Methods of embryo scoring in in vitro fertilization

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REVIEW PAPER

1_{Corresponding author: Clinic for Reproduction and Gynecology, Pomeranian Medical University,} 1 Unii Lubelskiej St., 71-252 Szczecin, Poland; e-mail: kurzawa@sci.pam.szczecin.pl

Methods of embryo scoring in

in vitro

fertilization

Tomasz Bączkowski2_{, Rafał Kurzawa}1,2_{, Wojciech Głąbowski}3

2_{Clinic for Reproduction and Gynecology;}

3_{Departmentof Histology and Embryology,}

3

Pomeranian Medical University, Szczecin, Poland Received: 3 November 2003; accepted: 12 January 2004

SUMMARY

It has been 25 years since the introduction of in vitro fertilization (IVF) for treatment of infertility. During this time very dynamic advances have taken place in all aspects of assisted reproductive technology (ART). The rapid improvement in embryological methods, especially these related to preimplantation embryo evaluation are of great importance. This article is a review of embryo classifi cation systems utilized in ART programs. The most widely used scoring systems of zygotes and embryos (includ-ing blastocysts) are described. Additionally, the advantages of advanced embryo classifi cations in relation to ART success rates are presented. Reproductive Biology 2004 4 (1): 5-22.

Key words: in vitro fertilization, intracytoplasmic sperm injection, embryo scoring, embryo culture

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INTRODUCTION

Non-invasive methods of embryo evaluation are useful in reproductive medicine. They help assess embryos without damage. Until recently they were only of scientifi c importance, but since rapid advances in the fi eld of assisted reproductive techniques (ART) they have gained more practical meaning. All ART specialists, particularly embryologists who handle hu-man germ cells and embryos, are now obliged to be familiar with precise, non-traumatic techniques of embryo evaluation. Moreover, due to ethical and legislative reasons the protocols of human embryo treatment are very restricted. Precise examination of embryos on particular days following in vitro fertilization (IVF) facilitate selection of the most potent embryos for transfer [1, 10, 22, 46, 47, 51, 53, 55, 57]. Such management improves suc-cess rates of IVF programs. Also, selection of the best embryo for transfer reduces the number of transferred embryos and subsequently the incidence of multifetal pregnancies.

The methods of embryo examination have been changing dramatically for the last 20 years. The routine embryo assessment has been supplemented with evaluation of numerous morphological features that enable prediction of developmental potential of particular embryo and subsequently a chance for pregnancy in infertile couples. Nowadays, a transfer of single embryo at day 3 following IVF or intracytoplasmic sperm injection (ICSI) is related with 10-30%, and a day 5 blastocysts transfer with 40-60% implantation rates [19, 21, 31, 55]. Obviously, such an improvement in success rates was related not only to the improved embryo examination and selection, but also to enhancement of ovarian stimulation, the techniques of oocyte insemination, micromanipulation and embryo transfer, preimplantation genetic diagnosis, and, fi nally the composition of culture media.

Non-invasive embryo examination is based on simple methods of ob-servation focused on morphology and dynamics of embryo development. The analysis is performed under contrast-phase microscope with Hoffmann modulation contrast (HMC) or difference-interference contrast (DIC), en-abling more precise assessment without fi xing and staining. Initially, the embryologists assessed only several parameters corresponding to embryo

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quality. Increasing number of retro- as well as prospective studies deter-mined a group of morphological features to be useful in predicting embryo quality. At present several classifi cations concerning many different criteria (embryo scores) are utilized in ART [10, 16, 34, 37, 51, 58]. Embryologi-cal scoring covers oocytes, zygotes, 2-12 cell embryos and blastocysts. Generally, the following parameters infl uence most often the selection of the good quality embryos:

• pronuclear morphology;

• polar body structure and placement;

• appearance of cytoplasm (pitting, vacuoles and halo effects) and zona pellucida;

• early cleavage;

• number of blastomeres in particular days of culture; • size, symmetry and fragmentation of blastomeres; • compaction and expansion of blastomeres;

