Sequential assessment of individually cultured human embryos as an indicator of subsequent good quality blastocyst development

E. Neuber1, P. Rinaudo2, J.R. Trimarchi3 and D. Sakkas1,4

1 Yale Center for Reproductive Medicine & Infertility, Yale University School of Medicine, New Haven, CT, USA, 2 Division of Reproductive Endocrinology and Infertility, University of Pennsylvania, Philadelphia, PA and 3 Women’s and Infants Hospital, Providence, RI, USA

4 To whom correspondence should be addressed. e-mail: denny.sakkas{at}yale.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: It is of fundamental importance for IVF clinics to determine the most viable embryos for transfer. The challenge for ART clinics is to transfer fewer embryos, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. In this study, an investigation was made to determine if developmental markers on the day of fertilization (day 1) can predict good subsequent blastocyst development. METHODS AND RESULTS: A total of 1550 individually cultured 2PN embryos from 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility from February to December 2001 was included. The results showed a significant positive relationship between early-cleaving 2-cell embryos and subsequent good quality >=4-cell, >=7-cell and blastocyst development (P < 0.05). PN symmetry (the relative size of the PN to each other), when checked at the time fertilization, is also a significant indictor of good quality >=4-cell, >=7-cell stage embryos and blastocysts. Combined, a developing embryo showing PN symmetry with early cleavage and subsequent good >=4-cell and >=7-cell cleavage, has a one in two chance of developing into a good-quality blastocyst. CONCLUSION: Early embryo assessment can be used as an indicator of subsequent good blastocyst development.

Key words: blastocyst development/early cleavage/embryo quality/in-vitro fertilization


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The ability to identify the most viable embryo for transfer is of fundamental importance to assisted reproduction techniques (ARTs). An early predictive assessment made in a non-invasive manner would allow ART clinics confidently to identify the best embryo and to limit the number of embryos transferred, thereby minimizing the risk of multiple-infant births, while still maintaining the greatest chance of pregnancy for their patients. Several European countries have legally restricted the number of embryos allowed for transfer because of the increased risks associated with multiple births. Although this approach would decrease the multiple-infant birth rates, it may also decrease the overall pregnancy rates and inadvertently subject patients to the increased medical risk of more cycles (Ozturk and Templeton, 2002Go).

The most commonly used embryo assessment technique is cell number and morphological appearance of the embryo on the day of transfer (Erenus et al., 1991Go; Steer et al., 1992Go). It has been proposed that characteristics of the 2PN zygote can be used to predict viability (Tesarik and Greco, 1999Go; Scott et al., 2000Go; Balaban et al., 2001Go; Montag and van der Ven, 2001Go). At the PN stage, the human oocyte is primed with maternal mRNA utilized by the embryo until it reaches the 4- to 8-cell stage (Braude et al., 1988Go); therefore, the quality of the 2PN embryo sets the stage for subsequent development. One group (Tesarik and Greco, 1999Go) have examined the presence and pattern of the nucleolar precursor bodies (NPB) in human 2PN zygotes and provided evidence that a higher implantation rate resulted if the number of NPBs was equal in both pronuclei, and the NPBs in the 2PN were either both polarized or both non-polarized. Others (Scott et al., 2000Go; Balaban et al., 2001Go) have shown that pronuclear morphology also correlates to good blastocyst formation. In addition, assessment of early cleavage to the 2-cell stage on day 1 has also been shown to be beneficial as a selection criterion for good embryo development and embryo viability (Shoukir et al., 1998Go; Lundin et al., 2001Go; Sakkas et al., 2001Go; Fenwick et al., 2002Go).

