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
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Abstract |
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Key words: embryo selection/ICSI/IVF/zygote
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Introduction |
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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, 1999), 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.
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Materials and methods |
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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 1418 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, 1999). The different patterns of pronuclear stage morphology are presented in Figure 1
. Scoring criteria of the zygotes were as previously described (Tesarik and Greco, 1999
) 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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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 2-test was used to compare proportions. The level of significance was set at P < 0.05.
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Results |
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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 II. 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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Discussion |
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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., 1997). Nuclear assessment of oocytes is possible using a cumulus spreading technique (Veeck, 1991
). 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., 1999
). 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., 1999
, 2000
). Concerning the cytoplasmic polarity in human oocytes, important questions about its potential effects on subsequent developmental events are still under debate (Fulka et al., 1998
).
At the pronuclear zygote stage, previous works have suggested a relationship between pronuclear morphology and implantation ability (Payne et al., 1997; Scott and Smith, 1998
). 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., 2000
). The further development up to the first cleavage division was also introduced into the embryo scoring system (Scott and Smith, 1998
). Other authors proposed a classification of pronuclear zygotes based upon a single static observation of the number and position of nucleoli (Tesarik and Greco, 1999
).
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., 1991; Shulman et al., 1993
; Roseboom et al., 1995
). 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., 1992
; Devreker et al., 1999
) or the timing of the first cell division (Shoukir et al., 1997
). 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., 1999
) or at the morula or blastocyst stage (McKiernan and Bavister, 1994
) 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, 1990), 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., 1994), 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., 1994; Gardner et al., 1998a
,b
) because of the high implantation rate associated with this type of transfer (Scholtes and Zeilmaker, 1996
). 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., 1998; Weissman et al., 1999
). 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.
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Acknowledgments |
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Notes |
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References |
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Ebner, T., Moser, M., Yaman, C. et al. (1999) Elective transfer of embryos selected on the basis of first polar body morphology is associated with increased rates of implantation and pregnancy. Fertil. Steril., 72, 599603.[ISI][Medline]
Ebner, T., Yaman, C., Moser, M. et al. (2000) Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum. Reprod., 15, 427430.
Erenus, M., Zouves, C., Rajamahendran, P. et al. (1991) The effect of embryo quality on subsequent pregnancy rates after in vitro fertilization. Fertil. Steril., 56, 707710.[ISI][Medline]
Evans, M.I., Kramer, R.L, Yaron, Y. et al. (1998) What are the ethical and technical problems associated with multifetal pregnancy gestation? Clin. Obstet. Gynecol., 4, 4754.
Fulka, J., Karnikova, L. and Moor, R.M. (1998) Oocyte polarity: ICSI, cloning and related techniques. Hum. Reprod., 13, 33033305.
Gardner, D.K., Vella, P., Lane, M. et al. (1998a) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril., 69, 8488.[ISI][Medline]
Gardner, D.K., Schoolcraft, W.B., Wagley, L. et al. (1998b) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod., 13, 34343440.[Abstract]
Garello, C., Baker, H., Rai, J. et al. (1999) Pronuclear orientation, polar body placement, and embryo quality after intracytoplasmic sperm injection and in-vitro fertilization: further evidence for polarity in human oocytes ? Hum. Reprod., 14, 25882595.
Gerris, J., De Neubourg, D., Mangelschots, K. et al. (1999) Prevention of twin pregnancy after in-vitro fertilization or intracytoplamsic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum. Reprod., 14, 25812587.
Hsu, M.I., Mayer, J., Aronshon, M. et al. (1999) Embryo implantation in in vitro fertilization and intracytoplamsic sperm injection: impact of cleavage status, morphology grade, and number of embryos transferred. Fertil. Steril., 72, 679685.[ISI][Medline]
Ludwig, M., Schöpper, B., Al-Hasani, S. et al. (2000) Clinical use of a pronuclear stage score following intracytoplasmic sperm injection: impact on pregnancy rates under the conditions of the German embryo protection law. Hum. Reprod., 15, 325329.
