The Journal of Clinical Embryology - Volume 7, Issue 1 - Spring 2004

The

Clinical Embryologist (Formerly The Embryologists’ Newsletter)

Volume 7, Issue 1

SPRING, 2004

D E D I C AT E D T O T H E D I S S E M I N AT I O N O F S H A R E D B E S T P R A C T I C E S A N D I S S U E S O F I N T E R E S T FOR PROFESSIONALS IN THE FIELD OF HUMAN ASSISTED REPRODUCTION.

M E D I C A L

M A R K E T S

C O N S U L T A N T S ,

I N C .

The Road to Single Embryo Transfer

T

David K. Gardner, Ph.D., Colorado Center for Reproductive Medicine

he benefits of single embryo transfer to the mother and child alike are evident. The major risks associated with a multiple gestation are the high rate of preterm delivery and low birth weights. In spite of improved antenatal care for multiple gestations, the perinatal mortality is ten fold higher than that of singleton gestations (1,2). Furthermore, the incidence of cerebral palsy is five fold higher in twins and twenty fold higher in triplets than in singleton pregnancies (3).

Selective embryo reduction has been introduced as a method of dealing with high order multiple gestations, but this is not without risk. Evans et al.

(4) observed that only 84% of pregnancies reached delivery after embryo reduction, and 5% of these suffered from extreme prematurity (25-28 weeks gestation). Furthermore, even after selective reduction, the problems of prematurity are not eliminated (5). This, therefore, implies that there are some abnormalities in implantation and placentation when multiple embryos are present.

There are also the psychological effects of embryo reduction to be considered (6). The economic consequences of multiple gestation represent a significant “hidden” cost of ART. The estimated medical cost of a triplet or greater gestation is around $340,000 (7).

The cost of twins is between $21,000 and $39,000.

Furthermore, such figures do not take into account the cost of long-term care for handicapped children resulting from prematurity.

There is an obvious need to reduce the number of embryos transferred following IVF, while still M E D I C A L

M A R K E T S

Optimizing Embryo Development in Culture How can we maximize the development of embryos in vitro? It is proposed that one needs to not only meet the requirements of the embryo as it develops and differentiates, but also to minimize the stress to which embryos are exposed in vivo. This leads us to the subject of how we can culture viable embryos. The term viable is used deliberately, as one can grow embryos to the blastocyst stage in a wide variety of culture conditions, but their subsequent viability, defined as their ability to implant and grow to term, is significantly different among different culture systems.

Meeting the needs of the embryo Within the female reproductive tract there exist gradients of nutrients. These gradients of nutrients mirror the changing physiology and requirements of Continued on Page 16

C O N S U L T A N T S ,

1 1 5 N . W. 8 4 Wa y / C o r a l S p r i n g s , F L 3 3 0 7 1

E-mail: embphil@bellsouth.net

maintaining acceptable pregnancy rates for our patients. Ultimately, the only way to ensure a singleton conception is to transfer a single embryo. What is required in order to make single embryo transfer a realistic and feasible procedure? There are three laboratory areas that warrant consideration and discussion: 1. Optimizing embryo development in culture.

2. Selecting the most viable/normal embryo for transfer.

3. Optimizing cryopreservation procedures.

These areas form the basis of this review.

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The Clinical Embryologist

Volume 7, Issue 1

EDITORIAL:

Opportunities for Readers of The Clinical Embryologist Herbert M. Phillips, Ph.D., HCLD

F

or the past two years, it has been my privilege to serve as Editor of this publication. At our last meeting with our Editorial Board (see box below in lower-left corner of this page), the Publisher, Fred Zander, the Associate Publisher and Business Manager, Mike Magee, and I joined in a consensus decision to change the title from The Embryologists’ Newsletter to The Clinical Embryologist. We wish briefly to review here the rationale for this decision – and its implications for improved communications for (and, we hope, increasingly by) readers of our quarterly periodical.

