Tews G, Sommergruber M et al. 2005 Cytoplasmic pitting has a negative influence on implantation outcome. Journal of Assisted Reproduction and Genetics 22, 239–244. Gardner DK, Schoolcraft WB 1999 In vitro culture of human blastocysts. In: Jansen R, Mortimer D (eds) Towards Reproductive Certainty: Infertility and Genetics Beyond 1999. Parthenon Press, Carnforth, pp. 378–388. Gardner DK, Lane M, Stevens J et al. 2003 Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertility and Sterility 79, 407–410. Gardner DK, Lane M, Stevens J et al. 2000 Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertility and Sterility 73, 1155–1158. Liebermann J, Tucker MJ 2006 Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertility and Sterility 86, 20–26. Mukaida T, Nakamura S, Tomiyama T et al. 2003 Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Human Reproduction 18, 384–391. Rienzi L, Ubaldi F, Iacobelli M et al. 2005 Developmental potential of fully intact and partially damaged cryopreserved embryos after laser-assisted removal of necrotic blastomeres and postthaw culture selection. Fertility and Sterility 84, 888–894. Rienzi L, Nagy ZP, Ubaldi F et al. 2002 Laser-assisted removal of necrotic blastomeres from cryopreserved embryos that were partially damaged. Fertility and Sterility 77, 1196–1201. Shu Y, Watt J, Gebhardt J et al. 2008 The value of fast blastocyst re-expansion in the selection of a viable thawed blastocyst for transfer. Fertility and Sterility 91, 401–406. Son WY, Yoon SH, Yoon HJ et al. 2003 Pregnancy outcome following transfer of human blastocysts vitrified on electron microscopy grids after induced collapse of the blastocoele. Human Reproduction 18, 137–139. Stehlik E, Stehlik J, Katayama KP et al. 2005 Vitrification demonstrates significant improvement versus slow freezing of human blastocysts. Reproductive BioMedicine Online 11, 53–57.
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Van den Abbeel E, Camus M, Verheyen G et al. 2005 Slow controlledrate freezing of sequentially cultured human blastocysts: an evaluation of two freezing strategies. Human Reproduction 20, 2929–2945. Vanderzwalmen P, Zech N, Greindl AJ et al. 2006 Cryopreservation of human embryos by vitrification. Gynecology Obstetrique é Fertilite 34, 760–769. Vanderzwalmen P, Bertin G, Debauche C et al. 2003 Vitrification of human blastocysts with the Hemi-straw carrier: application of assisted hatching after thawing. Human Reproduction 18, 1504–1511. Vanderzwalmen P, Bertin G, Debauche C et al. 2002 Births after vitrification at morula and blastula stages: effect of artificial reduction of the blastocoelic cavity before vitrification. Human Reproduction 17, 744–751. Veeck LL, Zaninovic N 2003 Human blastocysts in vitro. In: Veeck L, Zaninovic N (eds) An Atlas of Human blastocysts. Parthenon Publishing, Boca Raton, London, New York, Washington, pp. 99–137.
Youssry M, Ozmen B, Zohni K et al. 2008 Current aspects of blastocyst cryopreservation. Reproductive BioMedicine Online 16, 311–320. Zech NH, Lejeune B, Zech H et al. 2005 Vitrification of hatching and hatched human blastocysts: effect of an opening in the zona pellucida before vitrification. Reproductive BioMedicine Online 11, 355–361. Zhu SE, Zeng SM, Yu WL et al. 2003 Vitrification of in vivo and in vitro produced bovine blastocysts. Animal Biotechnology 12, 193–203.
Declaration: The authors report no financial or commercial conflicts of interest. Received 17 June 2008; refereed 12 August 2008; accepted 11 February 2009.
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Article Morphology of vitrified/warmed day-5 embryos predicts rates of implantation, pregnancy and live birth Thomas Ebner, PhD, graduated with honours from the Paris Lodron University of Salzburg, Austria, in 1992. His doctorate (1994) on cancer research was taken at the Institute for Pathology, General Hospital, Salzburg. After 2 years’ research at Salzburg University, he started in IVF in Linz. Completing his post-doctoral thesis, he became a university lecturer in Salzburg. He has published more than 60 papers. Research interests include non-invasive IVF selection processes, laser application, apoptosis, cryopreservation and culture media. He was certified as a senior clinical embryologist in 2008. Currently he is scientific director of the European School of Assisted Reproductive Technologies in Linz.
