1 The Fertility Clinic, Rigshospitalet, Section 4071, University Hospital of Copenhagen, Blegdamsvej 9, DK-2100 Copenhagen, 2 The Fertility Clinic, Brædstrup Sygehus, Sygehusvej 20, DK-8740 Brædstrup, Denmark
3 To whom correspondence should be addressed. Email: sziebe{at}rh.dk
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Abstract |
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Key words: blastomere size/computer-controlled multilevel morphological analysis/DNA staining/nuclear structures
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Introduction |
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Embryos with multinucleated blastomeres have been shown to be associated with decreased implantation, pregnancy and birth rates (Jackson et al., 1998; Pelinck et al., 1998
; Van Royen et al., 2003
). Furthermore, several studies have shown that multinucleate embryos correlate with increased rates of chromosomal abnormalities (Kligman et al., 1996
; Balakier and Cadesky, 1997
; Hardarson et al., 2001
).
It has therefore been suggested to exclude multinucleate embryos from transfer unless no other embryos are available (Kligman et al., 1996; Balakier and Cadesky, 1997
) and that assessment of nuclear status should be included in embryo scoring systems (Jackson et al., 1998
; Van Royen et al., 2003
).
However, the detection of nuclear structures in the embryo based on traditional light microscopic analysis is associated with several problems. Embryonic fragmentation may to some extent cover existing nuclear structures, leaving them undetected. Additionally, the morphology of nuclear structures may be very diverse and dynamic as a result of several normal and abnormal processes such as the breakdown and reassembly of the nuclear envelope associated with cell cleavage, fragmentation of nuclear structures or failure of karyokinesis (Hardy et al., 1993; Ellenberg et al., 1997
; Ellernberg and Lippincott-Schwartz, 1999
; Hardy, 1999
; Salina et al., 2001
; Burke and Ellenberg, 2002
; Erenpreisa et al., 2002
). This makes it difficult to define nuclear structures as well as to distinguish between nuclear structures and other circular embryonic structures such as vacuoles (Van Blerkom et al., 1987
).
In the present study, we used a computer-controlled system for multilevel and non-invasive embryo morphological analysis to detect nuclear structures in the intact embryo and to validate the findings by separating the embryos into individual blastomeres. Additionally, the morphological findings were verified by subsequent fixation of all nuclear structures and staining for the presence of DNA.
The aim of this study was to identify and characterize the nuclear structures in intact 2-cell and 4-cell embryos using traditional as well as computer-controlled multilevel analysis and to evaluate the findings as a predictive tool of the nuclear status in intact human embryos.
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Materials and methods |
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Patients were treated with the long protocol, using a GnRH agonist (Synarela®, Pharmacia, Denmark; or Suprefact®, Aventis Pharma, Denmark) for downregulation and recombinant FSH (Gonal-F®, Serono, Denmark; or Puregon®, Organon, Denmark) for ovarian stimulation. HCG (Profasi®, Serono, Denmark) was given 36 h before oocyte retrieval.
IVF and ICSI procedure
IVF and ICSI were performed according to the routine procedures of the clinic. Briefly, oocytes were aspirated 36 h after HCG injection and the IVF or ICSI procedure was performed 46 h later. On the following morning (18 h after insemination), the oocytes were checked for fertilization and cultured for a further 24 h. Embryo transfer was carried out 5052 h after aspiration. Immediately prior to transfer, embryos were selected for transfer by evaluating the cleavage stage and quality score in accordance with the normal procedures at our clinic. Embryos were considered suitable for donation based on this normal morphological evaluation. The selection of embryos for transfer was done independently of this study and prior to embryo donation.
Embryo donation
Only embryos that had developed to the 2- or 4-cell stage at transfer with <20% fragmentation were included in this study. The donated embryos were good quality surplus embryos that otherwise would have been frozen. Only patients having at least six good quality surplus embryos were asked to donate. Informed consent was obtained from all patients before donation.
