- Email: [email protected]
Embryo Quality Assessment
' ' ' '
0 RIG
N A L
RESEARCH
' ' ' '
EMBRYO QuALITY AssESSMENT Robert Hemmings, MD, FRCSC, 1 Tommaso Falcone, MD, FRCSC, 2 Pierre Miron, MD, FRCSC, 3 13 - Institut
de medecine de la reproduction de Montreal, and McGill University, 3 Universite de Montreal
ABSTRACT
Multiple pregnancy is a common complication of in vitro fertilization (IVF). Embryo quality assessment may allow us to reduce significantly the risk of multiple births by transferring two instead of three pre-embryos per cycle of IVF. So far, the assessment of embryo quality has been mainly morphological, which correlates with the chances of implantation. A more complete assessment might include the functional assessment of pre-embryos before they are transferred into the uterus. Two main methods exist to improve embryo quality, namely: coculture and improvement of culture media. The latter appears to have the most chances of improving, in a standardized way, in vitro development of pre-embryos. RESUME
La grossesse multiple est une complication courante de la fecondation in vitro (FIV) . L' evaluation de la qualite des embryons peut nous permettre de reduire significativement I.e risque de naissances multiples par I.e transfert de deux pre-embryons par cycle de FIV au lieu de trois. ]usqu'a present, !'evaluation de la qualite des embryons est demeuree essentiellement morphologique, presentant une bonne correlation avec la probabilite de nidation. Une evaluation plus complete pourrait comporter une evaluation fonctionnelle des pre-embryons, avant leur transfert dans l' uterus. Il existe deux methodes principales pour ameliorer la qualite des embryons : la co-culture et l' amelioration du milieu de culture. Cette demiere methode semble presenter une probabilite accrue d' ameliorer systematiquement I.e developpement in vitro des pre-embryons.
J SOGC 1995;17:372-5
KEY WORDS
Multiple pregnancy, in vitro fertilization, embryo quality assessment, pre-embryos, coculture, culture media.
number of transferred embryos increase, so does the incidence of multiple pregnancies with their poor outcomes. 2 These early observations have lead many IVF centres to limit to three the number of transferred pre-embryos. Despite such a limit, we still have a high percentage of multiple births, including a significant proportion of triplets. 1
INTRODUCTION
The success of in vitro fertilization depends essentially on the number and quality of embryos transferred, as well as on the uterine receptivity. The success rate of in vitro fertilization increases proportionally with the number of embryos transferred (Table 1 ). 1 Unfortunately, as the
JOURNAL SOGC
}
72
APRIL 1995
' ' ' The transfer of pre-embryos with good morphology has been linked to an increase in pregnancy rates and multiple pregnancies.4 It is, therefore, crucial to evaluate the embryo quality properly in order to transfer the optimal number of pre-embryos, allowing for the maximum pregnancy rate with the minimum incidence of multiple pregnancies. MORPHOLOGICAL CLASSIFICATIONS OF HUMAN PRE-EMBRYOS
It is unfortunate that no standard international classification exists yet regarding the morphological characteristics of pre-embryos. Most authors have classified the quality of pre-embryos in three or four gradesY Steer et al. 's classification is shown in Table 2. 5 A cumulative embryo scoring (CES) system (Table 3) has been developed from that classification in order to optimize the number of preembryos transferred, to avoid higher than twin gestation. These authors suggest that limiting the CES to a maximum of 42 would lead to no reduction in the overall pregnancy rate, would prevent most rriplet or higher number gestations while leaving more embryos for cryopreservation. A simpler classification has been used by Staessen et al. (Table 4). These authors, in a large retrospective study, correlate positively the embryo morphology and cleavage rate to the pregnancy rate and implantation rate. 6 Their data suggest that greater pregnancy rates are obtained when at least two good grade (A and B) embryos are transferred. The highest pregnancy rates (45%) result from the transfer of three high quality (A and B) embryos, but interestingly, in this latter situation, a significantly higher per embryo implantation rate is achieved. These data suggest that "inter-embryo helping" exists when good quality embryos are transferred. Such embryo interaction is not apparent when poor embryos are transferred. Schulman et al. have shown that the incidence of multiple gestation increases as the quality of embryos transferred increases. 4 It appears that once the
JOURNALSOGC
