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Towards single births after assisted reproduction treatment
RBMOnline - Vol 7. No 5. 506–508 Reproductive BioMedicine Online; www.rbmonline.com/Article/1119 on web 10 October 2003
Commentary Towards single births after assisted reproduction treatment RG Edwards Chief Editor, Reproductive BioMedicine Online, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK Correspondence: e-mail: [email protected]
Abstract The cause of increases in recorded multiple births is undoubtedly assisted human conception. Many arise when three or more embryos are replaced after IVF or ISCI. Others are due to multi-ovulation during artificial insemination or intrauterine inseminations, since complete control over numbers of ovulated eggs following ovarian stimulation is not fully possible. The frequency of multiple IVF births is best controlled by reducing numbers of transferred embryos to one or two. Methods to induce ovarian stimulation in amenorrhoeic and then in cyclic women 30 years ago have been greatly refined and extended in current practice. Curiously, a great many of them produce oocytes which fail to implant after fertilization, whether natural or induced cycles are being used. This massive failure of most embryos to implant poses numerous questions on the evolution of this situation in humans. Keywords: artificial insemination, implantation failure, multiple births, ovarian stimulation The drive to establishing singleton pregnancies after assisted conception is gaining momentum. Individual clinics and regulatory agencies are stressing the urgent need to restrict the number of embryos transferred. Some professionals claim that there is no decline in implantation rates as numbers of transferred embryos are reduced, which seems strange in view of the well-known statistical conclusions that embryos implant independently without interacting with companion embryos in the uterus. Moving to single embryo transfers demands greater understanding of the development of human embryos in vitro, in order to select the best for transfer. Remarkable progress along these lines depended on successive parameters being assessed and introduced into clinical practice. Rapid cleavage or polarization, the nature of blastomere fragments and the introduction of new culture media are slowly winning new converts to the small adaptations needed in laboratory practice to achieve this end.
506
A second approach to reducing multiple pregnancies is to pressurize clinics still transferring three, four or even more embryos. In some countries, legislation has insisted on the transfer of a maximum of three and then two embryos to reduce high-order multiples. This approach requires constant monitoring, by introducing auditing by outside agencies or returning detailed statistics to regulatory authorities. Pressures such as these will convince most professionals to declare truthfully the exact details of their transfers. Constant attention to this point in successive conferences is also influencing the attitudes of clinics to this need. A highly motivated international Expert Meeting on multiple pregnancies after assisted reproduction techniques will add its authority to restricting numbers of transferred embryos. Rightly, their paper is entitled ‘An Ongoing Epidemic’, since that is exactly what it is. The epidemic of multiple births has indeed been largely due to assisted reproduction. This can be seen from the various approaches to ovarian stimulation which were introduced successfully and became increasingly complex.
Even in its beginnings, some form of stimulation was considered to be essential to obtain sufficient oocytes for fertilization. At the time, it was recognized that mild multifolliculation was needed to produce several oocytes (Steptoe and Edwards, 1970). Studies on ovulation induction in animals, and especially mice, had shown how the number of ovulated oocytes in individual females could not be predicted, even within inbred strains or among F1 hybrids, which are usually considered as conferring enormous stability. Up to 100 oocytes were ovulated by some mouse females, and 50 or more fetuses competed for the limited uterine space available (Fowler and Edwards, 1957). Exactly the same situation arose when gonadotrophins extracted from human pituitaries, prepared from serum of pregnant mares, and lastly prepared from human postmenopausal urine were used to induce ovulation in amenorrhorrheic women. Press and TV reports, then in their infancy, described quintuplets, even octuplets, in some patients, despite the careful monitoring of urinary oestrogens as a measure of their response. Even with modern ultrasound and recombinant gonadotrophins, some mothers established octuplets quite against the desires of clinicians. Immense improvement in this technique developed from the introduction of IVF, where the very first clinical papers on human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) revealed variations in follicle numbers and the exact timing of follicle rupture (Steptoe and Edwards, 1970). Ovulation induction, artificial insemination, GIFT and IVF could now be practised with considerable accuracy, which increased pregnancy rates considerably, and also enabled the implantation of several embryos. Compared with today’s dosages, the amounts of gonadotrophins used were modest at 2–3 ampoules of HMG (i.e. 150–225 IU) three times during the follicular phase, followed by between 1000 and 12,500 IU HCG, which became standardized at 5000 IU. Even so, a mean of 6.6 oocytes were recovered per cycle and
Commentaries - Towards single births after ART - RG Edwards
most were fertilized in vitro, giving a mean of approximately four or five fertilized eggs. Half of the resulting embryos reached the blastocyst stage, some of them developing to day 9 in vitro (Edwards and Surani, 1997).
