Blastocyst formation and cell numbers in human frozen–thawed embryos following extended culture

Janell Archer1,3, Debra A. Gook1 and David H. Edgar1,2

1 Reproductive Services, Royal Women’s Hospital, Carlton, Victoria and 2 Melbourne IVF, East Melbourne, Victoria, Australia

3 To whom correspondence should be addressed at: Reproductive Services, Royal Women’s Hospital, 132 Grattan Street, Carlton, Victoria 3053, Australia. e-mail: janell.archer{at}rwh.org.au


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Blastomere loss following human embryo cryopreservation has been shown to be associated with reduced implantation potential. In order to elucidate the underlying mechanism, the present study was designed to investigate the consequences of blastomere loss on subsequent preimplantation development in vitro. METHODS: Cryopreserved embryos destined for disposal were thawed and cultured for 96 h prior to determination of total cell numbers in resultant blastocysts. RESULTS: The proportion of embryos which formed blastocysts in vitro was significantly reduced when blastomere loss was evident in thawed embryos (25 versus 41% in fully intact thawed embryos). In addition, blastocysts from partially intact thawed embryos exhibited a significant reduction in total cell number (45.0 versus 58.4 in blastocysts from fully intact thawed embryos). Development to the blastocyst stage was significantly less frequent in fully intact thawed embryos which had been generated using ICSI (19%) compared with standard IVF (47%), although cell numbers in the resultant blastocysts were similar (57.1 and 58.6 respectively). CONCLUSIONS: Blastomere loss following cryopreservation impairs preimplantation development and results in reduced cell numbers at peri-implantation stages. ICSI is associated with reduced potential for post-thaw development of cryopreserved embryos in vitro.

Key words: blastocyst/cryopreservation/embryo development/ICSI


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of ovarian stimulation in conjunction with IVF frequently results in the creation of more embryos than are considered safe to replace in a single embryo transfer procedure. Hence, cryopreservation of cleavage-stage embryos is now widely practised in human IVF (Mandelbaum et al., 1998Go) and enables high cumulative pregnancy rates per oocyte collection to be achieved while minimizing the risk of high-order multiple pregnancy (Jones et al., 1997Go; Cohen and Jones, 2001Go).

In contrast, however, cryopreservation can impact on the implantation potential of early cleavage-stage embryos when cryodamage (cell lysis) occurs. It has been shown clearly that partially intact thawed cleavage-stage embryos can result in pregnancies, but that the implantation potential drops in parallel with increasing degrees of blastomere lysis (Van den Abbeel et al., 1997Go; Burns et al., 1999Go; Edgar et al., 2000bGo; Guerif et al., 2002Go). In contrast, if cell lysis does not occur, the implantation rates of fresh and thawed cleavage-stage human embryos with equivalent blastomere numbers and morphology are not significantly different (Edgar et al., 2000bGo). Therefore, it is the resultant loss of blastomeres rather than cryopreservation per se which affects subsequent embryo development in vivo following embryo transfer. Whether the reduced implantation potential of these partially intact cleavage-stage embryos is a result of impaired preimplantation development and/or reduced cell number in the resultant blastocysts is not known.

Recent reports have suggested that fresh human embryos derived using ICSI develop to the blastocyst stage in vitro at significantly lower rates than those generated when standard IVF insemination techniques are used to achieve fertilization, although there appears to be no significant difference in the implantation rate of ICSI and IVF embryos when replaced in vivo at early cleavage stages (Griffiths et al., 2000Go; Miller and Smith, 2001Go; Plachot et al., 2002Go).