endometrium has allowed one embryo to implant, the other embryos implant more easily. There is growing evidence that the implantation process involves one or more signals from the embryo to the endometrial environment. 7 The possible candidates to act as signals by human preembryos are listed in Table 5. Recently, interleukin I was shown to up-regulate its own receptor in the endometrium while in a mouse model, blockage ofiL-IR was responsible for the inhibition of implantation. 8 These observations suggest that a more complete assessment of human pre-embryo quality should include an assessment of its functions. This functional assessment could be achieved by measuring pre-embryos' secretory products in the conditioned culture medium; alternative methods which have been shown to reflect embryos' functional quality in non-human mammals include measurements of oxygen consumption, glucose consumption or lactate production. 9 METHODS TO IMPROVE EMBRYONIC QUALITY
Two avenues have been proposed in order to improve in vitro embryonic quality: 1) the use of coculture with a number of feeder cells originating from reproductive or non-reproductive organs has been shown to improve in vitro development.10 One randomized study, using cells originating from reproductive organs, found a significant improvement in
373
APRIL1995
' ' ' a diminution in lactate production and decreased glucose uptake, were observed when the culture medium was enriched with vitamins, insulin, epidermal growth factor, and transferrin. 9 These changes in metabolism did not necessarily correlate with the morphological improvement of the embryos, suggesting that both physiological assessment of pre-embryos as well as a morphological assessment could be important to determine embryonic viability. In human pre-embryos, cultured in a medium containing low glucose (for the first 48 hours), high glutamine, high lactate/pyruvate ratio, and EDTA (chelating agent), a signifiqmt improvement in the percentage ofblastocysts obtained and a significant decrease in fragmentation have been reported. 14 These data suggest that bypassing the development block can be achieved as efficiently by altering the culture medium as by coculture. Further assessment of the value of these culture media on pregnancy rates remains to be performed.
the pregnancy rate and implantation rate when the preembryos were cocultured. A recent study, using vero cells originating from the green monkey kidney, did not find such a benefit. 12 It is likely that coculture has a beneficial effect which might vary depending on the feeder cells used. Its mechanism of action remains to be determined. The removal of embryotoxic factors or the addition of embryotrophic factors might explain such a positive effect. 2) An alternative to coculture consists of improving the standard culture medium used in IVF. The composition of most culture media used for the mammalian pre-implantation embryos are based on Krebs Ringer bicarbonate salt solution with energy sources consisting of pyruvate, glucose, and relatively high lactate concentrations compared with human fallopian tubal fluid composition. Protein sources usually consist of bovine serum albumin (BSA) or maternal serum. Gardner et al. have shown that high inorganic phosphate and glucose levels have an inhibitory effect on embryonic development. 9 In lower mammals, improvement in the culture medium ( CZB) has allowed the developmental block previously observed with standard in vitro culture medium to be bypassed. 13 Furthermore, recently, in mouse preembryos, profound alterations in metabolism, including
JOURNAL SOGC
SUMMARY
The morphological characteristics of the embryos transferred in the course of IVF-ET have important prognostic value and can limit the percentage of multiple gestation (in particular, triplets) by transferring only two good quality embryos when available. The good quality embryos have been shown to improve each others' chances of implantation in vivo. The mechanisms involved in such inter-embryo help remain to be clarified, but we have now strong evidence that embryonic secretory products are essential for the implantation process. It is, therefore, likely that the production of such products by pre-embryos could be used as a functional assessment of the physiological quality of pre-embryos. Two avenues have been explored in order to improve the embryo quality and should be assessed further to determine their effects on pregnancy rates. Coculture with different feeder cells has been shown to improve the in vitro development of human pre-embryos. The usefulness of coculture may vary, depending on the nature and origin of feeder cells tised. A more practical approach in improving embryo quality may consist of improving the embryo culture medium used by adding and/or removing elements of the embryonic culture medium used so far. This approach, coupled with varying culture conditions with each stage of development in vitro is likely to allow for the improvement of embryonic quality.