Dr Thomas Ebner T Ebner1,7, P Vanderzwalmen2,4, O Shebl1, W Urdl3, M Moser1, NH Zech5,6, G Tews1 1 Landes-Frauen-und Kinderklinik, IVF Unit, Linz, Upper Austria; 2Institute for Reproductive Medicine and Endocrinology, Bregenz, Vorarlberg; 3University of Graz, Klinische Abteilung für Gynäkologische Endokrinologie und Fortpflanzungsmedizin, Styria, Austria; 4Centre Hospitalier Inter Regional Cavell (CHIREC), Braine l´Alleud, Brussels, Belgium; 5Reproductive Genetics Institute, Chicago, Illinois, USA; 6University Hospital Zürich, Department of Obstetrics, Zürich, Switzerland 7 Correspondence: e-mail: [email protected]
Abstract Although some post-thaw morphological predictors of pregnancy have been investigated in slow freezing of blastocysts, no such data have been published for vitrified and warmed blastocysts. Therefore, a prospective four-part score was applied to vitrified/warmed day-5 embryos to evaluate whether certain morphological parameters could serve as predictors of implantation, pregnancy and live birth. All morulae/blastocysts that were considered to be viable after warming were scored according to a previously unpublished grading system based on re-expansion, hatching (out of an artificial gap in the zona pellucida), extensive cytoplasmic granulation and presence of necrotic foci. Overall, 74% (202/273) of the vitrified concepti were found to be viable after warming. Early blastocysts showed better survival versus extended/hatching blastocysts (P < 0.01). Of the morphological parameters analysed, immediate re-expansion (P < 0.05) and hatching (P < 0.001) were positive predictors of the rates of implantation, pregnancy and live birth. The opposite holds for extensive cytoplasmic granulation (P < 0.05), which was negatively related. Accurate scoring of warmed blastocysts (within the first 2 h) allows for prediction of pregnancy outcome, and thus will help to further reduce the number of transferred embryos. Keywords: blastocyst, blastocyst quality, cytoplasm, hatching, re-expansion, vitrification
Introduction
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More detailed blastocyst scoring systems allow for better prediction of implantation, and. as a consequence, better scoring systems should lead to a reduction in the number of blastocysts considered for transfer (Gardner et al., 2000). This strategy, however, increases the number of supernumerary blastocysts in culture, and if they are scored as having a good prognosis for cryosurvival, these day-5 concepti should be stored in liquid nitrogen. Approximately 15 years after the successful cryopreservation of a human blastocyst (Cohen et al., 1985), two major approaches have been developed and are currently applicable in routine IVF work.
Slow freezing is a safe and feasible option in human blastocyst cryopreservation, and results in adequate survival and pregnancy rates (Gardner et al., 2003; Van den Abbeel et al., 2005). However, as vitrification offers some obvious benefits compared with slow freezing, reports favouring this rapid freezing technique have become more frequent in the literature (Cho et al., 2002; Mukaida, 2003; Son et al., 2003; Vanderzwalmen et al., 2003, 2006), indicating that this technique for blastocysts is equivalent to (Liebermann and Tucker, 2006) or even better than (Stehlik et, 2004; Youssry et al., 2008) slow freezing.
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Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
Although some post-thaw predictive morphological parameters have been investigated in slow freezing of blastocysts (Desai and Goldfarb, 2005; Van den Abbeel et al., 2005; Shu et al., 2008), e.g. immediate re-expansion or 24-h survival, no such data have yet been published for vitrified blastocysts. This led to the introduction and prospective application of a four-part scoring system to vitrified/warmed blastocysts, in order to evaluate whether certain morphological parameters could serve as predictors of implantation, pregnancy, and live birth.
Materials and methods In the present study all consecutive transfers of the LandesFrauen und Kinderklinik Linz deriving from vitrified/warmed blastocysts (n = 129) were prospectively analysed (June 2006 to December 2007). As the routine strategy in the present laboratory is to cryopreserve only day-5 embryos, all patients involved in this study either showed an adequate response to ovarian stimulation, or had surplus embryos of good quality after transfer at cleavage stage. Institutional Review Board approval was not required.