The present study examined the effect of cryopreservation-associated cell loss and the impact of the original fertilization method on the subsequent in-vitro development of thawed human cleavage-stage embryos. Development to the blastocyst stage was assessed for intact and partially intact thawed embryos generated from both ICSI and IVF cycles, and the resultant blastocysts were analysed for total cell numbers.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Embryos and cryopreservation
Embryos included in this study were cryopreserved for patients undergoing IVF cycles between 1986 and 1996. Ovarian stimulation, oocyte collection, insemination, ICSI and embryo culture were as previously described (Bourne et al., 1995Go). Embryos were cryopreserved on day 2 of development and considered suitable for cryopreservation if <30% of their volume consisted of cytoplasmic fragmentation. Cryopreservation was carried out using 1,2-propanediol (PROH) and sucrose as cryoprotectants (Lassalle et al., 1985Go) as previously described (Edgar et al., 2000bGo). State Law in Victoria, Australia dictates that cryopreserved embryos cannot be stored for >5 years. Unless a specific extension is granted, patients are requested to consent either to discard supernumerary cryopreserved embryos or to donate them for use in the treatment of another couple. The vast majority (~95%) of patients, including all those whose embryos were used in the present study, consent to the discarding of their surplus embryos. The embryos were thawed, observed in culture and allowed to succumb according to State Law.

Thawing and culture
Straws containing embryos were rapidly thawed in air for 30 s followed by immersion for 40 s in a water bath at 30°C. The cryoprotectant was removed and embryos rehydrated in a three-step process at room temperature: 0.75 mol/l PROH + 0.2 mol/l sucrose for 7 min; 0.2 mol/l sucrose for 7 min; and HEPES-buffered medium for 10 min. The basal medium for all thaw solutions was Quinn’s Advantage HEPES-buffered human tubal fluid (HTF) containing 4 mg human serum albumin per ml (SAGE BioPharma, Bedminster, NJ, USA). After thawing, the embryos were washed and cultured in 0.5 ml Quinn’s Advantage stage-specific media with a 0.2 ml oil (Sigma-Aldrich, Sydney, Australia) overlay until day 6 (post-insemination) of development. Development was assessed on day 6 and embryos classified as either arrested prior to compaction, at compaction stages, or at blastocyst stages. The latter category included both early blastocysts and expanded blastocysts in which a distinct inner cell mass and trophectoderm were present and the diameter exceeded 150 µm. Embryos were allowed to succumb in accordance with State Law before staining for total cell number (TCN). This legislation does not permit the procedures required for differential staining of trophectoderm and inner cell mass in human embryos.

Assessment of TCN in blastocysts
Embryos were incubated in 0.9% sodium citrate at 37°C for 15–30 min, and then transferred to pre-cleaned glass slides. Cells were fixed and spread by the addition of cold methanol:acetic acid (3:1, v/v). Slides were air-dried, nuclei were stained with 10% Giemsa (ProSciTech, Queensland, Australia) in phosphate-buffered saline (TRACE Scientific, Victoria, Australia) for ~10 min and TCNs were counted.

Statistical analysis
Proportions of embryos were compared using a {chi}2-test, and mean TCNs were compared using ANOVA.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical results
Analysis of the characteristics of the treatment cycles which generated the cryopreserved embryos included in this study revealed no significant differences between standard IVF and ICSI. Cryopreserved embryos originated from 59 IVF and 52 ICSI cycles. The ICSI cycles included three cases of epididymal sperm injection and two cases of testicular sperm injection. The mean (± SD) maternal ages were 31.3 ± 3.8 and 32.1 ± 4.5 years for the IVF and ICSI groups respectively. Normal (two pronuclei) fertilization rates (72 versus 73%), mean cell numbers on day 2 (3.84 ± 0.11 versus 3.62 ± 0.98) and implantation (fetal heart detection) rates of freshly transferred embryos (30/115; 26% and 23/97; 24%) were not significantly different between the IVF and ICSI groups. The implantation rate of thawed embryos which had previously been replaced was 31% (41/131) in IVF cycles and 30% (32/107) in ICSI cycles.

Survival and development of thawed embryos
Of the 511 cleavage-stage embryos [335 (66%) IVF + 176 (34%) ICSI] which were thawed, 53 (10%) lysed completely. Of the 66 embryos (13%) in which <50% of the blastomeres survived, only two developed beyond the cleavage stages to compaction and, as they are not routinely used in the present authors’ clinical programme, these were excluded from the subsequent data. The remaining 392 embryos (77%) survived with at least 50% of their blastomeres intact following thawing (Table I). On the equivalent of day 6 of development, 47% of these embryos had arrested at the cleavage stages, 19% had reached compaction stages but failed to progress further, and 34% had formed blastocysts (Table I).