• multinucleation – more than one nucleus in each blastomere. ZYGOTES SCORING SYSTEMS

Routine embryo evaluation commences 16-18 hours following oocyte insemination (IVF) or ICSI [34, 47, 48, 50, 52, 53, 56]. In classic IVF procedure the follicular cells need to be removed (denudation) at time of examination to enable more precise examination. Different classifi cations are utilized to access the pronuclear stage zygote 16-18 hours after fertil-ization. The most popular, “zygote grading system” or “pronuclear scoring system” was introduced following the observation made by the pioneer in this area, Van Blerkom (1990), who found that symmetry and dimension of pronuclei as well as number and location of nucleoli were related to pregnancy rates. Since than, there were many attempts to standardize the pronuclear embryos grading. The system proposed by Scott et al. ([48]; see below) has been widely accepted and numerous reports confi rm its useful-ness in selection of good quality embryos, resulting in higher implantation rates per transfer. Needless to say, several modifi cations of the system were developed in the past few years, with scoring proposed by Tesaric at al. [52,

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53] to be commonly recognized. Generally, pronuclei grading is performed 16-18 hours after fertilization. Attention is paid to:

• pronuclear size and symmetry;

• size, number, equality and distribution of nucleoli; • appearance of cytoplasm.

Scott et al. [48] classifi ed zygotes into four groups according to pronuclear morphology labeled with grades corresponding to their quality (fi gs.1 and 5): Z1 - Equal pronuclei. Equal number and size of nucleoli, aligned in both

pronuclei at the pronuclear junction. The absolute number of nucleoli ranges between three and seven.

Z2 - Equal pronuclei. Equal number and size of nucleoli, scattered in both pronuclei. The absolute number of nucleoli ranges between three and seven.

Z3 - Equal pronuclei. Equal number and even or (and) uneven size of nu-cleoli, aligned in one pronucleus at the pronuclear junction. The other pronucleus with randomly scattered nucleoli. The absolute number of nucleoli ranges between three and seven.

Z4 - Unequal or separated pronuclei.

Practical implications of this simple method are highly benefi cial. Ac-cording to pronuclear morphology, the zygotes classifi ed as Z1 to Z2 usu-ally yield prospectively better quality embryos. Additionusu-ally, the transfers of such embryos are related to signifi cantly higher pregnancy rates. On the other hand, Z3 zygotes develop into embryos of worse quality and lower implantation potential. The zygotes classifi ed as Z4 are very often affected by chromosomal abnormalities and aneuploidy, so they should not be cul-tured and utilized in ART [25, 32, 36, 38].

The grading system recommended by Tesaric et al. ([52, 53]; fi g. 2) is quite similar to the previous one [48]. It classifi es the zygotes into six patterns of pronuclear morphology where pattern 0 corresponds to normal zygotes and patterns 1-5 represent varying irregularities of zygote morphology.

The described grading systems are widely used in ART and proved to be helpful in selection of embryos for single embryo transfer. Some

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Z2

Z3

Z4 Z1

Fig. 1. Zygote classifi cation according to Scott et al. [48].

previous studies confi rmed that the embryos of comparable morphology at the day of transfer may have signifi cantly different potential, which in turn, was closely associated with pronuclear morphology [4, 10, 14, 39, 47, 48, 52].

Appearance of cytoplasm is of at least equal importance. In normal zygotes the cytoplasm is heterogeneous, with a clear half a moon like zone at the peripheral part of the cell, often referred to as cortical halo effect. Additionally, the presence of peripronuclear halo and cytoplasmic fl are (a dense area of cytoplasm aggregated around the pronuclei) were also reported to be features of good quality zygotes [47, 48, 52].

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Pattern 0 Pattern 1 Pattern 2 Pattern 3 Pattern 4 Pattern 5

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Direction of second polar body extrusion and rotation of pronuclei and cytoplasm have also been reported to be important in embryo scoring [10, 22, 41]. Since the egg cell is polarized, it is postulated that proper location of second polar body on the long pronuclear axis determines different functions of two poles of zygote. Redistribution of cellular organelles infl uences the future of blastomeres of the dividing zygote. The cortical and peripronuclear halos are the signs of zygote polarization. The abnormalities in spatial relationship between pronuclei and the polar bodies are signs related to imminent cleavage disturbances. The usual effects are unequal blastomeres and slower cleavage [22]. The affected embryos are of worse quality and growth rate.

