Zygote evaluation: an efficient tool for embryo selection

C. Wittemer1,4, K. Bettahar-Lebugle1,2, J. Ohl1,2, C. Rongières1,2, I. Nisand1,2 and P. Gerlinger3

1 Centre d'Assistance Médicale à la Procréation, 2 Service de Gynécologie Obstétrique and 3 Service de Biologie de la Reproduction, Centre Médico-Chirurgical et Obstétrical (SIHCUS), 19 rue Louis Pasteur, BP 120, 67303 Schiltigheim, France


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the main problems concerning IVF units is the need to decrease the occurrence of multiple pregnancies in their practice without affecting the overall success rate. Different embryological parameters concerning every step of the early embryo development are known to have some predictive value for implantation potential. In this prospective study, a pronuclear scoring system was used to classify zygotes into six patterns from 0 to 5. Cleaved, day 3 embryos developed from pattern 0 zygotes, which was described as the normal pattern, were transferred when available. For each zygote pattern, the subsequent embryological development was analysed. Pattern 0 zygotes led to significantly more `good quality' embryos with higher implantation potential than embryos developing from the other zygote patterns (P < 0.01). Embryo transfers including at least one pattern 0 resulted in significantly more pregnancies than transfers without any pattern 0 zygotes (39.3 versus 19.7%, P < 0.01). No relationship between clinical parameters (age of female partner, infertility cause) and zygote pattern distribution was demonstrated.

Key words: embryo selection/ICSI/IVF/zygote


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
One of the main challenges to IVF units is the avoidance of multiple pregnancies without lowering overall success rate. Multiple pregnancies indeed are associated with worse obstetric and neonatal outcomes (Evans et al., 1998Go). Transferring only one embryo represents the ultimate goal and there have been many studies dedicated to the choice of the `right embryo' (Vilska et al., 1999Go; Gerris et al., 1999Go; Van Royen et al., 1999Go). Several parameters of early embryo development are known to have some predictive value for implantation potential. Numerous oocyte criteria including cumulus-coronal morphology (Ng et al., 1999Go), polar body aspect and position (Ebner et al., 1999Go, 2000Go), nuclear maturity or oocyte polarity (Garello et al., 1999Go) were extensively studied and could help to select oocytes with increased chances of fertilization. At the pronuclear stage, the genomes originating from both gametes are still physically separated within their respective pronuclei. Relationships between pronuclear morphology and the ability of an embryo to implant have also been suggested (Payne et al., 1997Go; Scott and Smith, 1998Go; Tesarik and Greco, 1999Go). The embryo morphology 2 or 3 days after oocyte retrieval is the most frequently used parameter to assess a patient's probability of success. Delaying embryo transfer until the blastocyst stage could also be envisioned with several expected advantages: embryonic selection, higher implantation rate, better embryonic-endometrial synchronization (Gardner et al., 1998aGo,bGo).

Numerous embryo grading systems have been proposed but the question concerning the choice of the right embryo remained open. In an attempt to settle this question, the present prospective study in which a combination of embryo evaluations was used: pronuclear stage morphology as previously described (Tesarik and Greco, 1999Go), classical cleavage stage morphology on days 2 and 3 and embryo development between days 2 and 3 after oocyte retrieval. Particular attention was given to the following clinical parameters: infertility indication, maternal age and type of assisted reproduction technique: IVF or intracytoplasmic sperm injection (ICSI). The objective was to evaluate whether the pronuclear scoring system would yield a more efficient selection of viable embryos, thereby increasing the implantation rate and allowing the reduction of number of embryos transferred in order to avoid multiple pregnancies.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
This study concerned 262 couples treated for infertility problems and having an embryo transfer between July and November 1999. Extensive counselling was given beforehand to the couples and the study was approved by the Internal Ethical Committee. Ovarian stimulation, oocyte recovery, IVF and ICSI procedures were carried out using standard protocols as previously described (Van Steirteghem et al.,1993Go; Wittemer et al., 2000Go).