Corresponding fresh cycles In their corresponding fresh cycles, patients (33.9 ± 4.2 years) were either stimulated with a long agonist (n = 40) or an antagonist (n = 89) protocol, both of which allowed collection of a comparable number of oocytes. The agonist protocol was performed with a combination of buserelin (Suprecur; Aventis Pharma, Vienna, Austria) and human menopausal gonadotrophin (Menogon; Ferring, Kiel, Germany); the antagonist approach used a gonadotrophin-releasing hormone antagonist (Cetrotide; Merck Serono, Vienna, Austria) and recombinant FSH (Puregon; Organon, Vienna, Austria). In 28% (n = 36) of the cycles conventional IVF was applied, whereas in the remaining patients intracytoplasmic injection of ejaculated spermatozoa was performed. Three patients had no embryo/blastocyst transfer procedure, due to high oestradiol concentration and the risk of ovarian hyperstimulation syndrome (OHSS). Of the remaining 126 cycles, almost half of the women had a blastocyst transfer (n = 60); the remainder had a cleavage stage transfer (day 3). Thus, depending on the outcome of the corresponding fresh cycle, embryos that were vitrified on day 5 were kept in liquid nitrogen for periods of time that ranged from 2 months (e.g. no fresh transfer due to OHSS) and 3 years (in case of pregnancy). The fresh cycles resulted in a clinical pregnancy rate of 43%. Although the time of storage differed slightly, all of the embryos that were vitrified were cultured under the same conditions, allowing for adequate comparison of their morphology. In more detail, embryos were initially incubated in groups in 80 μl drops of BlastAssist System Medium 1 (MediCult, Copenhagen, Denmark) for the first 2 days. On day 3, medium was changed to BlastAssist System Medium 2 (MediCult).
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Vitrification Prior to vitrification, all blastocysts were scored according to the guidelines of Gardner and Schoolcraft (1999), focusing on expansion, inner cell mass and trophectoderm. Only adequate morulae/blastocysts were considered for cryostorage. According to in-house definitions, an adequate quality was reached if morulae or early blastocysts had only minor fragmentation and fully expanded blastocysts showed either a perfect inner cell mass (ICM) and/or trophectoderm (TE) (with none of these cell types of poorest quality ‘C’ according to the Gardner score). Artificial reduction of the blastocoelic cavity prior to vitrification (Vanderzwalmen et al., 2002) was not performed in expanded blastocysts. In addition, images of the blastocysts considered for vitrification were prospectively stored (Octax EyeWare; MTG, Altdorf, Germany) in order to facilitate morphological comparison after warming. In all cases, vitrification was carried out utilizing a commercially available kit (VitriFreeze; FertiPro, Beernem, Belgium). All morulae/blastocysts were pre-incubated at room temperature in a medium containing phosphate-buffered saline (PBS) and human serum albumin (HSA) for 1 min. This step was followed by two incubations in vitrification media with different compositions. The duration of time that blastocysts were kept in the first medium [PBS, HSA, ethylene glycol and dimethyl sulphoxide (DMSO)] depended on the individual degree of expansion. In detail, morulae and early blastocysts were incubated for 1 min, full blastocysts for 2 min and expanded or hatching blastocysts for 3 min. As compared with vitrification medium 1, medium 2 had higher concentrations of cryoprotectant (PBS, HSA, ethylene glycol, DMSO and sucrose), and the exposure time was therefore kept to a minimum (30 s). Within this last crucial period, shrunken morulae/blastocysts had to be placed on the tip of a semi-straw (VitroPlug; Astro Med Tec, Salzburg, Austria) in very small volumes of vitrification medium 2 (<0.5 μl), followed by direct plunging into liquid nitrogen. Before storage in a special container (Arpege 170; Air Liquid, Vienna, Austria) all semi-straws were sealed with high security straws (Cryo Bio System, L’Aigle, France). It should be noted that blastocysts from fully expanded stage onwards were individually vitrified, whereas morulae and early blastocysts were placed as a pair on the semi-straw.
Warming Special care was taken to ensure very high warming rates, which required rapid separation of the semi- and the protective high security straw followed by immediate plunging into the first warming solution. Using the VitriThaw kit (FertiPro, Beernem, Belgium) at room temperature all concepti involved in this study were warmed in four thawing media (based on PBS and HSA) of descending concentrations of sucrose (0.5, 0.25, 0.125, 0 mol/l). This was followed by direct transfer into pre-warmed medium (BlastAssist; MediCult) for intrauterine blastocyst transfer.