Another parameter that may be analyzed in zygotes is thickness of zona pellucida [17, 18, 40]. Besides HMC, image analysis software may also prove helpful in such assessments. In 1989 Cohen proposed measurements of zona pellucida thickness variation (Z_VAR):

Z_VAR=[(Z_MAX_MAX_MAX–Z–Z_MEAN)÷Z_MEAN]×100%

The strict positive correlation between increased Z_VAR_VAR_VAR and pregnancy and pregnancy rates has been shown in the recent studies [18]. The Z_VAR_VAR_VAR higher than 20% higher than 20% predicts better IVF results and the value below 15% is related with very low pregnancy rates. In spite of relations between the zona pellucida thickness and ART success rates, this parameter, due to practical reasons, is not often used in selection of the best quality embryos for transfer.

Z_VAR>20% is a strong predictor of IVF outcome. The implantation correlates better with Z_VAR than thickness alone. The embryos with an irregularly thick zona pellucida have higher implantation rate than embryos with uniform thickness of zona pellucida. The embryos with thick zonae, greater than 15 microns have a smaller implantation rate than embryos with thinner zonae. For these embryos the assisted hatching (zona drilling) is highly recommended as an effective method to improve the outcome of IVF [5, 8, 9]. The ability to hatch blastocyst correlates negatively with the thickness of zonae.

The pronuclear stage embryo examination is common practice in IVF labs. It helps to select a group of embryos with prospectively good

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implan-tation potential and quality acceptable for cryopreservation. The problem of embryo selection has a legislative meaning in several countries. In Ger-many, for example, only pronuclear zygotes (not cleavage-stage embryos) selection and cryopreservation is allowed [34, 58].

CLEAVED EMBRYOS SCORING

The next step in embryo evaluation is made 24-28 hours after insemination or ICSI, when the presence of fi rst cleavage, blastomere symetry and the extent of fragmentation are examined [16, 29, 35, 45, 46, 57]. Especially the predictive value of the presence of early fi rst cleavage has been shown in numerous reports. Then, a good quality embryo has two equal and symetric blastomeres without or with negligible fragmentation. The fi rst cleavage failure 24-28 hours after fertilization is related to lower implantation rates even in embryos with good morphology on day of transfer [1, 29, 35, 46].

The rate of embryo growth (cleavage) was evaluated in many studies. It seems obvious that high number of blastomeres predicts the higher im-plantation rates. The majority of embryo quality classifi cations utilize the number of cells as the main parameter of the highest predictive value [1, 16, 50, 55, 57]. The frequency of mitotic divisions is related to developmental potential of the embryo. Four or less blastomeres on the third day of cul-ture indicates the extremely low developmental potential and subsequently very small chance for development after transfer. The extended culture of such embryos is often associated with the arrest of divisions, also called developmental block. The good quality embryo has at least 4 cells on the second day and at least 8 cells on the third day of culture.

Thereafter, the embryo inspections are routinely performed in daily intervals, 40-44 hours and 64-68 hours following the insemination or fer-tilization by ICSI [12, 16, 37, 49, 50, 51, 57]. Embryos are divided into classes, depending on several morphological parameters which are evalu-ated at that time. Beside the number of cells, the appearance of blastomeres and the presence of cytoplasm defects or fragmentation are the most often criteria used. The results of scoring are usually coded (fi gs. 3 and 5). Our laboratory has applied the following coding system:

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• fi rst symbol (digit) corresponds to the number of cells.

• second symbol (letter) corresponds to the structure of blastomeres. A - symmetric blastomeres;

B - distinctly asymmetric blastomeres; C - defects of cytoplasm;

• third symbol (digit) referrers to fragmentation; 1 - no fragmentation;

2 - fragmentation less than 20%; 3 - fragmentation between 20-50%; 4 - fragmentation above 50%.