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Table I. In-vitro development of frozen–thawed, cleavage-stage embryos
 
Development in relation to cell survival
Of the cryopreserved embryos which survived completely intact (i.e. no blastomere lysis), 41% formed blastocysts, a further 24% reached compaction stages, and 35% arrested prior to compaction at the cleavage stages (Table I). This contrasts with the development of the partially intact embryos where cell lysis had a significantly detrimental effect on in-vitro development following thawing. Development to the blastocyst stage was significantly lower (P < 0.01) in the partially intact embryos (25%) compared with intact embryos (41%), and developmental arrest occurred at earlier stages in the partial embryos, with 63% arresting at the pre-compaction cleavage stages compared with 35% in the intact embryos.

Development in relation to insemination method
A significant difference was found in the development of ICSI- and IVF-derived embryos, independently of blastomere survival (Table II). The proportion of ICSI embryos which reached the blastocyst stage was significantly lower, in both the intact (P < 0.001) and partially intact (P < 0.05) embryos, compared with the IVF embryos. When the morphology of the blastocysts was examined there was no difference in the proportion of ICSI-derived compared with IVF-derived blastocysts which had undergone expansion (11/20; 55% versus 58/113; 51%). However, post-thaw cell survival did have a significant effect on subsequent blastocyst morphology, with 61% of the blastocysts derived from fully intact thawed embryos having undergone expansion compared with only 32% of blastocysts derived from partially intact embryos (P < 0.05) (Table II).


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Table II. Impact of fertilization technique on development of frozen–thawed, cleavage stage embryos in vitro
 
Total cell numbers
In the embryos that reached the blastocyst stage there was no significant difference in TCN between ICSI and IVF embryos (Table III). When the ICSI and IVF results were combined, it could be seen that the there was a significant effect of cell lysis on the subsequent TCN in blastocysts on day 6. The blastocyts generated from intact embryos had a mean TCN of 58.4 ± 3.4, whereas that for blastocysts from partial embryos was significantly lower (45.0 ± 3.7) (P < 0.01). Representative micrographs of blastocysts with paired analysis of TCN are shown in Figure 1. Nuclei were detected in an early blastocyst (prior to expansion) (Figure 1b), and also in an expanded blastocyst (Figure 1d).


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Table III. Cell numbers in blastocysts derived from in-vitro culture of frozen–thawed IVF and ICSI cleavage-stage embryos
 


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Figure 1. Micrographs showing representative blastocysts (derived from culture of thawed embryos) with paired TCNs. (a) Early blastocyst with spread (b); TCN = 42. (c) Expanded blastocyst with spread (d); TCN = 106. Scale bars = 100 µm.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A reduction in the number of blastomeres as a result of cell lysis is a well-recognized consequence of the cryopreservation of human early cleavage-stage embryos. This phenomenon only occurs in a proportion of thawed embryos, but when it does it is associated with a reduced potential for in-vivo development following replacement in utero (Van den Abbeel et al., 1997Go; Burns et al., 1999Go; Edgar et al., 2000bGo; Guerif et al., 2002Go). The present study was designed to investigate the developmental consequences of blastomere loss at subsequent preimplantation stages by analysing the in-vitro development of thawed embryos to the blastocyst stage, and the resultant TCN.

Approximately 50% of non-frozen human IVF embryos have been reported to be capable of forming blastocysts in vitro when cultured in the presence of heterologous somatic cells (Kaufman et al., 1995Go) or in appropriately formulated sequential media (Gardner et al., 1998Go). The TCN observed in individual in-vitro-derived human blastocysts grown in media alone has been reported to range from <25 to >200 (Hardy et al., 1989Go; Devreker et al., 2001Go; Dumoulin et al., 2001Go). The TCN of blastocysts grown in co-culture has been reported to vary between 60 and >300, and also to be related to morphological characteristics of the individual blastocysts (Fong and Bongso, 1999Go). When human frozen–thawed embryos were cultured in sequential media, 30% (15/50) formed blastocysts with a mean TCN of ~80, although the proportion of blastocysts and the mean TCN were both increased by the addition of exogenous granulocyte-macrophage colony-stimulating factor to the culture media (Sjoblom et al., 1999Go). In-vitro development of frozen–thawed embryos to the blastocyst stage in the present study (34%; Table I) mirrored the above result. The slightly lower mean TCN (~60) observed among blastocysts in the present study might reflect the fact that the cleavage-stage embryos were frozen prior to 1996 and had not been cultured in more recently formulated media prior to cryopreservation.