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Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
All warmed blastocysts (except those that had started to leave the zona before vitrification) had their zonae opened by means of a laser in order to minimize any negative impact of cryopreservation on zona pellucida constitution (Vanderzwalmen et al., 2003; Liebermann and Tucker, 2006). In order to allow for an exact analysis of viability (e.g. showing distinct cell membranes, no discolouration) after vitrification (Figures 1, 2a,b), warmed blastocysts were kept in culture for 2 h prior to scoring and transfer. All blastocysts considered to be viable after warming were scored according to a previously unpublished grading system based on re-expansion (Figures 1, 2a), hatching out of the artificial gap in the zona pellucida (Figure 1), cytoplasmic granulation (Figure 2a) and presence of necrotic foci (Figure 1). It should be noted that morulae or early blastocysts were considered to be re-expanded when they reached their original size prior to vitrification. Cytoplasmic granulation should be homogeneous and never localized, whereas necrotic foci comprised a maximum of two or three cells.
Statistics Subsequently, these parameters were related to rates of implantation and pregnancy focusing exclusively on single blastocyst transfers and double transfers homogeneous for the prevailing morphology. Thus, predictive values of certain morphological attributes could be accurately estimated. Comparison was performed using chi-squared and Mann−Whitney U hypothesis tests. Statistical significance was defined as P < 0.05.
Results After fresh transfer, or in case of OHSS, a total of 400 surplus morulae/blastocysts were vitrified in 129 study patients (3.1 ± 2.0; range: 1–10). Of these cryopreserved day-5 embryos, 273 morulae/blastocysts were considered for warming during the study period. Although all of them (100%) could be retrieved, only 202 were found
Figure 1. Re-expanded blastocyst hatching out of an artificial gap created by laser pulses. One trophectodermal cell did not survive vitrification (9 o’clock position).
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Figure 2. Blastocyst showing suspicious cytoplasmic granulation after vitrification and warming. (a) Surviving blastocyst with cell membranes still identifiable (arrows). (b) Degenerating blastocyst with no functional cell membranes present. RBMOnline®
Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
to be viable immediately after warming (74%). As indicated in Table 1, a significant association between blastocyst size (before vitrification) and survival after warming was observed. In detail, morulae/early blastocysts (ca. 160 μm) showed a better survival rate versus expanded (ca. 180 μm) or hatched blastocysts (P < 0.01). Of all morulae/blastocysts found to be viable after warming, 147 (72.8%) showed re-expansion within 2 h in culture, and 75 (37.1%) hatched from the artificial gap. Less than 20% were found to be positive for cytoplasmic granulation (n = 38) and necrotic foci (n = 40). Table 1 indicates that hatching of embryos was related to the degree of blastocyst expansion (P < 0.001). In addition, morulae or early blastocysts revealed granular cytoplasm more frequently than expanded ones (P < 0.01). Overall, 57 pregnancies were achieved (44.2%). However, one ectopic pregnancy, three biochemical pregnancies, and five
delayed miscarriages reduced the live birth rate to 37.2%. The corresponding implantation rate was 33.7% (68/202). Focusing on single blastocyst transfers and on transfers of two blastocysts of equivalent blastocoele expansion (68 elective single and 45 double blastocyst transfers) it was demonstrated (Table 2) that there was no relationship between the size of the blastocyst and the rates of pregnancy and implantation. It was also found (Table 3) that re-expansion (P < 0.05) and spontaneous hatching after warming (P < 0.01) can be considered as positive predictors of outcome. When both positive prognostic markers, re-expansion and hatching, were combined, every second blastocyst (52.2%) implanted. In contrast, abnormal dense granulation after warming (P < 0.05) was a negative predictor. The presence of necrotic foci in either ICM or TE did not affect implantation behaviour.
Table 1. Survival and morphology of vitrified day-5 embryos of different expansion.
No. warmed Immediate survival Re-expansion Hatching Granulation Necrotic foci
Morula or early blastocyst
Full blastocyst
Expanded or hatching blastocyst
86 72 (83.7)a 52 (72.2) 6 (8.3)b 21 (29.2)d 14 (19.4)
53 39 (73.6) 25 (64.1) 8 (20.5)c 8 (20.5) 7 (17.9)
134 91 (67.9)a 70 (76.9) 61 (67.0)b,c 9 (9.9)d 19 (20.9)
Values in parentheses are percentages. Values with the same superscript letter are significantly different: aP < 0.01; bP < 0.001; c
P < 0.001; dP < 0.01.
Table 2. Rates of pregnancy, implantation and live birth in vitrified and warmed transfers homogeneous for blastocyst size (68 elective single and 45 double blastocyst transfers).