To further refine the established embryo assessment criteria, the effectiveness of these individual static assessment criteria was compared with a system in which all criteria are used sequentially to predict good embryo development on days 2, 3 and 5 after insemination. Therefore, the aims of this study were to investigate whether early PN assessment (PN symmetry, alignment and/or cytoplasmic quality) or late PN assessment (PN breakdown and/or early 2-cell development) can predict good embryonic development on days 2, 3 and 5 post insemination, and whether these parameters be used collectively to establish a predictive sequential embryo assessment model for routine use in IVF clinics.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
A total of 191 patients undergoing IVF/ICSI treatment at the Yale University Center for Reproductive Medicine and Infertility between February and December 2001 who had early embryo assessment performed on their oocytes was included in this study. A retrospective analysis of 1550 individually cultured 2PN embryos was performed. Overall in 2001, the 2PN fertilization rate was 56% (1685/3041), with a subsequent clinical pregnancy rate of 30%. The mean (± SD) age of the women treated was 36.1 ± 4.3 years (range: 26–47).

Embryo assessment
Embryo assessment was performed by scoring the following characteristics at specific time-points in development. Embryos were assessed only once for each time period. On day 1, at 16–19 h post insemination (PI), IVF- and ICSI-generated oocytes were assessed for: (i) the presence of 2PN; (ii) alignment of the PN to the two polar bodies (the orientation of the 2PN in respect to the polar bodies; (iii) PN symmetry (the relative size of the PN to each other); and (iv) the overall quality of the cytoplasm. Good-quality cytoplasm is defined as containing smooth, non-granular ooplasm with no darkening in the area surrounding the pronuclei. All measurements were made at the time of the fertilization check using a Nikon inverted microscope (x40 and x100 magnification). Later on day 1 the greatest majority of embryos was assessed between 23–28 h PI for the continued presence of 2PN, 2PN-breakdown or cleavage to the 2-cell stage. In some instances, due to conflicts in timing of egg retrievals, the assessment of fertilization and early cleavage was not always carried out in strict adherence to the above schedules. Embryos that had undergone 2-cell cleavage were designated ‘early-cleaving embryos’, and those which had not yet cleaved were designated ‘no early cleavage’. On day 2 (44–46 h PI) and day 3 (66–68 h PI), embryos were assessed for cleavage to the >=4-cell and >=7-cell stages. The individually cultured embryos were assessed for the number of cells per embryo in order to ascertain their cleavage rates, and were assigned a quality score based on the presence of fragments and the heterogeneity of the cytoplasm. The rating given to the embryos for cell number was 1 for one-cell, 2 for two-cell, etc. The rating given to embryos for quality was: grade 1 for regular blastomeres and no fragments; grade 2 for regular blastomeres and minor fragments; grade 3 for irregular blastomeres and no fragments; grade 4 regular or irregular blastomeres, many fragments; and grade 5 for few recognizable blastomeres and severe fragmentation (Veeck, 1999Go). The absence or presence of multinucleated blastomeres was also included for both days. Embryos were termed good quality (>=4 and >=7 cells) if they contained at least that number of blastomeres, the fragmentation grade was 1 or 2, and the embryos contained no multi-nucleated blastomeres.

Finally, on day 4 (94–96 h PI) and day 5 (116–118 h PI) embryos were assessed for morula and blastocyst stage development respectively. This translates to the assessment of embryos between 8:00 and 10:00 on days 4 and 5 for the majority of patients. Blastocysts were scored according to the expansion of the blastocoelic cavity and the number and cohesiveness of the inner cell mass and trophectodermal cells (Gardner et al., 2000Go). A good-quality blastocyst was defined, in this study, as one in which the blastocoele cavity fills at least 50% of the embryo, with a visible inner cell mass consisting of several grouped cells and a single layer of trophectoderm cells surrounding the cavity.

Media
Insemination and sperm preparation was performed in Scandinavian IVF20 medium (VitroLife, Gothenburg, Sweden). Insemination or ICSI was performed on the afternoon of the retrieval. On the following morning (day 1), 2 PN embryos were individually placed in 30 µl drops of Scandinavian G1 medium (VitroLife) under mineral oil (Sage BioPharma, Bedminster, NJ, USA) in Falcon Petri dishes (Macalaster Bicknell Co., New Haven, CT, USA). On day 3, the embryos were transferred to 30 µl drops of Scandinavian G2 medium (VitroLife) under mineral oil (Sage), until embryo transfer or cryopreservation. All cultures were performed in an atmosphere of 6% CO2 in air at 37°C.