In total, 87 embryos were donated comprising 46 two-cell embryos (consisting of 92 blastomeres) and 38 four-cell embryos (consisting of 152 blastomeres). Another three embryos had developed further from the 2-cell to the 3-cell stage within the time from routine embryo scoring to treatment of the donated embryos and were excluded from the study.
Recording of digital images
Using the FertiMorph computer system for multilevel embryo morphological analysis (Image House Medical A/S, Copenhagen, Denmark), image sequences were recorded of all included embryos immediately after donation. Additionally, image sequences of the individual blastomeres were recorded after dissolving the zona pellucida and segregation of the blastomeres. Each sequence consisted of 26 images of the same embryo or blastomere with the FertiMorph System automatically focusing in 5 µm intervals through the embryo or blastomere. The automatically controlled image recording and storing took 15 s per sequence. All recordings were performed at 400xmagnification with Hoffman modulation contrast illumination.
Measurements on the images are done in pixels and converted to actual physical units by knowing the distance between two adjacent pixels. The calibration is done by taking an image of a micrometer slide with the same magnification as the embryo images. A line is drawn on the micrometer slide. Knowing the outlined distance on the slide, the system calculates the physical distance between two pixels.
Evaluation of nuclear status based on morphological analysis
In total, 73 of the included embryos (39 two-cell and 34 four-cell embryos) could be analysed for the nuclear status of the intact embryos by both traditional and computer-controlled, multilevel morphological analysis as well as in their separated blastomeres and by subsequent DNA staining of the fixed nuclear structures (Figure 1). Of the 84 included embryos, 11 embryos were excluded for these subsequent steps of analysis (for four embryos, digital images were missing, for five embryos at least one blastomere got lost during blastomere segregation and fixation, one embryo had cleaved from a 2-cell to a 4-cell embryo after segregation of the blastomeres and for one embryo the blastomeres were not fixed).
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Traditional light microscopic evaluation
In accordance with the normal procedures at our clinic, the morphology of all the embryos included in this study was evaluated using light microscopy (400x magnification and a numerical aperture of 0.55) with Hoffman modulation contrast illumination prior to transfer and embryo donation. The traditional quality score assessment included the evaluation of the nuclear status.
Computer-controlled multilevel analysis of the blastomeres and nuclear structures
Based on the digital image sequences, blastomere size and nuclear structures were analysed in a semi-automatic manner using the morphology analysis software of the FertiMorph System. In the intact embryo, nuclear structures were defined as circular structures surrounded by a membrane containing nuclear precursor bodies. All images of one sequence could be viewed in detail, enabling us to select the pictures where the different structures were in focus. The number of blastomeres of the embryos were defined in accordance with the traditional light microscopic embryo evaluation and re-evaluated based on the image sequences. For all embryos, the outer border of all blastomeres and all visible nuclear structures were outlined. Thus, different structures in the same embryo could be outlined on different images, representing different focus depths. The same was done for the image sequences of the individually separated blastomeres.
Morphometric values describing the size of the blastomeres and nuclear structures (area, diameter and volume) were calculated automatically. The system calculated the area of the blastomere or nuclear structure. A radius (r) was calculated from a circle with the equivalent area. The cell volume (V) or volume of the nuclear structure was computed from the assumption that blastomeres and nuclear structures were spherical, therefore using the equation V = 4/3 r3.
The calculation of each individual morphological structure was based on the particular image(s) where this structure was outlined.
The degree of fragmentation was estimated based on the image sequences and each embryo was allocated to one of the three groups: group I, 0% fragmentation; group II, 110% fragmentation; group III, 1120% fragmentation.
Segregation of the individual blastomeres
After donation, the embryos were transferred for 1 min to culture medium containing pronase (5 mg/ml) (Sigma, USA) to dissolve the zona pellucida followed by incubation in Ca2+ /Mg2+ -free medium (EB-10; Vitrolife, Gothenburg, Sweden) for 14 min until segregation of the individual blastomeres. The individual blastomeres were transferred to separate wells containing IVF medium (Medicult, Denmark).