THE CLINICAL EMBRYOLOGIST EDITORIAL BOARD Christopher De Jonge, Ph.D., HCLD Reproductive Medicine Center University of Minnesota Minneapolis, MN • 612-627-4807 E-mail: dejonge@umn.edu Kenneth C. Drury, Ph.D., HCLD Department of Obstetrics and Gynecology University of Florida College of Medicine Gainesville, FL • 352-392-5680 E-mail: druryk@obgyn.ufl.edu Kathryn J. Go, Ph.D., HCLD Pennsylvania Reproductive Associates The Women’s Institute Philadelphia, PA • 215-922-3173, #335 E-mail: Kathy@womensinstitute.org David L. Hill, Ph.D., HCLD ART Reproductive Center Beverly Hills, CA • 310-246-2417 E-mail: embryonics@adelphia.net Thomas B. Pool, Ph.D., HCLD Fertility Center of San Antonio San Antonio, TX • 210-614 3232 E-mail: rpool@fertilitysa.com Herbert M. Phillips, Ph.D., HCLD Editor E-mail: embphil@bellsouth.net Friedel M. W. Zander Publisher E-mail: Fzander@zanderivf.com

Under the previous editor/ publisher, Stan Colquitt, The Embryologists’ Newsletter was a sort of hybrid -- part newsletter and part journal. Personnel from different laboratories were pictured and described, side by side with standard research and review articles.

I accepted my current position here with one basic objective in mind – namely, that any embryologist might find in each issue at least one idea or technique that would be of immediate practical value in his or her performance of ART procedures.

Of course, such an ambitious aspiration may not always be fully realized in every issue, but its formulation has nevertheless proved useful in the process of determining the overall course and specific goals toward which we aim.

This approach places premium value upon attracting articles both from leading investigators and, in general, from practicing clinical embryologists, plus physicians and basic scientists with related expertise. We have therefore endeavored to earn the confidence

INSTRUCTIONS FOR CONTRIBUTORS E-mail as Word documents, doublespaced, in any ART journal format, with all authors’ professional affiliations and e-mails. FAX or mail signed compliance with conventional standards of authorship and originality. Minimal illustrations.

Articles 1,500-3,000 words (6-12 pages), shorter for abstracts, lab tips and letters (signed, return mail and e-mail addresses).

Next article due dates: 5/14/04 for Vol. 7, Issue 2; 8/13/04 for Vol. 7, Issue 3.

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of our readers - and of other key prospective authors - that this publication has now become a reliable, convenient resource for sharing best practices in the field of clinical embryology. A new, more professional title was deemed unanimously to be a legitimate, timely step in the right direction.

This title is indeed more accurate now. From the beginning, I have attempted to move away from a newsletter ambience toward a standard clinical embryology journal format. However, another fundamental objective of my editorship has been to promote the welfare of all laboratory personnel involved in the care of human gametes and embryos – including clinical embryology technicians and technologists, as well as laboratory directors and associated fertility-program physicians. Thus, in our view, this publication has a service function as well as a strictly informational purpose.

Continued on Page 5

TABLE OF CONTENTS The Road to Single Embryo Transfer ................................................. 1 Opportunities for Readers of The Clinical Embryologist ................... 3 How Can Basic Science Help Human ART? ....................................... 6 Helpful Lab Tip: Embryo Biopsy Medium & PGD Pregnancy Rates..................................11 Cloned Human Embryo Stem Cell Breakthrough ......................................13

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The Clinical Embryologist

Volume 7, Issue 1

Continued from Page 3

I have therefore included in the previous Embryologists’ Newsletter -- now The Clinical Embryologist -- some extra features not always found in more formal journals – for example, in this and/or past issues: more detailed articles on techniques (e.g. see issue 5:3, p.13; Issue 6:3, p9; and this issue, p.11), articles about educational programs in this field (see Issue 5:3, p.3, 5, 7; Issue 5:4, p.3 and Issue 6:1, p.21), embryological opinion articles (e.g. “Musings” in Issue 6:4), relevant informational articles (e.g. see salary analyses, Issue 5:4, p.22; and see schedules of AAB meetings and courses, Issue 6:1, p.18 and Issue 6/4, p.8.), and republication (with permission) of interesting articles that, for one reason or another, many embryologists might not otherwise come across (see page 13 and numerous back issues), plus occasional humorous or inspirational “filler” articles (see Issue 6:2, p.17 and Issue 5:2, p.12).

We are extremely gratified by the excellence of the articles that we have been able to publish.

The Clinical Embryologist provides a unique opportunity to communicate directly with the great majority of clinical embryology laboratory personnel in the U.S. and Canada -- and with an increasing readership in six continents. To review the wide panorama of diverse articles that we have published, readers may visit our website, www.embryologists.com, where back issues are posted (up to now under our old banner, The Embryologists’ Newsletter). It should be noted that this website makes our authors’ articles instantly and permanently accessible to everyone.