Statistical analysis
All data were analysed using Microsoft Excel spreadsheet (MS Excel 2002) and SPSS package (version 10.0.5). PN assessment was compared using the chi-square test. A P-value < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Early 2PN results
A total of 1550 2PN embryos was observed on day 2 for their development to the >=4-cell stage (day 2), 1539 to the >=7-cell stage (day 3), and 898 to the blastocyst stage of development (day 5). Day 3 transfers were performed on 74% (205/277) of patients, with the remaining 26% (72/277) undergoing day 5 transfers.

PN symmetry at the time of fertilization check is a significant indicator of good quality (symmetrical) >=4-cell and >=7-cell embryo development and blastocyst development. PN symmetry resulted in 76% (669/1179) good-quality >=4-cell development, 44% (511/1171) >=7- cell development and 24% (164/673) blastocyst development (Table I).


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Table I. Early PN assessment and their relationship to subsequent good quality >=4-cell, >=7-cell stage embryos and blastocyst development
 
The alignment of the 2PN with the polar bodies was a significant indicator of good >=4-cell development (63%; 160/255). However, 2PN alignment was not a good indicator of >=7- cell or blastocyst development (Table I).

Good cytoplasmic quality at the PN stage was a significant indicator of good >=4- and >=7-cell development. Cytoplasmic quality was not a significant indicator of good blastocyst development (Table I).

The significance of these three parameters to subsequent development was ranked according to the P-values from the {chi}2 comparison. The order of highest significance is PN symmetry, cytoplasmic quality and lastly PN alignment.

2PN breakdown and early cleavage to the 2-cell stage
A total of 1303 2PN oocytes from 191 patients undergoing IVF or ICSI was assessed. The 2PN oocytes were observed late on day 1 (17–28 h PI) for the presence of PN breakdown and/or early 2-cell cleavage. The remaining fertilized 2PN embryos were not assessed due to schedule conflicts. Of the 2PN embryos assessed, 12.7% (165/1303) underwent PN breakdown, and 24.2% (315/1303) underwent early 2-cell cleavage at the time of observation. There was a significant positive relationship between early 2-cell development and PN breakdown with subsequent good quality >=4-cell, >=7-cell and blastocyst development (Table II).


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Table II. Late (day 1) PN assessments and their relationship to subsequent good quality >=4-cell, >=7-cell stage embryos and blastocyst development
 
Between 24–26 h PI, all three stages (PN intact, PN breakdown and 2-cell cleavage) were observed, and this was deemed the optimal time for observation (Figure 1). The present findings confirm the results of earlier investigations performed at different centres (Shoukir et al., 1997Go; Sakkas et al., 2001Go; Fenwick et al., 2002Go). However, as the present study was performed in a clinical setting, some observations were made outside these optimal times. The earliest PN breakdown was observed at 17.53 h PI, whereas 2-cell development was first observed by 20.1 h PI (Figure 1).



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Figure 1. Scatter graph assessing the development of 1303 embryos from 191 patients between 23 and 28 h post insemination, together with several outliers from this time period; these represent a minor number of assessments that were performed out of this range due to conflicts in clinical scheduling. The graph represents 2PN observation time versus 2PN, 2PN breakdown and 2-cell development. The earliest PN breakdown was observed at 17.53 h post insemination. Two-cell development was first observed by 23.05 h post insemination. Between 24 and 26 h PI, all three stages (2PN intact, 2PN breakdown and 2-cell cleavage) were observed, and this was deemed the optimal time for observation.

 
Embryos were again observed for development on day 2 (n = 1303) and day 3 (n = 1290). Embryos were assessed for cleavage to the >=4-cell and >=7-cell stages of development respectively. Good-quality >=4-cell and >=7-cell development was significantly increased in 2PN embryos having undergone early cleavage and PN breakdown at the time of observation (Table II).

Embryo fragmentation rates on day 3 are significantly reduced if the embryos underwent early 2-cell cleavage on day 1 compared to PN intact embryos (P = 0.015). Fragmentation was scored as positive if >20% of the embryo contained fragments (see Materials and methods).