Fixation
The nuclear structures from each blastomere were fixed separately on a silianized slide (Cat. No. S1308; Oncor, USA) in an HCl (0.01 mol/l)/Tween-20 (0.1%) solution (Coonen et al., 1994) using a microscope with 10x times magnification and Hoffman modulation contrast illumination. The fixed nuclear structures were located by drawing a circle around them, using a diamond objective. After fixation, the slides was washed in phosphate-buffered saline, followed by water and then dehydrated in a series of 70, 90 and 99% ethanol and dried at room temperature. The slides were packed in slide boxes with silica gel, covered with paraffin and stored at 20°C until thawing for 4',6-diamidino-2-phenylindole (DAPI) staining of the DNA.
DAPI staining of the DNA
The DNA was stained with DAPI (Cat. No. 32-804831, Vysis, Downers Grave, IL) and the presence of DNA was identified using a fluorescence microscope. Digital images were taken of all identified nuclear structures. The structures that became visible by DNA staining were defined as nuclear structures when (i) they were fully or partly surrounded by a membrane; or (ii) condensed chromosomes were observed despite the lack of an intact membrane.
Embryonic nuclear status based on DNA staining
In a subgroup of the included embryos (n=72), the nuclear status of all blastomeres in the embryos could be defined by DNA staining and related to the size of the separated blastomeres. Based on DNA staining, multinucleate embryos were defined as embryos having more than one visible nuclear structure in at least one blastomere. Mononucleate embryos were defined as embryos having no or one visible nuclear structure in at least one blastomere.
Ethical approval
Ethical approval for this study was obtained from the regional ethical committee for Copenhagen before initiation of the study.
Statistical analysis
For evaluating differences in mean sizes of blastomeres or nuclear structures, one-way analysis of variance (ANOVA) was done in the case of parametric data. For non-parametric data, KruskalWallis analysis of variance on ranks was performed. To assess differences in the percentage of blastomeres or embryos with the same nuclear status detected by morphological analysis of the intact embryos and the individually separated blastomeres, and by subsequent DNA staining, Pearson 2 test was performed. The same statistical test was performed to assess differences in the percentage of blastomeres with the same nuclear status in 2-cell and 4-cell embryos and when comparing computer-controlled, multilevel analysis with traditional analysis. Differences were considered significant when P<0.05.
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Results |
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Based on the findings in the separated blastomeres, 36 embryos were classified as mononucleated and 37 embryos were classified as multinucleated (Table I).
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Using computer-controlled multilevel analysis, all 36 mononucleated embryos were identified correctly (Table I). Of the multinucleated embryos, multilevel analysis correctly identified 36 embryos (97.3%). Overall, no statistically significant difference in the assessment of nuclear status between multilevel analysis of the intact embryos and of the separated blastomeres was found (P=1.0).
Using the computer-controlled multilevel analysis, 94% of all morphologically detectable nuclear structures in the separated blastomeres were detected already in the intact embryo. After DNA staining, all nuclear structures detected in the separated blastomeres were found to contain DNA. Additionally, three extra DNA-containing nuclear structures were located (Table II).
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Impact of fragmentation on morphological detection of nuclear structures
For embryos with no or <10% fragmentation, the same number of nuclear structures was detected in the separated bastomeres and in the intact embryos (Table IV). For embryos with 1120% fragmentation, 86% of the nuclear structures detected in the separate blastomeres were found in the intact embryos (Table IV).