In addition to those past kinds of articles described above, we have yet to publish instructive single-case reports, debates, letters-to-the-editor and noteworthy reminiscences, as well as reviews of books and critiques of articles. [We have published a review of oral presentations at a regional meeting (see Issue 5:4, p.1). We would be pleased to receive more of the same.] If other publication possibilities occur to you, I invite your further suggestions. In general, we appreciate any kinds of written communications that might help embryologists improve and perfect their strategies and methodologies for optimal nurturing of human gametes and embryos.

So we ask our readers, busy though you must be, to compose, and then to email to us, your manuscripts

throughout the coming months and years. Use your imaginations and (proven or latent) journalistic talents, however briefly [brief articles are especially valued], to the benefit of your colleagues in this important and challenging field -- and ultimately to the benefit of patients similar to those whom you do try so conscientiously to help throughout each and every work-day.

Kindly keep in mind that the future quality of The Clinical Embryologist will depend in large measure upon your response to these invitations. This carefully and laboriously constructed periodical, sent to you without charge (please patronize our advertisers) does have an unavoidable cost – i.e., steady, continuous, plentiful contributions of articles from our readers.

With every manuscript submitted to us, abundant, cordial, writer-friendly editorial assistance also comes without charge to all authors -- but ultimately, indirectly, with the same, above-mentioned literary price-tag to our readers.

Finally, on this occasion, it is a pleasure to take this opportunity to acknowledge those most responsible for getting my editorship off the ground during these last two years. They include the authors of articles that have appeared in this and the preceding seven issues of this publication, who have truly made superior contributions above and beyond my highest initial expectations. They also include the members of the Editorial Board, who have provided invaluable advice -- as well as articles and/or contacts with other prospective authors.

Most of all, they include the Publisher and Associate Publisher, who have made major sacrifices of their time and financial resources -- without which there could never be any Clinical Embryologist today -- who have found many of our authors for me, who have provided me with wise counsel on frequent occasions, and who have put up with the many idiosyncrasies of the truly dedicated but undeniably eccentric, perfectionistic and occasionally volatile editor of this publication. Readers will never know how much they owe to these two tireless supporters of The Clinical Embryologist – but, at least to a considerable extent, I do know -- and I thank them here profusely on behalf of all of their numerous laboratory ART beneficiaries. ■

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How Can Basic Science Help Human ART?

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Barry Bavister, Ph.D., and Carol Brenner, Ph.D., University of New Orleans and Tulane Institute for Reproductive Medicine

urrent status of human IVF success During the past decade, great strides have been made in improving the outcome of human IVF. Major advances have been made in ovarian stimulation protocols and the design of embryo culture media. According to the latest data [1], the U.S. average success rates for human IVF are 38% clinical pregnancy rate and 33% live baby rate.

There are three different ways to consider these data. First, the results appear to be remarkable, and similar to natural conception rates, so little or no further improvement is possible. Second, and alternatively, there is still room for improvement , because two-thirds of infertile patients are not rewarded with a live birth; moreover, these summary figures do not address the substantial frequency of multiple births underlying these successes. Third, these success rates based on embryo production data indicate that current human IVF is still rather inefficient. That is the focus of this article.

tion with exogenous gonadotropins produces cohorts of oocytes with inherent variations in quality and competence. This must play a major role in reducing the average quality of in vitro produced embryos, as well as in perturbing the normal uterine environment for implantation [4, 5].

The low average quality of human oocytes and embryos in a typical IVF cycle is somewhat compensated by increasing the numbers of embryos transferred in each cycle, at least in the U.S. It can be estimated from the year 2000 clinical data [1] that an average of 3 embryos is transferred in each treatment cycle. If the average clinical pregnancy rate (CPR) and live baby rate (LBR) are 38% and 33%, respectively [1], for 3 embryos transferred, then the actual rates per embryo transferred are only 13% and 11%, respectively. Although other factors are involved, these rates suggest that the average quality of embryos produced in human ART is low, in spite of the very best efforts of all concerned: the patients, the clinicians and the embryologists.