Table 1. Natural cycle IVF in Bourn Hall, 1982.
This number of oocytes seemed adequate for IVF, as indicated by experience in animals. Detailed experience with implantation rates per embryo in many mammalian species showed them to be 70% or greater. Consequently, in the opening clinical phases of human IVF, only a single embryo was chosen for transfer to its mother’s uterus even if four or five were available. The remainder were used for study or, in later years, cryopreserved for future use as the necessary techniques were understood. Emphasis was placed on the need for singleton pregnancies in attempts to spare mothers from multiple births. Human embryos were assumed to be the same, so transferring two was expected inevitably to lead to twins. As it transpired, this early research produced very few births, due to the very short luteal phases in stimulated IVF patients. Primulot depot was unwittingly used as a luteotrophin to overcome these luteal phase defects, but it turned out to be an abortifacient! Given just before or after blastocyst transfer, it caused the termination of many pregnancies by 1 week posttransfer, resulting in the term ‘biochemical pregnancies’.
Aspirations ≥One egg aspirated ≥One or more fertilized Replacements Clinical pregnancies % per replacement % per patients with eggs % per aspiration Biochemical pregnancies
Utilizing several other forms of ovarian stimulation, and avoiding Primulot, gave more, but still very few, pregnancies in response to transferring mostly single embryos. Rare transfers of two blastocysts, or replacing two mature oocytes plus spermatozoa in the oviduct, were performed with trepidation, since a primary target remained the avoidance of multiple births. Suspicions about low rates of implantation emerged even more strongly when natural cycle IVF was introduced. The final Oldham results produced four pregnancies, and perhaps one additional biochemical pregnancy, out of 32 transfers. There was no obvious reason for this low rate of success, since 8-cell embryos and blastocysts were healthy at transfer, natural cycles were regular, and transfers seemed completely normal and uncomplicated. Only when Bourn Hall opened in 1980 did sufficient data accrue to suggest the true underlying causes. A large study on natural cycle IVF revealed pregnancy rates optimizing at 25% per replacement (Table 1). Rising doubts about the quality of single embryos led to the resumption of mild forms of ovulation induction. The final clue came in a study selecting the highest quality embryos by timing those which cleaved first to 2-cell, and then to 4-cell, scoring them every 3 h. Decisive evidence emerged on immense variations
Items
1 Jan 1981– 14 Jan 1982
October 1982
382 312 236 232 37 16 12 9 5+
35 29 25 24 6 25 21 17 0
in quality between embryos growing in vitro, and perhaps in vivo (Edwards et al., 1984). Fast-cleaving embryos were three times more effective at implanting than their slow-growing siblings (Table 2) (Edwards et al., 1984). Yet, overall, 25% of embryos implanted per transfer, just as with the natural cycle. The obvious solution was to obtain several embryos and choose the best. As it transpired, more and more embryos were transferred in numerous clinics, until four and more were being replaced routinely. Transferring three or more certainly increased pregnancy rates and the frequency of twins and triplets, but also doubled the frequency of multiple pregnancies especially when some clinics moved to the transfer of seven, eight or even more embryos to maintain success rates. IVF is responsible for almost 50% of multiple births in developed nations today. Slightly fewer are due to ovulation induction and to artificial insemination (AI) combined with ovarian stimulation, and a few arise spontaneously. Ovulation induction and AI incur difficulties in controlling the numbers of Graafian follicles and the numbers of ovulated eggs. Numbers of growing follicles vary immensely, and many mature to ovulation. This situation leads inevitably to multiple pregnancies, as indeed discovered during the earliest days of ovulation induction when quintuplets and higher-order multiple pregnancies created disillusion and concern. Modifying stimulation regimens, better hormone assays and improving ultrasound provide some defence against multiovulatory cycles, and excess follicles can be aspirated if necessary without any need to cancel the cycle. Matters are very different with IVF, where control is exercised through the number of replaced embryos. Selecting the best embryos has
Table 2. Selecting high quality human embryos on the basis of an early first and second cleavage division (Edwards et al., 1984). No. of embryos transferred
Clinical pregnancies Day 1 transfer Day 2 transfer Fast Slow Fast Slow
1 2 3 All
2/6 2/5 9/18 13/29
1/8 1/5 0/11 2/24
Values in parentheses are percentages.
