Vol. 4, No. 1 5 REVIEW PAPER Methods of embryo scoring in in vitro fertilization Tomasz Bączkowski2, Rafał Kurzawa1,2, Wojciech Głąbowski3 2Clinic for Reproduction and Gynecology; 3Departmentof Histology and Embryology,Embryology, 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 1Corresponding author: Clinic for Reproduction and Gynecology, Pomeranian Medical University, 1 Unii Lubelskiej St., 71-252 Szczecin, Poland; e-mail: [email protected] Copyright © 2004 by the Society for Biology of Reproduction 6 The embryo scoring in IVF programs 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 Bączkowski et al. 7 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, 8 The embryo scoring in IVF programs 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. Additionally, 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 Bączkowski et al. 9 Z1 Z2 Z3 Z4 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]. 10 The embryo scoring in IVF programs Pattern 0 Pattern 1 Pattern 2 Pattern 3 Pattern 4 Pattern 5 Fig. 2. Zygote classifi cation according to Tesarik at al. [53]. Bączkowski et al. 11 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