}
74
APRIL 1995
' ' ' REFERENCES 1.
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Edwards RG, Fishel SB, Cohen J, Fehilly CB, Purdy JM, Slater JM. Factors influencing the success of in vitro fertilization for alleviating human infertility. J In Vitro Fert Embryo Transfer 1984; 1:3. Hershlag A, Floch JA, De Cherney AH, Lavy G. Comparison of singleton and multiple pregnancies in IVF and ET. J In Vitro Fert Embryo Transfer 1990;7:157-9. Plachot M. Viability of preimplantation embryos. Clin Obstet Gynaecol 1992;6:327 -38. Shulman a, Ben-Nun I, Ghetler Y, Kaneti H, Shilon M, Beyth Y. Relationship between embryo morphology and implantation rate after in vitro fertilization treatment in conception cycles. Fertil Steril1993;60:123-6. Steer CV, Mills CL, Tan SL, Campbell S, Edwards RG. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in vitro fertilization and embryo transfer programme. Hum Reprod 1992;7:117-9. Staessen C, Camus M, Bollen N, Devroey P, Van Steirtieghem AC. The relationship between embryo quality and the occurrence of multiple pregnancies. Fertil Steril1992,57:626-30. Hemmings R, Langlais J, Falcone T, Bourque J, Granger L, Miron P, Guyda H. Human embryos produce transforming growth factors a and insulin like growth factors II. Fertil Steril1992;58:1 01-4. Simon C, Piquette GN, Frances A. EI-Danassouri I, Polan ML. lnterleukin-1 receptor antagonist (IL-1ra): a novel immune mediator prevents embryonic implantation. Fertil Steril 1993 October suppl. Abstract# 003, pp. 82. Gardner OK, Sakkas D. Mouse embryo cleavage, metabolism and viability: role of medium composition. Hum Reprod 1993;8:288-95. Bongson A. Fong CY, Ng SC, Ratnam S. The search for improved in vitro systems should not be ignored: embryo coculture may be one of them. Hum Reprod 1993;8:1155-62. Wiemer KE, Hoffman 01, Maxson WS, Eager S, Muhlberger B, Fiore I, Cuervo M. Embryonic morphology and rate of implantation of human embryos following coculture on bovine oviductal epithelial cells. Hum Reprod 1993;8:97-101. Sakkas D, Jacquenoud N, Leppens G, Campana A. Comparison of results after in vitro fertilized human embryos are cultured in routine medium and in coculture on vero cells: a randomized study. Fertil Steril1994:61 :521-5. Chatot CL, Ziomek CA, BAvister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 1989;86:679-88. FitzGerald L, DiMattina M. An improved medium for long-term culture of human embryos overcomes the in vitro developmental block and increases blastocyst formation. Fertil Steril1992;57:641-7.