Assessment of pronuclear and embryo morphology
Each oocyte recovered was placed individually into a well (4-well dishes; NunclonTM Products, Roskilde, Denmark) containing 0.7 ml of G1.2 culture medium (G1.2TM; IVF Science Scandinavia, Gothenburg, Sweden) and checked for pronuclear morphology 14–18 h after in-vitro insemination or ICSI. At this stage, the number and distribution of nucleolar precursor bodies (NPB) in each pronucleus were assessed. Each zygote was then classified into one of the six previously described patterns (Tesarik and Greco, 1999Go). The different patterns of pronuclear stage morphology are presented in Figure 1Go. Scoring criteria of the zygotes were as previously described (Tesarik and Greco, 1999Go) and are summarized as follows. Zygotes with a `normal' pattern 0 presented the four following characteristics: (i) the number of NPB in both pronuclei never differed by more than three; (ii) NPB always polarized when fewer than seven and never polarized when more than seven in at least one pronucleus; (iii) the number of NPB in a pronucleus never fewer than three; (iv) the distribution of NPB either polarized or non-polarized in both pronuclei. Zygotes that did not conform to this morphological pattern were considered as abnormal and classified into one of the five following patterns: pattern 1 comprised zygotes that presented a large difference (>3) in the number of NPB between both pronuclei; pattern 2 included zygotes with a small number (<7) of NPB without polarization in at least one pronucleus; pattern 3 included zygotes with a large number (>7) of NPB with polarization in at least one pronucleus; pattern 4 zygotes had a very small number (<3) of NPB in at least one pronucleus and zygotes of pattern 5 presented a polarized distribution of NPB in one pronucleus and non-polarized in the other.



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Figure 1. Different patterns of pronuclear stage embryo morphology (adapted from Tesarik and Greco, 1999). See text for explanation.

 
Cleavage rate and embryo morphology were evaluated on the following 2 days of culture in G1.2 medium, by a system applied in our centre for several years. The morphological criteria considered were the number of blastomeres and the proportion of anucleate fragments inside the embryo. To evaluate the embryo development kinetics, the number of blastomeres was compared on day 2 and day 3. In our practice, all embryo transfers were performed on day 3.

Quantitative evaluations
For each zygote morphological pattern, the frequency of specific developmental fates was calculated: cleavage arrest (embryos with the same number of blastomeres 48 h and 72 h after insemination), degenerated embryos (high development of fragmentation between 48 h and 72 h after insemination) and embryos with delayed development (the number of blastomeres increased between 48 and 72 h but without reaching five). Zygotes that did not present one of these described developmental fates were considered as potentially `good quality' embryos.

The distribution among the six zygote patterns was evaluated according to infertility causes, maternal age and assisted reproduction technique.

For transfer, `good quality' embryos developed from pattern 0 zygotes were preferentially selected. Three types of embryo transfers were consequently distinguished: type A consisting of embryos developed from pattern 0 zygotes, type B consisting of embryos developed from different patterns with at least one pattern 0 zygote, and type C consisting of embryos developed from non-pattern 0 zygotes. For each type of transfer (A, B or C), infertility cause, maternal age, assisted reproduction technique as well as the number of transferred embryos, were analysed. The evolution of the initiated pregnancies at least 3 months after embryo transfer was noted. Initiated pregnancy was diagnosed by a positive ß-human chorionic gonadotrophin pregnancy test performed 20 days after embryo transfer; clinical pregnancy was defined by the visualization by ultrasound 6 weeks after embryo transfer of an intrauterine gestational sac with heart beat.

Statistics
Results were expressed as means (±SD) and percentages. Analysis of variance was used to compare group means while the {chi}2-test was used to compare proportions. The level of significance was set at P < 0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
General characteristics
For the 262 couples included in this study, general characteristics concerning causes of infertility (female factor only, male factor only, associated female and male factors and unexplained infertility), maternal age and assisted reproduction technique (IVF or ICSI) were noted and presented in Table IGo. The pregnancy rate per transfer was not significantly affected by the cause of infertility or assisted reproduction technique. In addition no significant difference could be found in pregnancy rate per transfer considering maternal age: 34.7% for women 38 years old or less (239 cases) compared to 30.4% for women older than 38 years (23 cases). The mean (±SD) ages of pregnant and non-pregnant women were not different (32.5 ± 4.21 and 32.9 ± 4.08 years respectively).


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Table I. General characteristics of the studied population
 
Pronuclear zygotes study
One thousand zygotes obtained during the 262 attempts were individually observed and followed from 18 to 72 h after insemination or ICSI.