The in-vitro preimplantation developmental consequences of blastomere loss following cryopreservation are clear from the study results (see Tables I, II and III). First, a significantly reduced proportion of partially intact thawed embryos developed to the blastocyst stage (25% compared with 41% of fully intact embryos; Table I). This represents a 39% reduction in the ability to form blastocysts in vitro—a figure which is, notably, equivalent to the reduction in clinical implantation rate previously reported for partially intact thawed embryos in vivo (6.9 versus 11.3%; Edgar et al., 2000bGo). The negligible potential for in-vitro blastocyst formation in embryos with <50% blastomere survival (see Results section) was also consistent with previous clinical findings (Edgar et al., 2000bGo). In addition to developmental arrest in partially intact thawed embryos, a reduction was also observed in TCN (Table III) and a lower incidence of full expansion of the blastocyst (Table II). The physical removal of a single blastomere from mouse 4-cell embryos prior to continued culture has been reported to be associated with lower cell numbers in resultant blastocysts (Somers et al., 1990Go). The close correlation between the extent of reduction in blastocyst formation and reduced implantation potential, however, suggests that impaired preimplantation development may be the predominant mechanism by which blastomere loss impacts on clinical outcome. Lending support to this interpretation is a limited analysis of pregnancies resulting from the transfer of exclusively partially intact thawed embryos (Edgar et al., 2000aGo), which suggested that the frequency of early pregnancy loss was not significantly increased.

Studies in which sibling oocytes were inseminated by ICSI or conventional IVF have demonstrated no difference in embryonic development during the subsequent 2 days in vitro, nor in the implantation rate following transfer in utero (Staessen et al., 1999Go; Plachot et al., 2002Go). However, a number of studies have reported a reduced ability of ICSI-derived embryos to develop to the blastocyst stage in vitro (Shoukir et al., 1998Go; Dumoulin et al., 2000Go; Ménézo and Barak, 2000Go; Miller and Smith, 2001Go). The paternal contribution to early embryo development may play a role in this phenomenon (Sakkas et al., 1998Go). However, this has also been reported in a sibling oocyte study (Griffiths et al., 2000Go), suggesting that it might be related to the injection procedure rather than to the origin of the male gamete. The latter interpretation is supported by a study on the effects of injection volume on blastocyst development following ICSI (Dumoulin et al., 2001Go). In agreement with the above observations on non-frozen embryos, the present results demonstrate that thawed ICSI-derived cleavage-stage embryos are significantly less likely to form blastocysts in vitro than their IVF-derived counterparts (Table II). It is also clear from the results shown in Table II that this is not a consequence of reduced blastomere survival in ICSI embryos as the difference persists when the development of only fully intact embryos from IVF and ICSI is examined. Interestingly, there was no significant difference in the number of cells in blastocysts (Table III) or the frequency of blastocyst expansion (see Results section) in relation to the means of fertilization. It appears, therefore, that any perturbation of subsequent embryo development, which is a result of the ICSI procedure, only manifests under in-vitro conditions. It is tempting to hypothesize that distortion of cellular processes due to ICSI renders the early cleavage-stage embryo vulnerable to any subsequent insult such as exposure to a suboptimal environment. The marked difference between the continued in-vitro development of thawed IVF and ICSI embryos suggests that the additional insult of cryopreservation may further magnify this phenomenon, although thawed ICSI-derived embryos which are able to form blastocysts in vitro appear similar in terms of morphology and cell number to blastocysts derived from thawed IVF embryos.

In conclusion, the consequences of blastomere loss in thawed embryos include impaired preimplantation development in vitro and reduced TCN in resultant blastocysts. The decreased potential for in-vitro blastocyst formation reported for ICSI embryos is also evident, and may be magnified following cryopreservation at early cleavage stages, further emphasizing the need for caution in the application of ICSI (Oehninger and Gosden, 2002Go).


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 Materials and methods
 Results
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Submitted on February 13, 2003; accepted on April 30, 2003.