But the embryos for transfer are selected by a skilled embryologist from a cohort that may average approximately 10 embryos. When considered on a per-embryo produced per-cycle basis, the ART success rate is 3/10 of the above or 4% CPR and 3% LBR. Even this does not fully represent the efficiency of human ART. If an average of 15 oocytes is retrieved and inseminated per cycle, then the “bottom line” ART success rates are 3/15 of the reported success rates, i.e., 2-3 % CPR and LBR per oocyte.

Viewed in this way, the average success rates of human ART leave much room for improvement.

To be sure, a few clinics are reporting much higher outcomes than the U.S. average, but even these are achieved using multiple embryo transfers, a factor that needs to be taken into account in deriving the true “per embryo” success rates. If much higher average oocyte and embryo quality could be achieved, then it might be possible to transfer one or at most two embryos and obtain success rate at least double the current reported U.S. average.

Analysis of human IVF outcome data In higher primates (humans and, for example, macaque monkeys), reproductive efficiency is quite low, compared to some non-primate species. In natural human and rhesus monkey menstrual cycles, it is estimated that only about 25% of potential conceptions support clinical pregnancies; most likely, defects in oocytes and the resulting anomalies of fertilization and/or embryonic development are major contributors to this loss. This inefficiency is in marked contrast to natural cycles in some rodents, where 98% of ovulated oocytes develop into blastocysts and almost all of these become viable fetuses [2]. In dairy cattle, which like humans are generally monovular, pregnancy rates of 65% are expected. Studies in laboratory animals and other species (e.g., [3]) have demonstrated that exogenous gonadotropin stimulation lowers the viability of embryos, doubtless resulting from recruitment of potentially atretic follicles containing defective oocytes.

Most likely, in human ART cycles ovarian stimula-

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How could such a quantum improvement be achieved? There are several factors to consider. First, much more emphasis needs to be placed on oocyte quality and less on quantity (numbers per retrieval).

At present, by recruiting a heterogeneous cohort of oocytes using high dose FSH stimulation regimens, not only is oocyte quality compromised but the job of the clinical embryologist is made more difficult because he/she has few tools to help select the best oocytes/embryos. The bottom line: the higher the numbers of oocytes collected, the broader the range of quality, and the lower the chances of producing and selecting a small number of viable embryos from the available cohort. Second, the intrinsic quality of women’s oocytes declines with age of the patient, and third, the artificial environment (culture medium) in which oocytes and embryos are cultured most likely alters their properties and behavior. Fourth, these two factors (age and culture environment) probably interact, because experimental studies show that stressed or lower quality oocytes and embryos perform less

well in vitro than healthier cells. The performance of cryopreserved oocytes and embryos attests to this problem. Thus, there is still much need to improve culture media used for ART. Adverse effects of age on the quality of human oocytes and impact on fertility.

Superimposed on the inherent variability of oocyte quality are diverse epigenetic effects of oocyte/ embryo culture that can alter gene expression, mitochondrial localization and/or activity, and affect embryo development/viability [6-18]. However, these subcellular alterations are unlikely to be manifested as changes in morphological appearance, which is currently the principal criterion for selecting embryos for transfer.

It is well known that embryo morphology, even at the blastocyst stage, is not a strong predictor or correlate of viability. For example, infertile women of advanced reproductive age exhibit very low success in ART, yet morphologically normal-looking blastocysts can be produced at rates comparable to those seen in Continued on Page 8

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The Clinical Embryologist

Volume 7, Issue 1 Continued from Page 7

younger women [19]. The same result was found with IVF embryos derived from oocytes of old rhesus monkeys [20].

The quality or health of human eggs is progressively diminished due to: (i) inherent or genetic factors as part of the natural aging process: older women on average show reduced fertility, and decreased success in ART; and (ii) epigenetic factors such as nutrition, smoking and environmental effects. While reduced fertility in women derives in large part from diminished oocyte quality or “health�, there is currently no reliable way to measure this attribute.

We need ways to measure oocyte quality that could lead to improved selection of oocytes/embryos in ART clinics, which would increase pregnancy success rates, especially in low-outcome patients such as women aged 40 or over.

Such methods will come from better understanding of the cellular and molecular properties of oocytes and embryos.