The good quality embryo has many blastomeres and no, or negligible fragmentation. The optimal numbers of blastomeres are 4 to 6 on the second day and 8-12 on the third day of culture. For example, the optimal quality embryos are described as 4A1 on the second day and 8A1 on the third day of culture.

It has been recently apparent, that a Day 3 embryo is more likely to implant than a Day 2 one. This is why the third day classifi cations includ-ing varyinclud-ing criteria has recently gained on signifi cance. A Day 3 embryo

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quality score (D3EQ) is based on the assessment of cell number, pattern of fragmentation and the presence of fi ve additional morphological fea-tures: 1/ blastomeres of equal size, 2/ signs of compaction, 3/ good blas-tomere expansion (blasblas-tomeres touching the zona pellucida with minimal perivitelline space), 4/ clear cellular cytoplasm without vacuoles, and 5/ presence of cytoplasmic pitting [12]. Number of cells, fragmentation and equality of blastomeres are the most important parameters [1, 2, 15, 27]. The cytoplasmic pitting for instance, has a minor predictive value or have not the infl uence of embryo quality [43]. This system defi nes fi ve types of fragmentation patterns (FP):

FP-I - minimal fragments, usually in association with a single blastomere; FP-II - some small fragments, localized in the perivitelline space;

FP-III - many small fragments can be seen throughout the cleavage cavity and perivitelline space;

FP-IV - many fragments, usually in association with uneven-sized blasto-meres. The fragments are often large, almost of the size of a single blastomere;

FP-V - fragmentation is so extensive that blastomeres can not be distin-guished.

The score may be calculated according to following formula: D3EQ score = number of blastomeres – 2(if FP>II) + 0.4 x n

(n – number of additional morphological features present; out of 5 features described above). A perfect 8-cell embryo with all fi ve positive features would be scored as 10 points.

Another classifi cation for embryo assessment on the third day of culture (day 3 scoring) was introduced by Veeck [28]. The number of blastomeres represents the cleaving status of the embryo. Additionally the embryos are graded as following:

Grade 1 - embryo with blastomeres of equal size, no cytoplasmic frag-ments;

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frag-ments or blebs;

Grade 3 - embryo with blastomeres of distinctly unequal size; none or few cytoplasmic fragments;

Grade 4 - embryo with blastomeres of equal or unequal size; signifi cant cytoplasmic fragmentation;

Grade 5 - embryo with few blastomeres of any size; severe or complete fragmentation.

The described system pays particular attention to the presence of multinucleated blastomeres. It was suggested that the mutlinuclation was related to chromosomal abnormalities of the embryo. It was also reported that embryos with multinuclear blastomeres presented lower developmental potential [27, 30, 42].

The third day embryo examination may have a high predictive value if several parameters are considered simultaneously. Although, only the cumulative analysis of all observations carried out during the whole culture period (from fertilization to transfer) may be helpful to choose the embryos of the highest developmental potential. The third day transfer of embryos, which are selected based on such analysis, is related to 30% chance of im-plantation per transfer per 1 embryo. This imim-plantation rate is two to three folds higher than in cycles with simplifi ed embryo evaluation on the day of transfer [16, 57]. Therefore, there are attempts to standardize the embryo scorings performed at different moments of their development. Fisch et al. [16] proposed graduated embryo score to achieve this goal. It accounts for the three embryo examinations performed 16-18 hours, 25-27 hours and 64-67 hours following fertilization. Each of several parameters such as pronuclear morphology, the presence of early cleavage and advanced day 3 evaluation, is rated in point scale. The highest result (100 points) identifi es the embryo of the best quality.