JOURNAL SOGC
51st Annual Clinical Meeting June 23-28, 1995 in Calgary, Alberta The Scientific Programme will include Satellite Symposia entitled "Adolescent Sexuality" and "Imaging in the 90's: a Window into Women's Health", ten Post Graduate Courses, the Cannell Lecture, ten Clinical Updates, six International Symposia including the President's symposium, and ten Workshops. Other activities will include Paper, Poster and Video Presentations, the President's Award Programme, and the "Stump the Professor" session. Three four-hour concurrent sessions for the following sub-specialties will be presented this year: Oncology, Reproductive Endocrinology and Infertility, and Perinatology. Seminars on Presentation Skills· for CME Providers, Patient/Physician Communication, and Human Resources and Economics are also scheduled. This programme will focus mainly on the health of women over 40. A special programme including social and cultural activities for the whole family will enable participants to enjoy everything that the Calgary region has to offer. For complete programme and registration information, please contact Linda Huskins, CME Coordinator, 774 Echo Drive, Ottawa, ON K1S 5N8 Tel. (613) 730-4192/1-800-561-2416 or FAX (613) 730-4314
}
75
APRIL 1995
0 RIG
N A L
RESEARCH
' ' ' '
EMBRYO QuALITY AssESSMENT Robert Hemmings, MD, FRCSC, 1 Tommaso Falcone, MD, FRCSC, 2 Pierre Miron, MD, FRCSC, 3 13 - Institut
de medecine de la reproduction de Montreal, and McGill University, 3 Universite de Montreal
ABSTRACT
Multiple pregnancy is a common complication of in vitro fertilization (IVF). Embryo quality assessment may allow us to reduce significantly the risk of multiple births by transferring two instead of three pre-embryos per cycle of IVF. So far, the assessment of embryo quality has been mainly morphological, which correlates with the chances of implantation. A more complete assessment might include the functional assessment of pre-embryos before they are transferred into the uterus. Two main methods exist to improve embryo quality, namely: coculture and improvement of culture media. The latter appears to have the most chances of improving, in a standardized way, in vitro development of pre-embryos. RESUME
La grossesse multiple est une complication courante de la fecondation in vitro (FIV) . L' evaluation de la qualite des embryons peut nous permettre de reduire significativement I.e risque de naissances multiples par I.e transfert de deux pre-embryons par cycle de FIV au lieu de trois. ]usqu'a present, !'evaluation de la qualite des embryons est demeuree essentiellement morphologique, presentant une bonne correlation avec la probabilite de nidation. Une evaluation plus complete pourrait comporter une evaluation fonctionnelle des pre-embryons, avant leur transfert dans l' uterus. Il existe deux methodes principales pour ameliorer la qualite des embryons : la co-culture et l' amelioration du milieu de culture. Cette demiere methode semble presenter une probabilite accrue d' ameliorer systematiquement I.e developpement in vitro des pre-embryons.
J SOGC 1995;17:372-5
KEY WORDS
Multiple pregnancy, in vitro fertilization, embryo quality assessment, pre-embryos, coculture, culture media.
number of transferred embryos increase, so does the incidence of multiple pregnancies with their poor outcomes. 2 These early observations have lead many IVF centres to limit to three the number of transferred pre-embryos. Despite such a limit, we still have a high percentage of multiple births, including a significant proportion of triplets. 1
INTRODUCTION
The success of in vitro fertilization depends essentially on the number and quality of embryos transferred, as well as on the uterine receptivity. The success rate of in vitro fertilization increases proportionally with the number of embryos transferred (Table 1 ). 1 Unfortunately, as the
JOURNAL SOGC
}
72
APRIL 1995
' ' ' The transfer of pre-embryos with good morphology has been linked to an increase in pregnancy rates and multiple pregnancies.4 It is, therefore, crucial to evaluate the embryo quality properly in order to transfer the optimal number of pre-embryos, allowing for the maximum pregnancy rate with the minimum incidence of multiple pregnancies. MORPHOLOGICAL CLASSIFICATIONS OF HUMAN PRE-EMBRYOS
It is unfortunate that no standard international classification exists yet regarding the morphological characteristics of pre-embryos. Most authors have classified the quality of pre-embryos in three or four gradesY Steer et al. 's classification is shown in Table 2. 5 A cumulative embryo scoring (CES) system (Table 3) has been developed from that classification in order to optimize the number of preembryos transferred, to avoid higher than twin gestation. These authors suggest that limiting the CES to a maximum of 42 would lead to no reduction in the overall pregnancy rate, would prevent most rriplet or higher number gestations while leaving more embryos for cryopreservation. A simpler classification has been used by Staessen et al. (Table 4). These authors, in a large retrospective study, correlate positively the embryo morphology and cleavage rate to the pregnancy rate and implantation rate. 6 Their data suggest that greater pregnancy rates are obtained when at least two good grade (A and B) embryos are transferred. The highest pregnancy rates (45%) result from the transfer of three high quality (A and B) embryos, but interestingly, in this latter situation, a significantly higher per embryo implantation rate is achieved. These data suggest that "inter-embryo helping" exists when good quality embryos are transferred. Such embryo interaction is not apparent when poor embryos are transferred. Schulman et al. have shown that the incidence of multiple gestation increases as the quality of embryos transferred increases. 4 It appears that once the