A key component of all cells including oocytes is the mitochondrion, which provides most of the cell’s energy and also possesses its own DNA for encoding protein production. Defects in either of these functions could lead to loss of oocyte competence in the short term (spindle defects leading to aneuploidy, and/or failure of fertilization or of embryo development) or in the long term (non-viable fetuses or incompetent placentas, leading to pregnancy loss, or possibly post-natal health defects). Human oocytes would be the optimal subjects for undertaking studies on oocyte quality and the embryos derived from them; however, practical and ethical constraints preclude intensive studies with human oocytes and embryos, so suitable animal models must be used instead.

Problems facing clinical embryologists Central problems in human ART laboratories are (i) how to minimize epigenetic and genetic aberrations, and (ii) how to select the most competent oocytes or embryos.

How can these goals be met? First, there are ongoing efforts to develop culture media that support increased embryo development to the blastocyst stage, and provide higher pregnancy/live birth rates (e.g. [21, 22]). As mentioned above, this may be especially helpful with oocytes and embryos that are developmentally compromised.

However, these attempts alone will not solve the problems in human ART because of the underlying, inherent variability of oocyte characteristics, so that oocyte/embryo selection remains a major concern.

There are few, if any, methods available that can discriminate among competent and non-competent oocytes,

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so most emphasis is placed on selecting the embryos after IVF. However, because of the poor predictive value of embryo morphology for viability, virtually all human ART clinics employ multiple embryo transfers to improve clinical outcomes. While this practice increases pregnancy and live birth rates [1], it also incurs a substantial frequency of multiple births, associated with pre- or peri-natal complications [23].

The ideal clinical situation would be to transfer only one, or at the most, two embryos possessing very high viability. Thus, “single embryo” transfer could avoid multiple high risk pregnancies [22].

Unfortunately, at present there are no highly reliable, objective methods for identifying the most viable embryos within a cohort. Markers of competence, exhibited before, during or soon after fertilization, would be very helpful. Useful markers could be of two kinds: (i) invasive or destructive markers that provide basic information on oocyte or embryo biology so the underlying causes of incompetence can be better understood; (ii) non- or minimally-invasive markers that could be used in ART practice to select the most competent oocytes or embryos derived from them.

Currently, preimplantation genetic diagnosis (PGD) is being used as a diagnostic tool to detect chromosomal aneuploidies using either polar body or single-cell blastomere biopsies [24] but this seems impractical as a routine diagnostic tool applied to all oocytes or embryos. To date, no definitive marker has been demonstrated that can be used to predict viability of individual oocytes or embryos, although the use of metabolic markers such as amino acid turnover appears promising [25]. There is one exception to this poor state of affairs. It is now abundantly clear that the timing of cleavage, particularly the time elapsed from insemination of oocytes to completion of the third cleavage division, is a strong indicator of embryo viability, in animal and in human embryos [26, 27, 28, 29, 30]. The average success rates of human ART would most likely improve significantly if this simple, non-invasive marker of embryo quality were routinely used to select embryos for transfer.

technology is low when assessed by the denominator, i.e., the number of embryos transferred or of oocytes retrieved and inseminated. This conclusion emphasizes the need for improvements in key aspects of human ART, especially: (1) examination of the consequences of high dose gonadotropin stimulation regimens; (2) derivation of non-invasive methods for identifying viable oocytes and/or embryos; (3) emphasis on the particular problems underlying greatly diminished ART success rates in older women patients; (4) increased emphasis on understanding the biology of Continued on Page 10

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Conclusions While the current success rates of human ART are commendable, we believe that the efficiency of this

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Continued from Page 9

human oocytes and embryos, essentially using representative animal models, in order to reveal their particular characteristics and requirements in vitro.

All of these goals are readily achievable if clinical and basic research laboratories work on them collaboratively. Such collaborations should be encouraged and supported. ■ Barry Bavister, Ph.D., and Carol Brenner, Ph.D., University of New Orleans, and Tulane Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Tulane University Medical School, New Orleans. [E-mail addresses: bbaviste@uno.edu; cbrenner@uno.edu]

stimulation of donor females decreases post-implantation viability of cultured 1-cell hamster embryos. Hum Reprod 13:724-729.

4. Kolb BA, Paulson RJ. (1997) The luteal phase of cycles utilizing controlled ovarian hyperstimulation and the possible impact of this hyperstimulation on embryo implantation. Am J Obstet Gynecol. 176:1262-7.

5. Kolb BA, Najmabadi S, Paulson RJ. (1997) Ultrastructural characteristics of the luteal phase endometrium in patients undergoing controlled ovarian hyperstimulation. Fertil Steril.