Relationship between the zygote pattern and subsequent embryonic development
Considering all cleaved embryos subsequently developed from the 1000 studied zygotes, 170 presented cleavage arrest, 42 spontaneously degenerated, 144 had delayed development and the majority of them (644) were considered as good morphology embryos.

According to the six pronuclear patterns, the developmental fate of the 1000 pronuclear zygotes is presented on Table IIGo. The proportion of spontaneously arrested embryos varied from 14.1 to 33.3% and was significantly different among the six zygote patterns (P < 0.05), with a markedly low rate for pattern 0 zygotes (14.1%) and high rate for pattern 3 zygotes (33.3%). The proportion of degenerated embryos remained low: from 2.4 to 5.4% irrespective of the zygote pattern, without any significant difference. The proportion of embryos with delayed development varied between 11.3 and 20.3% without significant difference between the six zygote patterns. Good morphology embryos represented at least 50% of all the embryos, from 52.4% for pattern 3 zygotes to 68.0% for pattern 0 zygotes (no significant difference). To sum up, pattern 0 zygotes generated significantly fewer arrested embryos (P < 0.01) and more `good morphology' embryos (P < 0.02) than pooled zygotes of all other patterns (respectively 14.1 versus 20.0% arrested embryos and 68.0 versus 60.5% `good morphology' embryos). On the contrary, pattern 3 zygotes led to significantly more arrested embryos (P < 0.01) (33.3 versus 16.3% for the zygotes of other patterns). Although pattern 3 zygotes had fewer good morphology embryos than the zygotes of all other patterns, the difference (52.4 versus 64.9%) was not significant.


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Table II. Developmental fate of zygotes according to their pronuclear stage morphology
 
Influence of infertility causes, age of female partner and assisted reproduction technique on zygote pattern
The distribution of the six zygote patterns was evaluated according to the four previously described infertility causes, maternal age and type of assisted reproduction technique (Table IIIGo). No significant difference was found between the distribution of different zygote patterns in relation to these parameters.


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Table III. Relationship between zygote patterns and infertility causes, maternal age and assisted reproduction technique
 
Embryo transfer study
For the three types of embryo transfers: type A (involving only pattern 0 embryos), type B (involving at least one pattern 0 embryo) and type C (without any pattern 0 embryo), several parameters were studied. Distribution of infertility causes, maternal age and assisted reproduction technique were not different between the three types of embryo transfer (data not shown). Other characteristics are presented on Table IVGo. The pregnancy rate per embryo transfer was significantly different (P < 0.01) between the three groups: 42.2% in type A embryo transfer, 35.6% in type B and 19.7% in type C. No specific zygote pattern or embryo transfer type was related to the occurrence of miscarriage. Considering the whole studied population (irrespective of the type of embryo transfer), women suffering from early miscarriage seemed to be older than women presenting a developing pregnancy (33.8 ± 6.6 versus 32.2 ± 3.9 years old), but the difference was not significant. In addition, 12 out of 17 miscarriages occurred in ICSI cycles but the difference in miscarriage rates between IVF and ICSI cycles was not statistically significant (respectively 12.8 and 23.5% of the initiated pregnancies). Concerning embryonic implantation rate, a significant difference was detected between the three types of embryo transfer: when only pattern 0 embryos were transferred (type A) 25.8% implanted, when embryo transfer included at least one pattern 0 embryos together with other embryo patterns (type B), the implantation rate was 18.6% and when only non-pattern 0 embryos were transferred (type C), only 11.8% of them implanted (P < 0.01). Within each embryo transfer group (A, B and C), the impact of zygote pattern on implantation rate according to young (<=38 years old) and older (>38 years) patients was studied (Table VGo). Allowing for the small number of cases in the group >38 years, no significant difference was observed; therefore using pattern 0 zygotes seemed to allow older women to reach the same implantation rate than young patients.