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Detection of the embryonic nuclear status based on DNA staining
In 72 embryos with nuclear status defined by DNA staining, multinucleated blastomeres were significantly larger in volume than the mononucleated blastomeres [on average 21.9% larger in 2-cell embryos (P=0.03) and 29.7% larger in 4-cell embryos (P=0.002)], with an average volume of 0.345±0.09x106 and 0.283±0.07x106 µm3, respectively, in multinucleate 2-cell embryos, and 0.201±0.06x106 and 0.155±0.04x106 µm3 in multinucleate 4-cell embryos (Table V). Furthermore, in multinucleate 2-cell embryos, the mean blastomere volume of anucleated blastomeres was 24.4% smaller than the mean volume of mononucleated blastomeres (P=0.09) and 46.7% smaller in multinucleate 4-cell embryos (P<0.001). For mononucleate embryos, the mean blastomere volume of anucleated blastomeres was 43.7% smaller than the mean volume of mononucleated blastomeres for 4-cell embryos (P<0.001) but only 6% for 2-cell embryos (P=0.7).
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Ten 4-cell blastomeres contained no DNA. Their average blastomere size was 52.3±7.4 µm in diameter, corresponding to a volume of 0.0867±0.04 x 106 µm3. Furthermore, none of the 4-cell blastomeres <50 µm in diameter (n=5) contained DNA, whereas 96% (138 out of 144) of the blastomeres with a diameter >50 µm contained DNA.
In nine 2-cell blastomeres, no DNA could be detected. The average size of these blastomeres was 76.2±13.6 µm in diameter, corresponding to a volume of 0.249±0.13 x 106 µm3, ranging from the smallest diameter of 58.9 µm to the largest diameter of 97.6 µm.
Blastomeres detected at a state between late interphase and early mitosis
A total of 15.4% (12 out of 78) of the analysed 2-cell blastomeres compared with 3.1% (two out of 64) of the 4-cell blastomeres showed morphologically invisible or hardly detectable nuclear structures in addition to visible, condensed chromosomes and breakdown of the nuclear envelope detected by DNA staining. Furthermore, for 57.1% (eight out of 14) of these blastomeres, their remaining sibling blastomeres showed the same nuclear characteristics.
Size of nuclear structures
The size of nuclear structures was significantly decreased from a mean diameter of 22.1±4.0 µm (corresponding to 26% of the mean blastomere diameter) and a volume of 0.00606±0.003 x 106 µm3 (n=26) in blastomeres from mononucleate 2-cell embryos to a mean diameter of 18.7±2.4 µm (corresponding to 29% of the mean blastomere diameter) and a volume of 0.00356±0.001 x 106 µm3 (n=72) in blastomeres from mononucleate 4-cell embryos (P<0.001) (Table VI).
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Discussion |
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Further, our data showed a high level of agreement between the total number of nuclear structures detected by computer-controlled multilevel morphological analysis of the intact embryos and their individually separated blastomeres, and by their DNA staining, respectively. Ninety-nine percent of the nuclear structures detected by DNA staining were found by morphological analysis of the individually separated blastomeres. Further, 94.3% of the nuclear structures detected morphologically in the separated blastomeres, where the potential visible disturbance of fragmentation and overlapping blastomeres was removed, could be found by morphological analysis of the intact embryos. These findings additionally support our suggestion that computer-controlled multilevel analysis is a powerful method to assess nuclear status when evaluating embryo quality in assisted reproduction. This was supported further by the significant agreement found between percentages of multinucleated, mononocleated and anucleated blastomeres assessed by morphological analysis of the intact embryo, the individual blastomeres and by their DNA staining, respectively.
We find that the presence of <10% embryonic fragmentation does not compromise the morphological evaluation of nuclear status. However, our data indicate some impact of fragmentation on the detection rates of nuclear structures for embryos with 1120% fragmentation. This impact will probably increase with increasing degree of fragmentation. However, as embryos with >20% fragmentation normally are excluded for transfer, this may not affect the evaluation of potential transferable embryos.