67:625-30.

6. Bavister BD. (1995) Culture of preimplantation embryos: facts and artifacts. Hum. Reprod. Update 1: 91-148.

7. Barnett DK, Bavister BD. (1996) What is the relationship between the metabolism of preimplantation embryos and their development in vitro? Molec. Reprod. Dev. 43:105133.

8. Barnett DK, Clayton MK, Kimura J, Bavister BD. (1997) Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol. Reprod. Dev. 48: 227-237.

9. Van Blerkom J, Davis P, Alexander S. (2000) Differential mitochondrial distribution in human pronuclear embryos leads to disproportionate inheritance between blastomeres: relationship to microtubular organization, ATP content and competence. Hum Reprod. 15: 2621-2633.

10. Squirrell JM, Wokosin DL, White JG, Bavister BD. (1999) Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nature Biotech.

17:763-767.

11. Squirrell JM, Lane M, Bavister BD. (2001) Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos. Biol Reprod 64:18451854.

12. Squirrell JM, Schramm RD, Paprocki AM, Wokosin DL, Bavister BD. (2002) Imaging mitochondrial organization in living primate oocytes and embryos using multiphoton microscopy. Microscopy and Microanalysis 9:190-201.

13. Niemann H, Wrenzycki C. (2000) Alterations of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development. Theriogenology 53:21-34.

14. Wrenzycki C, Herrmann D, Carnwath JW, Niemann H. (1999) Alterations in the relative abundance of gene transcripts in preimplantation bovine embryos cultured in medium supplemented with either serum or PVA. Mol. Reprod. Dev. 53:8-18.

15.Wrenzycki C, Herrmann D, Keskintepe L, Martins AJ, Sirisathien S, Brackett B, Niemann H. (2001) Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum. Reprod. 16:893901.

16. Doherty AS, Mann MR, Tremblay KD, Bartolomei MS,

References 1. Wright VC, Schieve LA, Reynolds MA, Jeng G. (2003) Assisted reproductive technology surveillance--United States, 2000. MMWR Surveill Summ. 52:1-16. Erratum in: MMWR 52:942.

2. Gonzales DS, Bavister BD. (1995) Zona pellucida escape by hamster blastocysts in vitro is delayed and morphologically different compared with zona escape in vivo. Biol. Reprod.

52:470-480.

3. McKiernan SH and Bavister BD. (1998) Gonadotropin

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Schultz RM. (2000) Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol.

Reprod. 62:1526-35.

17. Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. (1995) Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression.

Mol. Reprod. Dev. 41:232-8.

18. Poueymirou WT, Conover JC, Schultz RM. (1989) Regulation of mouse preimplantation development: differential effects of CZB medium and Whitten’s medium on rates and patterns of protein synthesis in 2-cell embryos. Biol.

Reprod. 41:317-22.

19. Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munne S. (2001) Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage.

Hum Reprod 16:1954-8.

20. Schramm RD, Paprocki AM and Bavister BD. (2002) Features associated with reproductive ageing in female rhesus monkeys. Hum. Reprod. 17: 1597-1603.

21. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, Hesla J. (1998) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum. Reprod., 13:3434-40.

22. Gardner DK, Vella P, Lane M, Wagley L, Schlenker T, Schoolcraft WB. (1998) Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertil. Steril. 69:84-8.

23. Wimalasundera R, Fisk NM. (2002) In-vitro fertilisation and risk of multiple pregnancy. Lancet 360:414.

24. Munne S, Cohen J. (1998) Chromosome abnormalities in human embryos. Hum Reprod Update 4:842-55.

25. Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, Leese HJ. (2002) Non-invasive amino acid turnover predicts human embryo developmental capacity. Hum Reprod. 2002 17:999-1005.

26. McKiernan SH,.Bavister BD. (1994) Timing of development is a critical parameter for predicting successful embryogenesis. Hum. Reprod. 9:2123-9.

27. Racowsky C, Jackson KV, Cekleniak NA, Fox JH, Hornstein MD, Ginsburg ES. (2000) The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. Fertil. Steril. 73:558-64.

28. Bavister BD (2002) Timing of embryo development. In: Assessment of mammalian embryo quality: Invasive and noninvasive techniques. Eds. Van Soom A, Boerjan ML., Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 139155.