McKiernan, S.H. and Bavister, B.D. (1994) Timing of development is a critical parameter for predicting successful embryogenesis. Hum. Reprod., 12, 21232129.[Abstract]
Nagy, P., Liu, J., Joris, H et al. (1994) Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum. Reprod., 9, 17431748.[Abstract]
Ng, S.T., Chang, T.H. and Wu, J.T.C. (1999) Prediction of the rates of fertilization, cleavage, and pregnancy success by cumulus-coronal morphology in an in vitro fertilization program. Fertil. Steril., 72, 412417.[ISI][Medline]
Olivennes, F., Hazout, A., Lelaidier, C. et al. (1994) Four indications for embryo transfer at the blastocyst stage. Hum. Reprod., 9, 23672373.[Abstract]
Payne, D., Flaherty, S., Barry, M. et al. (1997) Preliminary observation on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography. Hum. Reprod., 12, 532541.[ISI][Medline]
Roseboom, T.J., Vermeiden, J.P.W., Schoute, E. et al. (1995) The probability of pregnancy after embryo transfer is affected by the age of the patient, cause of infertility, number of embryos transferred and the average morphology score, as revealed by multiple logistic regression analysis. Hum. Reprod., 10, 30353041.[Abstract]
Salle, B., Bied-Damon, V., Benchaid, M. et al. (1998) Preliminary report of an ultrasonography and color Doppler uterine score to predict uterine receptivity in an in-vitro fertilization program. Hum. Reprod., 13, 16691673.[Abstract]
Scholtes, M. and Zeilmaker, G. (1996) A prospective randomized study of embryo transfer results after 3 or 5 days of embryo culture in in vitro fertilization. Fertil. Steril., 65, 12451248.[ISI][Medline]
Scott, L. and Smith, S. (1998) The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod., 13, 10031013.[Abstract]
Shoukir, Y., Campana, A., Farley, T. and al (1997) Early cleavage of in vitro fertilized human embryos to the 2-cell stage: a novel indicator of embryo quality and viability. Hum. Reprod., 12, 15311536.[Abstract]
Shulman, A., Ben-Nun, I., Ghetler, Y. et al. (1993) Relationship between embryo morphology and implantation rate after in vitro fertilization treatment in conception cycles. Fertil. Steril., 60, 123126.[ISI][Medline]
Staessen, C., Camus, M., Bollen, N. et al. (1992) The relationship between embryo quality and the occurrence of multiple pregnancies. Fertil. Steril., 57, 626630.[ISI][Medline]
Tesarik, J. and Kopecny, V. (1990) Assembly of the nucleolar precursor bodies in human male pronuclei is correlated with an early RNA synthetic activity. Exp. Cell. Res., 191, 153156.[ISI][Medline]
Tesarik, J.and Greco, E. (1999) The probability of abnormal preimplantation development can be predicted by a single static observation on pronuclear stage morphology. Hum. Reprod., 14, 13181323.
Tesarik, J., Junca, A.M., Hazout, A. et al. (2000) Embryos with high implantation potential after intracytoplasmic sperm injection can be recognized by a simple, non-invasive examination of pronuclear morphology. Hum. Reprod., 15, 13961399.
Van Blerkom, J., Antczak, M. and Schrader, R. (1997) The developmental potential of the human oocyte is related to the dissolved oxygen content of follicular fluid: association with vascular endothelial growth factor levels and perifollicular blood flow characteristics. Hum. Reprod., 12, 16101614.[ISI][Medline]
Van Royen, E., Mangelschots, K., De Neubourg, K. et al. (1999) Characterization of a top quality embryo, a step towards single-embryo transfer. Hum. Reprod., 14, 23452349.
Van Steirteghem, A., Nagy, Z., Joris, H. et al. (1993) High fertilization and implantation rates after intracytoplasmic sperm injection. Hum. Reprod., 8, 10611066.[Abstract]
Veeck L. (1991) Atlas of the Human Oocyte and Early Conceptus, Vol. 2. Williams & Wilkins, Baltimore.
Vilska, S., Tiitinen, A., Hydén-Granskog, C. et al. (1999) Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth. Hum. Reprod., 14, 23922395.
Weissman, A., Gotlieb, L. and Casper, R.F. (1999) The detrimental effect of increased endometrial thickness on implantation and pregnancy rates and outcome in an in-vitro fertilization program. Fertil. Steril., 71, 147149.[ISI][Medline]
Wittemer, C., Ohl, J., Bettahar-Lebugle, K. et al. (2000) A quantitative and morphological analysis of oocytes collected during 438 IVF cycles. J. Assist. Reprod. Genet., 17, 4450.[ISI][Medline]
Submitted on April 7, 2000; accepted on August 30, 2000.