SCORING OF EMBRYOS

DR.RENUKADEVI SENIOR EMBRYOLOGIST

ARC INTERNATION FERTILITY AND RESEARCH CENTER

INTRODUCTION

• It has been 25 years since the introduction of in vitro fertilization (IVF)for treatment of infertility.

• Non-invasive methods of embryo evaluation are useful in reproductive medicine.• They help assess embryos without damage. Until recently they were only of scientific

importance, but since rapid advances in the field of assisted reproductive techniques (ART) they have gained more practical meaning.

• All ART specialists, particularly embryologists who handle human germ cells and embryos, are now obliged to be familiar with precise,non-traumatic techniques of embryo evaluation

• Precise examination of embryos on particular days following invitro fertilization (IVF) facilitate selection of the most potent embryos for transfer .

• selection of the best embryo for transfer reduces the number of transferred embryos and subsequently the incidenceof multifetal pregnancies.

METHODS OF EXAMINATION

• Embryological scoring covers oocytes, zygotes, 2-12 cell embryos and blastocysts.Generally, the following parameters influence 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 zonapellucida; 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.

pronuclear morphology

• Different classifi cations are utilized to access the pronuclear stage zygote 16-18 hours after fertilization.

• The most popular, “zygote grading system” or “pronuclear scoring system”

Steps of zygote scoring pronuclear size and symmetry; size, number, equality and distribution of nucleoli; appearance of cytoplasm

Zygote scoring system

1. 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.

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

3. Z3 - Equal pronuclei. Equal number and even or (and) uneven size of nucleoli,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.

4. Z4 - Unequal or separated pronuclei.

REPRESENTATION

CLEAVED EMBRYOS SCORING ( Day 2 to Day 5)

• 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

• a good quality embryo has two equal and symmetric blastomeres without or with negligible fragmentation

• The rate of embryo growth (cleavage) was evaluated in many studies.It seems obvious that high number of blastomeres predicts the higher implantation rates.

• The frequency of mitotic divisions is related to developmental potential of the embryo

Day -2 scoring

• The first cleavage failure 24-28 hours after fertilization is related to lower implantation rates even in embryos with good morphology on day of transfer.

• The good quality embryo has at least 4 cells on the second day• Embryos are divided into classes, depending on several morphological

parameters which are evaluated at that time.1. Division of blastomeres2. Fragmentation status• Beside the number of cells, the appearance of blastomeres and the presence

of cytoplasm defects or fragmentation are the most often criteria used.• The optimal quality embryos are described as 4A1 on the second day culture.

Day 2 embryo grading with blastomeres and fragmentation criteria

Day -3 embryo scoring

• The good quality embryo has atleast 8 cells on the third day of culture.• The optimal numbers of blastomeres are 4 to 6 on the second day and

8-12 on the third day of culture.• The optimal quality embryos are described as 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• The third day classifications including varying criteria has recently

gained on significance.

• 1/ blastomeres of equal size,• 2/ signs of compaction, • 3/ good blastomere expansion

(blastomeres touching the zona pellucida with minimal perivitelline space),

• 4/ clear cellular cytoplasm without vacuoles

• 5/presence of cytoplasmic pitting

• 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 blastomeres.

• The fragments are often large, almost of the size of a single

• blastomere;• FP-V - fragmentation is so extensive that

blastomeres can not be distinguished.

CRITERIA -1 ( DIVISION OF BLASTOMERES)

CRITERA -2 ( FRAGMENTATION STATUS)

REPRESENTATION OF D3 EMBRYOS

BLASTOCYSTS SCORING FROM ( DAY 4 TO DAY 5)

• The non-invasive evaluation of embryos at the early stages of their development (until day three following fertilization)

Parameters of blast scoring• 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.

EARY BLAST STAGE SCORING BLAST STAGE SCORING

LARGE EXPANDED BLAST SCORINGHATCHING BLAST

REFERENCE

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Bahce M Cohen J Munne S 1999 Preimplantation genetic diagnosis of aneuploidy: were we looking at the wrong chromosomes? Journal of Assisted Reproduction and Genetics 16 176-181.

Balaban B Urman B Isiclar A Alatas C Aksoy S Mercan R Mumcu A Nuhoglu A 2001 The effect of pronuclear morphology on embryo quality parameters and blastocyst transfer outcome. Human Reproduction 16 2357-2361.

Balaban B Urman B Alatas C Mercan R Mumcu A Isiklar A. 2002 Comparison of four different techniques of assisted hatching. Human Reproduction 17 1239-1243.

Battaglia C Ciotti P Notarangelo L Fratto R Facchinetti F de Aloysio D 2003 Embryonic production of nitric oxide and its role in implantation: a pilot study. Journal of Assisted Reproduction and Genetics 20 449-454.

Butcher L Coates A Martin KL Rutherford AJ Leese HJ. 1998 Metabolism of pyruvate by the early human embryo. Biology of Reproduction 58 1054-1056.

Cohen J Elsner C Kort H Malter H Massey J Mayer MP Weimer K. 1990 Impairment of the htaching process following IVF in the human and improvement of implantation by assisted hatching using micromanipulation. Human Reproduction 5 7-13.