29. Warner CM, Brenner CA. (2001) Genetic regulation of preimplantation embryo survival. Curr Top Dev Biol.

2001;52:151-92.

30. van Soom A, Ysebaert MT, de Kruif A. (1997) Relationship between timing of development, morula morphology, and cell allocation to inner cell mass and trophectoderm in in vitroproduced bovine embryos. Mol Reprod Dev. 47:47-56.

Helpful Lab Tip:

Embryo Biopsy Medium & PGD Pregnancy Rates David L. Hill, Ph.D.. and Man Li, M.D., Ph.D., ART Reproductive Center, Beverly Hills, CA [E-mail: embryonics@adelphia.net]

I

f I’ve learned anything in my 17 years of involvement in ART, I’ve learned this: “everything matters!” If this comes as an epiphany to you, then you have not spent much time in an IVF lab. This brief communication, which is the essence of materials we’re going to present at the upcoming annual meeting of the Pacific Coast Reproductive Society in April, describes yet another instance of the above axiom, and hopefully will assist you in bumping up the “win column” in your programs.

Embryo biopsy requires a hole or slit to be made in the zona pellucida large enough to allow removal of a blastomere. Two popular methods of “zona opening”-- partial zona dissection (PZD) and acidified Tyrode’s solution (ATS) -- have been described by several authors; and it appears they are similarly efficient and safe in experienced hands. We believe that the “embryo biopsy medium” (EBM) used in the process of zona opening and blastomere extraction is just as important as the method employed for zona opening; and its long-term effects on the embryo may be under-appreciated. This retrospective data analysis compared the pregnancy rates using two kinds of EBM, both in use at our center from 03/01/2002 to 10/31/2003 for aneuploidy screening cases using Fluorescent in Situ Hybridization (FISH).

To briefly describe the materials and methods, we use Sequential media G1, G2 (Vitrolife, Sweden) and a triple gas (5%O2, 6%CO2, balance nitrogen) incubator internal environment for all of our insemination and embryo culture. Biopsy medium 1 (EBM1) was Hank’s Balanced Salt Solution without Ca++ & Mg++ (HBSS, Sigma H6648) supplemented with 5% Synthetic Serum Supplement (Irvine Scientific) and 0.05M Sucrose. Medium 2 (EBM2) was similar to Continued on Page 12

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Continued from Page 11

EBM1 but was supplemented with alanyl-glutamine, EDTA, sodium pyruvate and sodium lactate (DL).

A total of 165 cases of PZD embryo biopsy for aneuploidy screening were done from 03/01/02 to 03/31/03 in EBM1 (group 1). At the same time, 744 age-matched, non-biopsy cases were done at our center, and we used them as “controls” for group 1 medium. From 04/01/03 to 10/31/03, another 146 biopsy cases were done in EBM2 (group 2). The biopsies were done using ATS to open the zonas on these cases, and during the same period 280 nonbiopsy cases served as controls for the group 2 biopsy medium.

The results are shown in Table 1. After looking at the results, we’ve concluded that micromanipulation medium for embryo biopsy, and possibly for all micromanipulative procedures, may be as important as the embryo culture medium itself in its effects on embryonic growth and implantation. Because the embryos are in the biopsy medium for just a brief time, it may not be intuitive, so to speak, that this could be a problem like, say, leaving embryos in a poor pH or warm or cold situation for the same period of time. Embryo biopsy procedures generally are not done by just removing one embryo from the incubator and working on it – up to five embryos may be exposed to the extra-incubator environment at a time, and even if you’re fast at it, it takes several minutes to

biopsy and handle these embryos. “Brief”, therefore, is a most relative term!

That said, development of biopsied embryos was significantly impaired if the embryos were exposed even briefly in medium lacking amino acids and their necessary nutrients, such as sodium pyruvate and sodium lactate (DL). Supplementation with SSS can not compensate for the depletions, even when the embryos were subsequently cultured in the presence of amino acids and nutrient rich medium, such as G2 medium. Very interesting. We believe this step -- that of using embryo biopsy media that contain essential amino acids and nutrients found in the primary culturing milieu -- to be critical in maximizing clinical outcomes.