BLASTOCYSTS SCORING SYSTEMS

Although the non-invasive evaluation of embryos at the early stages of their development (until day three following fertilization) is the main subject of

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1

Early blastocyst

Blastocoel less then half of the blastocyst

1AA 2

Blastocyst

Blastocoel more then half of the blastocyst

2AA 3

Blastocyst

Blastocoel fills the blastocyst

3AA 4

Expanded blastocyst

The embryo is large and the zona is thin

4AA 4BB 4CC

Inner cell mass

A

Numerous and tightly packed cells

B

Several and loosely packed cells

C

Few cells

Trophoectoderm

A

Many cells organized in epithelium

B

Several cells organized in loose epithelium

C

Few cells

Fig. 4. Blastocyst grading according to Gardner et al. [20].

this paper, it should be mentioned that since the extended culture combined with day 5 embryo transfer were introduced, also the specifi c blastocyst quality classifi cation was developed [19, 31, 48, 49]. According to it the thin zona pellucida, smooth trophoectoderm, equality and close adhesion of blastomeres, clearly visible blastocyst cavity and the well developed inner cell mass with many closely aggregated cells are the most important parameters correlating to the top blastocyst quality. The blastocyst grading (fi g. 4) was proposed by Gardner et al. [20].

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The non-invasive methods of embryo evaluation do not supply adequate information on chromosomal abnormalities or genetic disorders. The genetic studies on embryos which were not transferred nor cryopreserved revealed the relationship between certain morphological features and chromosomal aberrations. The grade 5 of pronuclear morphology and unequal embryo blastomeres have the most predictive value in this area, indicating high rates of aneuploidy and other chromosomal abnormalities [2, 27, 32, 36, 38, 44, 47, 53].

In last two decades the development of advanced genetic methods as well as micro-manipulation enabled genetic preimplantation diagnosis (PGD). The genetic evaluation of material obtained from one blastomere enables

optimal zygote

Z2(Scott)

Pattern 0(Tesarik) (also note cytoplasmic halo

and 2 polar bodies)

suboptimal zygote

Z3(Scott)

Pattern 1(Tesarik) (also note exocentric location

of PN and fragmented polar bodies)

suboptimal zygote

Z3(Scott)

Pattern 5(Tesarik) (also note exocentric location of PN and uneven outline of

the zygote)

2 cell embryo

2C3

(second day of culture, two uneven blastomeres with cytoplasmic defects, 30-50%

fragmentatation)

2 cell embryo

2A1

(second day of culture)

4 cell embryo

4A2

(second day of culture, minor fragmentation located at one

pole)

6 cell embryo

6A1

(third day of culture)

8 cell embryo

8A1

(third day of culture)

Fig. 5. Examples to illustrate zygote and embryo scoring (see details in text); PN - pronuclei.

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detection of the most frequent chromosomal abnormalities and gene muta-tions. The fl uorescent in situ hybridization (FISH) and polymerase chain reaction (PCR) are very effective screening tests in this area [3, 11, 23, 24, 33, 48]. FISH involves hybridization of fl uorescently labeled probes to chromosomes to detect their most common abnormalities. For instance FISH may help detect the trisomy of chromosome 21 (Down’s syndrome), while PCR a common gene disease like cystic fi brosis. In several countries the PGD has been introduced and accepted in many ART centers. It helps detect abnormalities especially in high risk couples. On the other hand the moral and ethical aspects of these methods should not be underestimated.

There are also attempts to evaluate embryos metabolic performance. The measurement of embryo consumption of oxygen, uptake of pyruvate and glucose, production of lactate and fi nally secretion of enzymes would allow for functional evaluation of the embryos [6, 7, 13, 21, 26, 54]. However, the methods as expensive, time and labor-consuming are still at initial stages of development, awaiting their verifi cation in daily laboratory practice.

To conclude, the non-invasive, microscopic embryo assessment is the most important method to select the most potent embryos in ART. The utilization of simple observation may be very helpful to predict the chance of particular embryo to implant. It is well known that the relatively low success rates in ART programs is a reason for the great number of stud-ies concerning many different aspects of assisted reproduction. Because of its simplicity, cost-effectiveness and correlation with implantation and pregnancy rates the advanced examination of embryo morphology seems to be one of the most important method enabling the improvement of IVF treatment results. Embryologists! Observe the embryos! - said two years ago, the pioneer of IVF, professor Edwards during the opening lecture at the annual ESHRE meeting in Vienna.

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