JOURNALSOGC
endometrium has allowed one embryo to implant, the other embryos implant more easily. There is growing evidence that the implantation process involves one or more signals from the embryo to the endometrial environment. 7 The possible candidates to act as signals by human preembryos are listed in Table 5. Recently, interleukin I was shown to up-regulate its own receptor in the endometrium while in a mouse model, blockage ofiL-IR was responsible for the inhibition of implantation. 8 These observations suggest that a more complete assessment of human pre-embryo quality should include an assessment of its functions. This functional assessment could be achieved by measuring pre-embryos' secretory products in the conditioned culture medium; alternative methods which have been shown to reflect embryos' functional quality in non-human mammals include measurements of oxygen consumption, glucose consumption or lactate production. 9 METHODS TO IMPROVE EMBRYONIC QUALITY
Two avenues have been proposed in order to improve in vitro embryonic quality: 1) the use of coculture with a number of feeder cells originating from reproductive or non-reproductive organs has been shown to improve in vitro development.10 One randomized study, using cells originating from reproductive organs, found a significant improvement in
373
APRIL1995
' ' ' a diminution in lactate production and decreased glucose uptake, were observed when the culture medium was enriched with vitamins, insulin, epidermal growth factor, and transferrin. 9 These changes in metabolism did not necessarily correlate with the morphological improvement of the embryos, suggesting that both physiological assessment of pre-embryos as well as a morphological assessment could be important to determine embryonic viability. In human pre-embryos, cultured in a medium containing low glucose (for the first 48 hours), high glutamine, high lactate/pyruvate ratio, and EDTA (chelating agent), a signifiqmt improvement in the percentage ofblastocysts obtained and a significant decrease in fragmentation have been reported. 14 These data suggest that bypassing the development block can be achieved as efficiently by altering the culture medium as by coculture. Further assessment of the value of these culture media on pregnancy rates remains to be performed.
the pregnancy rate and implantation rate when the preembryos were cocultured. A recent study, using vero cells originating from the green monkey kidney, did not find such a benefit. 12 It is likely that coculture has a beneficial effect which might vary depending on the feeder cells used. Its mechanism of action remains to be determined. The removal of embryotoxic factors or the addition of embryotrophic factors might explain such a positive effect. 2) An alternative to coculture consists of improving the standard culture medium used in IVF. The composition of most culture media used for the mammalian pre-implantation embryos are based on Krebs Ringer bicarbonate salt solution with energy sources consisting of pyruvate, glucose, and relatively high lactate concentrations compared with human fallopian tubal fluid composition. Protein sources usually consist of bovine serum albumin (BSA) or maternal serum. Gardner et al. have shown that high inorganic phosphate and glucose levels have an inhibitory effect on embryonic development. 9 In lower mammals, improvement in the culture medium ( CZB) has allowed the developmental block previously observed with standard in vitro culture medium to be bypassed. 13 Furthermore, recently, in mouse preembryos, profound alterations in metabolism, including
JOURNAL SOGC
SUMMARY
The morphological characteristics of the embryos transferred in the course of IVF-ET have important prognostic value and can limit the percentage of multiple gestation (in particular, triplets) by transferring only two good quality embryos when available. The good quality embryos have been shown to improve each others' chances of implantation in vivo. The mechanisms involved in such inter-embryo help remain to be clarified, but we have now strong evidence that embryonic secretory products are essential for the implantation process. It is, therefore, likely that the production of such products by pre-embryos could be used as a functional assessment of the physiological quality of pre-embryos. Two avenues have been explored in order to improve the embryo quality and should be assessed further to determine their effects on pregnancy rates. Coculture with different feeder cells has been shown to improve the in vitro development of human pre-embryos. The usefulness of coculture may vary, depending on the nature and origin of feeder cells tised. A more practical approach in improving embryo quality may consist of improving the embryo culture medium used by adding and/or removing elements of the embryonic culture medium used so far. This approach, coupled with varying culture conditions with each stage of development in vitro is likely to allow for the improvement of embryonic quality.