1/5 3/7 5/11 9/23
3/16 1/14 3/16 7/46
Combined Fast Slow 3/11 4/24 5/12 2/19 14/29 3/27 22/52 9/70 (42.3) (12.9)
507
Commentaries - Towards single births after ART - RG Edwards
become increasingly refined, and will improve even more as better diagnostic methods are introduced using chips and proteomics. Some clinics offer single embryo transfers as a routine. Natural cycle IVF could receive an enormous boost if simple measures were developed to aspirate extra oocytes from small follicles combined with the use of anti-apoptotic agents. One day, perhaps, the causes leading to such immense numbers of chromosomal and other anomalies in human embryos failing to implant will be understood. Why does Nature block pregnancy among 80% of its ovulated eggs, whether conception occurs in vivo or in vitro? The situation could easily get even worse. Global disasters such as the spread of pollutants, contraceptive steroids or greenhouse gases are reported to add even more impairments to human implantation. Assisted reproduction may be needed even more than today to maintain anything like the birth rates typical of the twentieth century. Last but far from least, multiple pregnancies have legal, ethical, social and economic consequences. The shock experienced by many couples having multiple births is all too
508
familiar. Legal cases have arisen where doctors were sued because they failed to discuss the risks, stress and costs of raising triplets or higher-order pregnancies. These are the sort of undesired consequences of IVF and its associated forms of assisted conception. Let us hope that soon they will be matters of the past.
References Edwards RG, Surani, MAH 1978 The primate blastocyst and its environment. Science (suppl. 22), 39–50. Edwards RG, Fishel SB, Cohen J et al. 1984 Factors influencing the success of in vitro fertilization for alleviating human infertility. Journal of In Vitro Fertilization and Embryo Transfer 1, 3–23. Fowler RE, Edwards RG 1957 Induction of superovulation and pregnancy in mature mice by gonadotrophins. Journal of Endocrinology 15, 374–384. Proceedings of an Expert Meeting 2003 Infertility therapy-associated multiple pregnancies (births): an ongoing epidemic. Reproductive BioMedicine Online 7, 515–542. Steptoe PC, Edwards RG 1970 Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet i, 683–689.
Commentary Towards single births after assisted reproduction treatment RG Edwards Chief Editor, Reproductive BioMedicine Online, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK Correspondence: e-mail: [email protected]
Abstract The cause of increases in recorded multiple births is undoubtedly assisted human conception. Many arise when three or more embryos are replaced after IVF or ISCI. Others are due to multi-ovulation during artificial insemination or intrauterine inseminations, since complete control over numbers of ovulated eggs following ovarian stimulation is not fully possible. The frequency of multiple IVF births is best controlled by reducing numbers of transferred embryos to one or two. Methods to induce ovarian stimulation in amenorrhoeic and then in cyclic women 30 years ago have been greatly refined and extended in current practice. Curiously, a great many of them produce oocytes which fail to implant after fertilization, whether natural or induced cycles are being used. This massive failure of most embryos to implant poses numerous questions on the evolution of this situation in humans. Keywords: artificial insemination, implantation failure, multiple births, ovarian stimulation The drive to establishing singleton pregnancies after assisted conception is gaining momentum. Individual clinics and regulatory agencies are stressing the urgent need to restrict the number of embryos transferred. Some professionals claim that there is no decline in implantation rates as numbers of transferred embryos are reduced, which seems strange in view of the well-known statistical conclusions that embryos implant independently without interacting with companion embryos in the uterus. Moving to single embryo transfers demands greater understanding of the development of human embryos in vitro, in order to select the best for transfer. Remarkable progress along these lines depended on successive parameters being assessed and introduced into clinical practice. Rapid cleavage or polarization, the nature of blastomere fragments and the introduction of new culture media are slowly winning new converts to the small adaptations needed in laboratory practice to achieve this end.
506
A second approach to reducing multiple pregnancies is to pressurize clinics still transferring three, four or even more embryos. In some countries, legislation has insisted on the transfer of a maximum of three and then two embryos to reduce high-order multiples. This approach requires constant monitoring, by introducing auditing by outside agencies or returning detailed statistics to regulatory authorities. Pressures such as these will convince most professionals to declare truthfully the exact details of their transfers. Constant attention to this point in successive conferences is also influencing the attitudes of clinics to this need. A highly motivated international Expert Meeting on multiple pregnancies after assisted reproduction techniques will add its authority to restricting numbers of transferred embryos. Rightly, their paper is entitled ‘An Ongoing Epidemic’, since that is exactly what it is. The epidemic of multiple births has indeed been largely due to assisted reproduction. This can be seen from the various approaches to ovarian stimulation which were introduced successfully and became increasingly complex.