Patients Pregnancy rate Clinical pregnancy Live birth rate Implantation rate
Morula or early blastocyst
Full blastocyst
Expanded or hatching blastocyst
42 20 (47.6) 19 (45.2) 17 (40.5) 22/60 (36.7)
23 7 (30.4) 7 (30.4) 6 (26.1) 10/33 (30.3)
48 25 (52.1) 23 (47.9) 23 (47.9) 29/65 (44.6)
Values in parentheses are percentages. There were no statistically significant differences.
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Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
Table 3. Morphological aspects of vitrified blastocysts after thawing and 2-h culture. Only single blastocyst transfers and homogeneous double blastocyst transfers were considered. n Re-expansion Alla Nonea Precocious hatching All None Granulation Allf Nonef Necrotic foci All None
β-HCG
Clinical PR Live birth
Implantation rate
81 21
45 (55.6) 6 (28.6)
39 (48.2) 5 (23.8)
38 (46.9) 4 (19.0)
53/121 (43.8) 6/29 (20.7)
30 77
21 (70.0)b 27 (35.1)b
19 (63.3)c 22 (28.6)c
19 (63.3)d 22 (28.6)d
23/39 (59.0)e 31/125 (24.8)e
18 102
4 (22.2) 50 (49.0)
3 (16.7) 43 (42.2)
3 (16.7) 42 (41.2)
4/26 (15.4) 59/156 (37.8)
19 93
8 (42.1) 42 (45.2)
7 (36.8) 35 (37.6)
7 (36.8) 35 (37.6)
8/25 (32.0) 47/143 (32.9)
Values in parentheses are percentages. Values with the same superscript letter are significantly different: a,fP < 0.05; bP < 0.01; cP < 0.001; d,eP < 0.0001. All = patients with transfer of day-5 embryos that were exclusively positive for the given phenomenon. None = patients with transfer of day-5 embryos that were exclusively negative for the given phenomenon. HCG = human chorionic gonadotrophin; PR = pregnancy rate.
Discussion
difficult to assess and score a vitrified blastocyst after warming than a fresh one (Shu et al., 2008).
Several factors (unrelated to the method of cryopreservation) are known directly to influence the fate of a cryopreserved blastocyst after warming/thawing and transfer. It is important to realize that survival rates in the literature may not be comparable, due to the fact that some embryologists focus on immediate survival while others suggest an additional waiting period of 24 h to monitor survival and growth (Vanderzwalmen et al., 2003). Differences in implantation rates may be attributed to the fact that not all groups apply assisted hatching to the thawed blastocysts (whilst collapsed), although this was found to improve outcome (Vanderzwalmen et al., 2003).
Re-expansion of the blastocoele (and consequently the blastocyst) after warming is expected within 24 h after thawing (Vanderzwalmen et al., 2003; Van den Abbeel et al., 2005). However, immediate re-expansion, e.g. within the first 2 h after warming, has not been used for prognostic purposes in vitrified blastocysts. In slow freezing, however, approximately half of the frozen blastocysts (197/402) did re-expand after 2–4 h in culture (Shu et al., 2008). Present results indicate that a higher rate of re-expansion might be observed using vitrification.
Most importantly, the morphology of the blastocyst will have a significant impact on survival; therefore, only blastocysts with good to moderate cell lineages will usually be considered for cryostorage. Nevertheless, cryosurvival of morphologically inconspicuous blastocysts may also fail if they derive from a cohort of poor quality day-3 embryos (Vanderzwalmen et al., 2003). There is some evidence in the literature that the efficiency of the vitrification method may depend on the expansion of the blastocyst, with better survival in morulae or early blastocyst stages compared with full or expanded blastocysts (Vanderzwalmen et al., 1999; 2002; Son et al., 2003; Zech et al., 2005). This phenomenon could, theoretically, be related to the size of the blastocoele, which in turn is related to the volume of watery fluid within the blastocyst. Present data are in line with this hypothesis, and routinely bringing forward the day of vitrification to compaction stage (exclusively vitrifying adequate concepti) could be considered (Ebner et al., 2008).