Blastocyst development was also assessed for these three categories. If ‘no early cleavage’ took place on late day 1, only 19% (82/444) developed to blastocysts by day 5 PI. In contrast, if PN breakdown or early cleavage was observed, then 26% (52/197) and 39% (44/113) respectively of these embryos developed to blastocyst on day 5. A three-way {chi}2 analysis indicated that this trend was also statistically significant (Table II).

Sequential embryo assessment
All the parameters mentioned above were combined in a sequential embryo assessment data base, and the percentage of embryos developing to the blastocyst stage was determined. A number of scenarios for sequential embryo assessment predicting blastocyst development are shown in Figure 2. The use of specific assessment criteria showed a greater predictive power for selecting a good blastocyst. For example, if an embryo showed PN symmetry, PN breakdown and subsequent good quality >=4 and >=7-cell embryo on day 2 and 3 respectively, it had a 47.9% chance of forming a blastocyst by day 5. If in addition an embryo showed the above criteria and has undergone ‘early cleavage’ on day 1, it had a 54.2% chance of developing into a good quality blastocyst. In contrast, if an embryo showed none of the above criteria it had only a 5.6% chance of developing into a good quality blastocyst (Figure 2).



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Figure 2. Sequential assessment of individual embryos and their ability to reach the blastocyst stage on day 5. Percentage development to the blastocyst stage is calculated from the number of blastocysts divided by the number of embryos showing the sequential cleavage characteristics. (A) All embryos showing PN alignment, early cleavage, good quality >=4- and >=7-cell development. (B) All embryos showing early cleavage, good quality >=4- and >=7-cell development. (C) All embryos showing PN alignment, PN breakdown and good quality >=4- and >=7-cell development. (D) All embryos showing good quality >=4- and >=7-cell development only. (E)All embryos showing intact PN, poor quality >=4- and >=7-cell development. For assessment parameters, see Materials and methods.

 
Pregnancy results
This was a retrospective study, and therefore the sequential embryo assessment data presented here were not incorporated in selection of the embryos used in the embryo transfer procedure. Nonetheless, a significant increase in pregnancy and implantation rates (P = 0.05 and P = 0.03) was still observed from patients who had at least one early 2-cell embryo [40% (25/63) and 21.9% (40/183)], over patients that did not [26% (33/128) and 14.3% (53/371)]. The mean age of the patients in these two groups was not significantly different. However, there was a tendency for the mean number of zygotes and the mean number of embryos produced to be increased in the patients that had at least one early-cleaving embryo (9.8 and 8.2 versus 7.5 and 6.1 respectively); this failed to reach statistical significance (P = 0.08 and P = 0.09).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As ARTs have become used worldwide there has also been an increase in the reported number of multiple-infant births. Such births result in greater problems for both mother and infants, including an increased rate of Caesarean sections, premature birth, low birth weight, infant death and disability (Bergh et al., 1999Go). The challenge has been to identify a method to select the best-quality embryo to select for transfer. The present findings, which characterize the individual morphological development of embryos from day 1 to day 5, identify key patterns that will aid in predicting which embryos to select for transfer.

It has been shown in animal models that early embryo assessment is positively correlated to good subsequent development (McLaren and Bowman, 1973Go). The various techniques proposed to evaluate embryo viability in humans, including assessment at the PN stage (Tesarik and Greco, 1999Go; Scott et al., 2000Go), assessment of early cleaving 2-cell embryos (Shoukir et al., 1997Go; Bos-Mikich et al., 2001Go; Sakkas et al., 2001Go) and the blastocyst stage (Gardner et al., 2000Go) have all been largely examined on a cohort of embryos, and not individually cultured embryos.