Even in its beginnings, some form of stimulation was considered to be essential to obtain sufficient oocytes for fertilization. At the time, it was recognized that mild multifolliculation was needed to produce several oocytes (Steptoe and Edwards, 1970). Studies on ovulation induction in animals, and especially mice, had shown how the number of ovulated oocytes in individual females could not be predicted, even within inbred strains or among F1 hybrids, which are usually considered as conferring enormous stability. Up to 100 oocytes were ovulated by some mouse females, and 50 or more fetuses competed for the limited uterine space available (Fowler and Edwards, 1957). Exactly the same situation arose when gonadotrophins extracted from human pituitaries, prepared from serum of pregnant mares, and lastly prepared from human postmenopausal urine were used to induce ovulation in amenorrhorrheic women. Press and TV reports, then in their infancy, described quintuplets, even octuplets, in some patients, despite the careful monitoring of urinary oestrogens as a measure of their response. Even with modern ultrasound and recombinant gonadotrophins, some mothers established octuplets quite against the desires of clinicians. Immense improvement in this technique developed from the introduction of IVF, where the very first clinical papers on human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) revealed variations in follicle numbers and the exact timing of follicle rupture (Steptoe and Edwards, 1970). Ovulation induction, artificial insemination, GIFT and IVF could now be practised with considerable accuracy, which increased pregnancy rates considerably, and also enabled the implantation of several embryos. Compared with today’s dosages, the amounts of gonadotrophins used were modest at 2–3 ampoules of HMG (i.e. 150–225 IU) three times during the follicular phase, followed by between 1000 and 12,500 IU HCG, which became standardized at 5000 IU. Even so, a mean of 6.6 oocytes were recovered per cycle and
Commentaries - Towards single births after ART - RG Edwards
most were fertilized in vitro, giving a mean of approximately four or five fertilized eggs. Half of the resulting embryos reached the blastocyst stage, some of them developing to day 9 in vitro (Edwards and Surani, 1997).
Table 1. Natural cycle IVF in Bourn Hall, 1982.
This number of oocytes seemed adequate for IVF, as indicated by experience in animals. Detailed experience with implantation rates per embryo in many mammalian species showed them to be 70% or greater. Consequently, in the opening clinical phases of human IVF, only a single embryo was chosen for transfer to its mother’s uterus even if four or five were available. The remainder were used for study or, in later years, cryopreserved for future use as the necessary techniques were understood. Emphasis was placed on the need for singleton pregnancies in attempts to spare mothers from multiple births. Human embryos were assumed to be the same, so transferring two was expected inevitably to lead to twins. As it transpired, this early research produced very few births, due to the very short luteal phases in stimulated IVF patients. Primulot depot was unwittingly used as a luteotrophin to overcome these luteal phase defects, but it turned out to be an abortifacient! Given just before or after blastocyst transfer, it caused the termination of many pregnancies by 1 week posttransfer, resulting in the term ‘biochemical pregnancies’.