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As a result of the presence and size of the blastocoelic cavity, vitrified−warmed blastocysts experience several morphological changes and collapse during cryopreservation. Thus, it is more
Table 3 indicates that failure of the re-expansion process in vitrified/warmed blastocysts is associated with significantly reduced rates of implantation (P = 0.022) and clinical pregnancy (P = 0.021), suggesting that rapid blastocoelic re-expansion may also be an indicative marker of viability after vitrification. Even if it might be assumed that all viable blastocysts will re-expand after several more hours, any delay in this process could be a manifestation of altered osmotic and/or metabolic conditions. These events are comparable with the situation found during blastocoele development, when water enters the blastocoelic cavity via tight junctions, either diffusing passively or being pumped actively (Veeck and Zaninoviv, 2003). It may be postulated that in blastocysts with delayed reexpansion, vitrification may have influenced their permeability to water. This could either be due to a cryoartefact per se, eventually causing a delay in active and efficient pumping, or to laser manipulation during assisted hatching, although the latter assumption is less likely, since zona drilling is usually performed immediately after warming, when the blastocyst is completely shrunk. One morphological feature of warmed blastocysts that often goes with re-expansion is precocious hatching of the blastocyst RBMOnline®
Article - Predictive morphology of vitrified/warmed embryos - T Ebner et al.
through the artificial gap created by laser pulses. Quite logically, the probability that a blastocyst will hatch is likely to be increased if the conceptus is already re-expanded. However, even shrunken blastocysts tend to leave their outer shell if the position within the zona is close to the opening. Present data suggest that double clinical pregnancy rates can be achieved (63%) if transferred blastocysts have already started to hatch as compared with blastocysts without (29%) this morphological attribute (Table 3). Blebbing out of the artificial gap (2 h after thawing) obviously characterizes a subgroup of blastocysts that can hatch completely without being trapped within the zona. In contrast to previous positive predictors, necrotic foci in the blastocyst are considered to have a negative impact on further development. Similar to cryopreservation at earlier cleavage stages, areas of necrosis in warmed blastocysts mark cells that did not survive vitrification. There is general agreement that at earlier stages at least 50% of the blastomeres have to survive in order to assess a thawed embryo as viable (Rienzi et al., 2002; Archer et al., 2003). Recently, it was shown that blastomere loss after thawing in day-2 embryos has a detrimental effect on blastocyst formation and cell number (Archer et al., 2003). Partially damaged day-3 embryos can be rescued if the necrotic blastomeres are removed prior to transfer (Rienzi et al., 2002, 2005). However, prior to compaction, cell−cell adhesion can be neglected and does not reflect cell fusion in blastocysts. In other words, removal of necrotic cells is impossible on day 5 and would possibly harm the affected cell lineages. It has to be stressed that in the present data set, only minor injuries were observed (one to three cells affected by necrosis), thus removal of necrotic cells would not bring any benefit. This is the first evidence demonstrating that partial damage of the blastocyst caused by vitrification does not reduce rates of implantation and pregnancy. This is made all the more interesting by the fact that whether ICM or TE was affected played no role. Obviously, the blastocyst can compensate for minor injuries as a result of vitrification. This seems not to be the case if the whole cytoplasm is harmed by vitrification. Occasionally, blastocysts show extensive granular cytoplasm immediately after thawing instead of an expected homogeneous appearance. Although these blastocysts could be considered for transfer once they have recovered, their implantation potential seems to be limited. This conspicuous morphological feature is different from previous cytoplasmic irregularities such as cytoplasmic pitting, the manifestation of which is probably culture dependent (Ebner et al., 2005). The granular cytoplasm sometimes observed after vitrification is characterized by a halo-like structure in the periphery of the cells (which is most likely to consist of water) and a dominant accumulation of some cytoskeletal components (Figure 2a). Warmed blastocysts showing this phenomenon have a reduced capacity to survive vitrification. In the presence of this cytoplasmic anomaly, blastocysts with a good prognosis for subsequent survival may be distinguished from those with a bad prognosis (Figure 2b) by a healthy appearance of the cell membranes (Zhu et al., 2001), which should appear as a small intact line (Figure 2a).
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It should be clearly stated that the present morphological data are exclusively based on vitrification of day-5 embryos (asthis is the routine technique in the authors’ laboratories), and theoretically, morphology of thawed day-5 embryos after slow freezing might differ slightly. However, this is the first evidence that accurate scoring of vitrified and warmed day-5 embryos allows adequate prediction of pregnancy outcome. Subsequent scoring, based on the suggested morphological parameters, could improve cumulative pregnancy rate and help to further reduce the number of transferred embryos, thus limiting multiple pregnancy rates.
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Declaration: The authors report no financial or commercial conflicts of interest. Received 17 June 2008; refereed 12 August 2008; accepted 11 February 2009.
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