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Table IV. Outcomes from the three different embryo transfer types
 

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Table V. Implantation rate in the three types of embryo transfer according to maternalage
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite the considerable development of assisted reproductive technologies, the clinical result in terms of take-home baby rate remains disappointing. The present work was conducted in our centre in order to improve clinical results of assisted reproduction, especially concerning the adverse outcome which often accompanies multiple pregnancies. To address these issues, different embryonic criteria were used: pronuclear morphology as previously described by Tesarik et al. (Tesarik and Greco, 1999Go; Tesarik et al., 2000Go), cleaved embryo morphology (Hsu et al., 1999Go) and kinetic parameters (Shoukir et al., 1997Go). These criteria would allow us to improve the selection of embryos for transfer. This prospective work was aimed at confirming the clinical value of the classification of Tesarik et al. (2000) who defined a normal pattern of pronuclear morphology which was shared by all the embryos capable of implantation after uterine transfer. The zygotes giving rise to embryos of the 100% implantation group (i.e. clinical pregnancies in which the number of gestational sacs equals the number of embryos transferred) were defined as pattern 0. Zygotes that did not conform to this normal morphological pattern were classified into five patterns according to distinct specific features. In our study, the pronuclear zygotes obtained in 262 IVF or ICSI cycles were classified and pattern 0 embryos (when available) with good morphology on day 3 and normal cleavage rate from day 2 to day 3, were transferred preferentially to good morphology embryos of other morphological patterns. When only low quality embryos (day 3) developed from pattern 0 zygotes, good quality embryos derived from other patterns were preferentially transferred.

The daily practice of assisted reproduction allows the morphological assessment of gametes and embryos prior to transfer. Some of the numerous studies dedicated to the evaluation of these parameters were mentioned here according to the studied developmental stage: oocyte, zygote, early cleavage and blastocyst stages. Concerning oocyte quality, different techniques have been developed to assess accurately the status of the oocyte. Heterogeneity of human oocytes exists even before ovulation and analysis of follicular fluid has been shown to provide much information concerning oocyte developmental competence (Van Blerkom et al., 1997Go). Nuclear assessment of oocytes is possible using a cumulus spreading technique (Veeck, 1991Go). The grading of cumulus-coronal morphology according to the aspect of corona radiata and cumulus cells has also been proposed and seems to be a useful predictor of success (Ng et al., 1999Go). The first polar body could also provide useful information: firstly, its presence in the perivitelline space proves the nuclear maturity of the oocyte and secondly its morphology has been shown to be related to embryo quality (Ebner et al., 1999Go, 2000Go). Concerning the cytoplasmic polarity in human oocytes, important questions about its potential effects on subsequent developmental events are still under debate (Fulka et al., 1998Go).

At the pronuclear zygote stage, previous works have suggested a relationship between pronuclear morphology and implantation ability (Payne et al., 1997Go; Scott and Smith, 1998Go). A reliable scoring system for pronuclear stage oocytes has been proposed by several authors. This embryo scoring was done 14 to 18 h after insemination or ICSI. The main parameters were the respective location of the pronuclei, the distribution of the nucleoli and appearance of cytoplasm (Ludwig et al., 2000Go). The further development up to the first cleavage division was also introduced into the embryo scoring system (Scott and Smith, 1998Go). Other authors proposed a classification of pronuclear zygotes based upon a single static observation of the number and position of nucleoli (Tesarik and Greco, 1999Go).

The morphological evaluation of embryos on day 2 after oocyte retrieval is usually based on the following parameters: number of blastomeres, relative size and shape of blastomeres and evidence of fragmentation. These parameters are used to grade the embryos and the value of these classifications in predicting pregnancy after embryo transfer has been investigated (Erenus et al., 1991Go; Shulman et al., 1993Go; Roseboom et al., 1995Go). In addition to these morphological parameters, embryo viability was also evaluated in relation to the cleavage rate 48 h after oocyte retrieval (Staessen et al., 1992Go; Devreker et al., 1999Go) or the timing of the first cell division (Shoukir et al., 1997Go). All these studies indicated that embryonic kinetic parameters provide a good indication of the likelihood of pregnancy. Estimation of embryo quality after 3 days in-vitro culture (Hsu et al., 1999Go) or at the morula or blastocyst stage (McKiernan and Bavister, 1994Go) has also been done.

Considering the subsequent embryonic development of each zygote pattern, good morphology embryos were obtained in every pattern of pronuclear morphology but were most frequently associated with pattern 0 zygotes [54.5% (351/644) of all good morphology embryos]. This finding demonstrated that the zygote scoring system described could be more accurate than the classical morphological evaluation of cleaved embryos. Indeed, between two good morphology embryos on day 2 or 3 after oocyte retrieval, the zygote score detected irregularities in zygote development possibly harmful for embryo implantation.