Aspirations ≥One egg aspirated ≥One or more fertilized Replacements Clinical pregnancies % per replacement % per patients with eggs % per aspiration Biochemical pregnancies
Utilizing several other forms of ovarian stimulation, and avoiding Primulot, gave more, but still very few, pregnancies in response to transferring mostly single embryos. Rare transfers of two blastocysts, or replacing two mature oocytes plus spermatozoa in the oviduct, were performed with trepidation, since a primary target remained the avoidance of multiple births. Suspicions about low rates of implantation emerged even more strongly when natural cycle IVF was introduced. The final Oldham results produced four pregnancies, and perhaps one additional biochemical pregnancy, out of 32 transfers. There was no obvious reason for this low rate of success, since 8-cell embryos and blastocysts were healthy at transfer, natural cycles were regular, and transfers seemed completely normal and uncomplicated. Only when Bourn Hall opened in 1980 did sufficient data accrue to suggest the true underlying causes. A large study on natural cycle IVF revealed pregnancy rates optimizing at 25% per replacement (Table 1). Rising doubts about the quality of single embryos led to the resumption of mild forms of ovulation induction. The final clue came in a study selecting the highest quality embryos by timing those which cleaved first to 2-cell, and then to 4-cell, scoring them every 3 h. Decisive evidence emerged on immense variations
Items
1 Jan 1981– 14 Jan 1982
October 1982
382 312 236 232 37 16 12 9 5+
35 29 25 24 6 25 21 17 0
in quality between embryos growing in vitro, and perhaps in vivo (Edwards et al., 1984). Fast-cleaving embryos were three times more effective at implanting than their slow-growing siblings (Table 2) (Edwards et al., 1984). Yet, overall, 25% of embryos implanted per transfer, just as with the natural cycle. The obvious solution was to obtain several embryos and choose the best. As it transpired, more and more embryos were transferred in numerous clinics, until four and more were being replaced routinely. Transferring three or more certainly increased pregnancy rates and the frequency of twins and triplets, but also doubled the frequency of multiple pregnancies especially when some clinics moved to the transfer of seven, eight or even more embryos to maintain success rates. IVF is responsible for almost 50% of multiple births in developed nations today. Slightly fewer are due to ovulation induction and to artificial insemination (AI) combined with ovarian stimulation, and a few arise spontaneously. Ovulation induction and AI incur difficulties in controlling the numbers of Graafian follicles and the numbers of ovulated eggs. Numbers of growing follicles vary immensely, and many mature to ovulation. This situation leads inevitably to multiple pregnancies, as indeed discovered during the earliest days of ovulation induction when quintuplets and higher-order multiple pregnancies created disillusion and concern. Modifying stimulation regimens, better hormone assays and improving ultrasound provide some defence against multiovulatory cycles, and excess follicles can be aspirated if necessary without any need to cancel the cycle. Matters are very different with IVF, where control is exercised through the number of replaced embryos. Selecting the best embryos has
Table 2. Selecting high quality human embryos on the basis of an early first and second cleavage division (Edwards et al., 1984). No. of embryos transferred
Clinical pregnancies Day 1 transfer Day 2 transfer Fast Slow Fast Slow
1 2 3 All
2/6 2/5 9/18 13/29
1/8 1/5 0/11 2/24
Values in parentheses are percentages.
1/5 3/7 5/11 9/23
3/16 1/14 3/16 7/46
Combined Fast Slow 3/11 4/24 5/12 2/19 14/29 3/27 22/52 9/70 (42.3) (12.9)
507
Commentaries - Towards single births after ART - RG Edwards
become increasingly refined, and will improve even more as better diagnostic methods are introduced using chips and proteomics. Some clinics offer single embryo transfers as a routine. Natural cycle IVF could receive an enormous boost if simple measures were developed to aspirate extra oocytes from small follicles combined with the use of anti-apoptotic agents. One day, perhaps, the causes leading to such immense numbers of chromosomal and other anomalies in human embryos failing to implant will be understood. Why does Nature block pregnancy among 80% of its ovulated eggs, whether conception occurs in vivo or in vitro? The situation could easily get even worse. Global disasters such as the spread of pollutants, contraceptive steroids or greenhouse gases are reported to add even more impairments to human implantation. Assisted reproduction may be needed even more than today to maintain anything like the birth rates typical of the twentieth century. Last but far from least, multiple pregnancies have legal, ethical, social and economic consequences. The shock experienced by many couples having multiple births is all too
508
familiar. Legal cases have arisen where doctors were sued because they failed to discuss the risks, stress and costs of raising triplets or higher-order pregnancies. These are the sort of undesired consequences of IVF and its associated forms of assisted conception. Let us hope that soon they will be matters of the past.
References Edwards RG, Surani, MAH 1978 The primate blastocyst and its environment. Science (suppl. 22), 39–50. Edwards RG, Fishel SB, Cohen J et al. 1984 Factors influencing the success of in vitro fertilization for alleviating human infertility. Journal of In Vitro Fertilization and Embryo Transfer 1, 3–23. Fowler RE, Edwards RG 1957 Induction of superovulation and pregnancy in mature mice by gonadotrophins. Journal of Endocrinology 15, 374–384. Proceedings of an Expert Meeting 2003 Infertility therapy-associated multiple pregnancies (births): an ongoing epidemic. Reproductive BioMedicine Online 7, 515–542. Steptoe PC, Edwards RG 1970 Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins. Lancet i, 683–689.