Based on data from this study, the percentage of multinucleated blastomeres was significantly higher among multinucleate 2-cell embryos compared with multinucleate 4-cell embryos as a high frequency of multinucleate 2-cell embryos showed multinuclearity in both their blastomeres. This may be a result of major errors during the first embryonic division that may be associated with delayed or arrested embryo development, as suggested by Roux et al. (1995a,b
) and Balakier and Cadesky (1997)
. In 4-cell embryos, multinucleation may also start at the second cleavage.
Evaluating the nuclear status assessed by DNA staining showed a significant correlation between multinuclearity and increased blastomeric size as well as decreased size of the mononucleated sibling blastomeres. This is in line with the findings from a previous study (Hnida et al., 2004), where the definition of nuclear status was based solely on morphological analysis of intact embryos using the FertiMorph system.
Our finding that anucleated blastomeres were smaller than mononucleated blastomeres may indicate that some of the anucleated blastomeres may be large fragments. The number of blastomeres in the embryos was assessed by normal embryo evaluation prior to inclusion of the embryos in this study. However, this blastomere assessment is subjective and may have misjudged some large fragments as blastomeres. For the multinucleate embryos, the small size of anucleated blastomeres may be a result of asymmetric cytokinesis associated with multinucleation (Hardy et al., 1993). However, the precise cut-off in size between blastomeres and fragments at different cleavage stages is still unknown. As the diameters measured in the intact 4-cell embryos were 2.9% smaller than in the separated blastomeres (P<0.05), we suggest a cut-off limit of
4550 µm between blastomeres and fragments in 4-cell stage embryos, which is in line with the study of Johansson et al. (2003)
. In 2-cell embryos, the size of blastomeres with no detectable DNA showed a huge range from a diameter of 58.9 µm to a diameter of 97.6 µm, making it difficult to define a cut-off limit for 2-cell embryos.
Data from the present study showed that the mean size of nuclear structures in mononucleate embryos decreases significantly from an average diameter of 22.1 µm at the 2-cell stage to 18.7 µm at the 4-cell stage. This decrease was concomitant with a significant decrease in blastomere diameter. Based on these findings, we suggest that the nucleuscell ratio remains fairly constant, with the nuclei comprising 2629% of the blastomere volume during the first embryonic cleavage stages.
Improving individual factors such as contrast systems, wavelength or increasing numerical aperture may improve the resolution of traditional microscopy to some extent. However, Hoffman modulation contrast illumination is generally used when evaluating embryos by light microscopy, as this optic provides an improved image of oocytes and embryos when cultured in plastic dishes. Our reason for using computer-assisted multilevel analysis was to implement the system with the type of microscope that is normally used for evaluation of embryos and to use the multilevel system to detect nuclear status in relation to traditional embryo evaluation.
When using standard culture techniques with plastic dishes and Hoffman modulation, contrast illumination detection of nuclear structures may be improved using computer-controlled multilevel analysis. Extensive exposure for transmitted light as well as time-dependent changes of temperature and pH are detrimental for the embryo. However, our digital imaging process takes 15 s per embryo, which equals the time used for an experienced laboratory technician to evaluate an embryo in the traditional manner. Thus our new technique has no additional negative impact compared with traditional analysis. As we culture several embryos in one culture dish, the average time an embryo is out of the incubator and under the microscope stage is
2 min.
In conclusion, the results of this study indicate that the use of computer-controlled, multilevel, morphological analysis can improve the detection of nuclear structures in human embryos and thus is a good alternative to traditional evaluation. Embryonic fragmentation of <10% had no impact on detection rate. However, embryonic fragmentation of 1020% had a minor impact on the morphological detection of nuclear structures in the intact embryo. This impact may increase with increasing degree of fragmentation. We suggest a cut-off limit between fragments and blastomeres of 4550 µm for 4-cell embryos. Finally, it could be shown that the nuclear structures in mononucleate embryos decrease significantly from the 2-cell stage to the 4-cell stage.
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References |
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Submitted on March 8, 2004; resubmitted on June 22, 2004; accepted on November 2, 2004.
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