Considering embryos with developmental arrest, their presence was markedly increased in zygote pattern 3 compared to other zygote patterns. Pattern 3 of pronuclear stage morphology was characterized by a large number of nucleoli precursor bodies (NPB) with polarization in at least one pronucleus. This situation probably indicated a delayed growth and fusion of NPB compared to the normal morphological picture. Timing of these events was well known in human zygotes and described to be dependent on an early wave of pronuclear transcriptional activity (Tesarik and Kopecny, 1990Go), so the disturbance of these parameters could perhaps affect the developmental potential in embryos derived from pattern 3 zygotes.

Careful examination of the 1000 studied zygotes demonstrated that the breakdown by pronuclear morphology into the six described patterns was independent of cause of infertility, maternal age and assisted reproduction technique. Although it has been shown that the fertilization process after ICSI is accelerated compared to conventional IVF (Nagy et al., 1994Go), no difference in the timing of pronuclear coming out was observed here.

The implantation rate of the transferred embryos according to the pronuclear pattern confirmed that embryos generated from pattern 0 zygotes have the best implantation potential. An embryo transfer strategy based on the choice of embryos developed from pattern 0 zygotes known to have higher implantation potential, would allow the improvement of pregnancy rate and reduce the risk of multiple pregnancies by the transfer of fewer embryos. Different parameters in our own infertility programme have been improved since the zygote scoring system was introduced in July 1999 to select embryos in addition to classical evaluation: compared with results obtained during the 6 months prior to starting this work, overall pregnancy rate per embryo transfer has gone up from 28.3 to 34.4% (P < 0.05), overall implantation rate has increased from 13.4 to 19.8% (P < 0.001) and the multiple pregnancy rate has been reduced from 13.5 to 11.1% (not significant) without any triplet pregnancy.

When embryo transfers involving at least one pattern 0 embryo (type B) were compared to transfers without any pattern 0 embryos (type C), the only statistically significant difference (P < 0.03) consisted in the pregnancy rate which was increased almost 2-fold in the first case (35.6 versus 19.7%). The difference in implantation rate (18.6 versus 11.8%) was not significant. Despite the limitation on the number of transferred embryos (mean 1.9, SD 0.6), 10 twin pregnancies were initiated. Consequently the only way of avoiding multiple pregnancies was to transfer a single embryo. Culturing embryos to the blastocyst stage has often been presented as an adequate strategy to avoid multiple pregnancies (Olivennes et al., 1994Go; Gardner et al., 1998aGo,bGo) because of the high implantation rate associated with this type of transfer (Scholtes and Zeilmaker, 1996Go). Considering discrepancies between data from the numerous studies devoted to this subject, the clinical pregnancy rate initiated after transferring a single blastocyst could not easily be evaluated. Referring to the number of pronuclear zygotes obtained per cycle, transfer of a single blastocyst has not yet proved its efficiency compared with transfer of one cleaved embryo on day 3 selected as described here. It remains to be determined whether the developmental rate to the blastocyst stage is higher for pattern 0 zygotes than for the other pronuclear morphological patterns.

Of course the occurrence of pregnancy could not be predicted by considering only embryological factors (as illustrated in our series where three pregnancies were initiated following the uterine transfer of pattern 3 embryos). Clinical parameters, e.g. those concerning the uterine receptivity have also proved to be important (Salle et al., 1998Go; Weissman et al., 1999Go). For this reason, a prospective study is presently being conducted in our centre in order to determine a global scoring system based on embryological and uterine parameters.

In conclusion, it appears that the zygote scoring system described, associated with morphological and kinetic evaluation of the embryos on day 2 and 3 after oocyte retrieval, allows selection of embryos with a high implantation potential. Use of this strategy would lead to a strict limitation of the number of transferred embryos and therefore could avoid the occurrence of multiple pregnancies with their important drawbacks.


    Acknowledgments
 
We would like to express our gratitude to the laboratory staff, B.Davoine, B.Laborde, C.Revil and N.Ruch, who enthusiastically helped us to study zygote patterns and to enter patient data into the computer database.


    Notes
 
4 To whom correspondence should be addressed at: Centre d'Assistance Médicale à la Procréation, Centre Médico-Chirurgical et Obstétrical (SIHCUS), 19 rue Louis Pasteur, BP 120, 67303 Schiltigheim, France. E-mail: christiane.wittemer{at}wanadoo.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on April 7, 2000; accepted on August 30, 2000.