The results of the present study offer evidence of a significant positive relationship between either PN symmetry at time of fertilization check and early cleaving 2-cell embryos with subsequent good-quality >=4-cell, >=7-cell and blastocyst development. Combining these observations, a single developing embryo that shows early cleavage and subsequent good >=4-cell and >=7-cell cleavage, has a one in two chance of developing into a good quality blastocyst. It is believed that sequential embryo assessment is a valuable tool in allowing IVF clinics to select the best embryos from the patient’s cohort of embryos to be used for the transfer. Sequential scoring systems take into account multiple developmental stages, beginning with PN stage assessments, first cell cycle, cleavage stages and finally blastocyst development. The model proposed herein takes into account all the possible pathways that the developing embryo can undergo. For example, at the time of fertilization a 2PN embryo will either have the two PN aligned with the polar body, or not. In contrast, if the developing embryo shows no PN symmetry, no early cleavage and poor >=4-cell and >=7-cell development it has only a 6% chance of developing into a good quality blastocyst. Applying this model to developing embryos should provide sufficient information to allow us to predict on day 3 of development which embryos are most likely to develop into a good quality blastocyst. This may ultimately allow IVF clinics a greater flexibility in transferring embryos to patients on either day 3 or day 5.

One possible concern with a sequential embryo assessment is the potential damage done to the embryo by performing multiple viewings. Precautions need to be taken to limit the viewing time, as the observations need to be as non-invasive as possible. In the present authors’ laboratory, the embryos are cultured individually under mineral oil, with only five embryos being cultured per dish. The assessment is ideally performed by two embryologists working together, during which time one embryologist assesses the embryos under the microscope and the other records the results, thereby reducing the time that the embryos are out of the incubator. Furthermore, for blastocyst (day 5) transfers the day 4 observation could be eliminated. Eliminating this time point would bring the total number of observations made on each blastocyst embryo to five during the course of its incubation. The embryos are viewed using an inverted microscope, and no manipulations are carried out at the time of viewing. The viewing of the embryo is made as a ‘snapshot’ image. For consistency, the embryos were viewed at approximately the same time PI for all the patients, although in some cases this was inevitably influenced by clinical scheduling constraints. The importance of timing when viewing embryos as a snapshot image has been previously highlighted as a crucial factor in embryo assessment (Bavister, 1995Go; Sakkas et al., 1998Go; 2001). A second possible concern might be that embryos need to be cultured individually. In animal models there is good evidence that developing embryos benefit from being cultured together (Wiley et al., 1986Go; Paria and Dey, 1990Go; Lane and Gardner, 1992Go). This indeed may be a drawback with a sequential embryo assessment model, though reports have been made in human embryo culture where clinics have used individual culture techniques (Balaban et al., 2001Go).

A sequential embryo assessment method can be used as a valid indicator of good subsequent development, especially if the patient has more than three equal-looking blastocysts on the day of the transfer. In the future, it would allow for differentiation of the blastocyst by taking into account the parameters it underwent during its development. Ultimately, this sequential embryo assessment technique may allow selection of the patient’s best one or two embryos for transfer and still maintain a high pregnancy rate while limiting the major complication of multiple infant births. The adoption in clinics of either single- or two-embryo transfer is already being recognized in Europe (De Sutter et al., 2002Go), whereas in the United States there is still a tendency for higher rates of multiple births (SART, 2000Go). The implementation of techniques such as sequential embryo transfer, nuclear precursor body scoring (Tesarik and Greco, 1999Go; Scott et al., 2000Go) or more technical procedures such as metabolic assessment (Leese, 1987Go) will assist the movement towards single- or two-embryo transfer more confidently.

The present study has provided valuable data on the cleavage characteristics of individual embryos in relation to their development potential in vitro. The basic mechanisms involved in the cleavage characteristics of human embryos have yet to be fully established, although there are indications that certain proteins may be involved in determining rates of cleavage (Cao et al., 1999Go). A prospective trial to ascertain whether sequential embryo assessment will improve the ability to limit the number of embryos transferred and to select the most viable embryos will very much help clinics to achieve pregnancies when transferring fewer embryos on day 3 or day 5.


    Acknowledgements
 
The authors gratefully acknowledge the assistance of Westley Anderson, Kathi Greco, the MDs and nursing staff of the Yale Center of Reproductive Medicine and Infertility.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 19, 2002; resubmitted on December 31, 2002; accepted on March 11, 2002.