}
74
APRIL 1995
' ' ' REFERENCES 1.
2.
3. 4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Edwards RG, Fishel SB, Cohen J, Fehilly CB, Purdy JM, Slater JM. Factors influencing the success of in vitro fertilization for alleviating human infertility. J In Vitro Fert Embryo Transfer 1984; 1:3. Hershlag A, Floch JA, De Cherney AH, Lavy G. Comparison of singleton and multiple pregnancies in IVF and ET. J In Vitro Fert Embryo Transfer 1990;7:157-9. Plachot M. Viability of preimplantation embryos. Clin Obstet Gynaecol 1992;6:327 -38. Shulman a, Ben-Nun I, Ghetler Y, Kaneti H, Shilon M, Beyth Y. Relationship between embryo morphology and implantation rate after in vitro fertilization treatment in conception cycles. Fertil Steril1993;60:123-6. Steer CV, Mills CL, Tan SL, Campbell S, Edwards RG. The cumulative embryo score: a predictive embryo scoring technique to select the optimal number of embryos to transfer in an in vitro fertilization and embryo transfer programme. Hum Reprod 1992;7:117-9. Staessen C, Camus M, Bollen N, Devroey P, Van Steirtieghem AC. The relationship between embryo quality and the occurrence of multiple pregnancies. Fertil Steril1992,57:626-30. Hemmings R, Langlais J, Falcone T, Bourque J, Granger L, Miron P, Guyda H. Human embryos produce transforming growth factors a and insulin like growth factors II. Fertil Steril1992;58:1 01-4. Simon C, Piquette GN, Frances A. EI-Danassouri I, Polan ML. lnterleukin-1 receptor antagonist (IL-1ra): a novel immune mediator prevents embryonic implantation. Fertil Steril 1993 October suppl. Abstract# 003, pp. 82. Gardner OK, Sakkas D. Mouse embryo cleavage, metabolism and viability: role of medium composition. Hum Reprod 1993;8:288-95. Bongson A. Fong CY, Ng SC, Ratnam S. The search for improved in vitro systems should not be ignored: embryo coculture may be one of them. Hum Reprod 1993;8:1155-62. Wiemer KE, Hoffman 01, Maxson WS, Eager S, Muhlberger B, Fiore I, Cuervo M. Embryonic morphology and rate of implantation of human embryos following coculture on bovine oviductal epithelial cells. Hum Reprod 1993;8:97-101. Sakkas D, Jacquenoud N, Leppens G, Campana A. Comparison of results after in vitro fertilized human embryos are cultured in routine medium and in coculture on vero cells: a randomized study. Fertil Steril1994:61 :521-5. Chatot CL, Ziomek CA, BAvister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 1989;86:679-88. FitzGerald L, DiMattina M. An improved medium for long-term culture of human embryos overcomes the in vitro developmental block and increases blastocyst formation. Fertil Steril1992;57:641-7.
JOURNAL SOGC
51st Annual Clinical Meeting June 23-28, 1995 in Calgary, Alberta The Scientific Programme will include Satellite Symposia entitled "Adolescent Sexuality" and "Imaging in the 90's: a Window into Women's Health", ten Post Graduate Courses, the Cannell Lecture, ten Clinical Updates, six International Symposia including the President's symposium, and ten Workshops. Other activities will include Paper, Poster and Video Presentations, the President's Award Programme, and the "Stump the Professor" session. Three four-hour concurrent sessions for the following sub-specialties will be presented this year: Oncology, Reproductive Endocrinology and Infertility, and Perinatology. Seminars on Presentation Skills· for CME Providers, Patient/Physician Communication, and Human Resources and Economics are also scheduled. This programme will focus mainly on the health of women over 40. A special programme including social and cultural activities for the whole family will enable participants to enjoy everything that the Calgary region has to offer. For complete programme and registration information, please contact Linda Huskins, CME Coordinator, 774 Echo Drive, Ottawa, ON K1S 5N8 Tel. (613) 730-4192/1-800-561-2416 or FAX (613) 730-4314
}
75
APRIL 1995