We hope this helps. Both myself and Man Li, MD, PhD, Director of the Division of Preimplantation Genetics and Research at our Center would be happy to discuss these results or any aspect of embryo biopsy-PGD. We do about 30 PGD cases a month now, so we’re gaining a fair amount of practical experience. The last 40 cases or so have been performed with a micro-operative laser for zona opening, and as might be predicted the results appear promising. And finally, I’d like to thank my friend and colleague, Ken Drury, Ph.D., for his insight into the specific issue of micromanipulation media, and for his patience in helping me learn PGD itself. Thanks, Ken. ■

TABLE 1.

Cases

Average age (yrs)

Clinical Pregnancy Rate Per Retrieval

Clinical Pregnancy Rate Per Embryo Transfer

Group 1

165

38.36

53/165 (32.1%)

53/138 (38.4%)

Control 1

744

37.41

272/744 (36.6%)

272/653 (41.7%)

Group 2

146

37.42

64/146 (43.8%)*

64/127 (50.4%)

Control 2

280

37.45

89/280 (31.8%)

89/231 (38.5%)

*Significant difference (p<0.05) from group 1.

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Cloned Human Embryo Stem Cell Breakthrough

S

By Dr. Kirsty Horsey, 2/12/04. BioNews 23:17

cientists in South Korea have extracted and grown stem cells from cloned, early human embryos, a breakthrough in ‘therapeutic cloning’ research. Using a modified version of the technique used to clone Dolly the sheep, the team, based at the Seoul National University, created 30 cloned human embryos. The researchers managed to extract stem cells from 20 of these, from which they managed to grow one human embryo stem (hES) cell line in the laboratory. Their work, which will be published in the journal Science, has been hailed as “a landmark paper” by other scientists, and could pave the way for research into new treatments for many diseases.

The stem cells present in very early embryos are the body’s “master” cells, capable of growing into any type of tissue. Since the unveiling of Dolly, the cloned sheep, in February 1997, scientists have been investigating the possibility of using stem cells from cloned, early human embryos to develop tissue-matched therapies for diseases such as Parkinson’s disease and diabetes: an approach known as therapeutic cloning.

To create Dolly, scientists at the Roslin Institute in Scotland used a technique called somatic cell nuclear transfer (SCNT), which involved transferring the nucleus of an adult sheep mammary gland cell into a donor egg stripped of its own genetic material. Since then, many other species of animal have been cloned using this technique, or modified versions of it. But creating SCNT embryos from humans (or any primate) has proved very difficult, although several groups have managed to isolate embryo stem cells from IVF (in vitro fertilization) embryos. Such cells are being used to carry out research into new therapies, although if used in human patients, they would face rejection by the body’s immune system.

Stem cells isolated from a person’s own cloned (or reprogrammed) body cells would be an exact genetic match, however, and would not face transplant rejection problems.

Scientists at the US firm Advanced Cell Technology (ACT) first reported the creation of cloned human embryos in November 2001, but the team, lead by Dr Robert Lanza, did not manage to extract any stem

cells from them. Commenting on the new research, Lanza said: “you now have the cookbook; you have a methodology that’s publicly available.” The South Korean team, lead by Drs Woo Suk Hwang and Shin Yong Moon, carried out their work on 247 unfertilised eggs donated by 16 women. The researchers removed the genetic material from 176 of them, choosing those at the most suitable stage of development. They removed the nuclei of these eggs, and replaced them with the genetic material from cumulus cells (cells that surround a developing egg), taken from each of the egg donor women, so that each clone was an exact genetic copy. “They had an incredible amount of eggs and an opportunity to perfect the protocols; they tried 14 different protocols,” said Dr Jose Cibelli, formerly of ACT. Their method yielded blastocysts (five or six day old embryos that contain ES cells) around 26 per cent of the time.

The discovery will provide scientists with a “unique Opportunity” to study human disease, says stem cell scientist Ron McKay. However, the Editor-in-chief of Science, Donald Kennedy, cautions that “it may be years yet before embryonic stem cells can be used in transplantation medicine.” The breakthrough is also likely to reignite the debate over regulating attempts to clone human beings, while allowing therapeutic cloning research to continue. ■ http://www.BioNews.org.uk. BioNews@progress. org.uk © Copyright 2003 Progress Educational Trust Reproduced from BioNews with permission, a weband email-based source of news, information and comment on assisted reproduction and human genetics, published by Progress Educational Trust.

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The Clinical Embryologist

Volume 7, Issue 1

The Road to Single Embryo Transfer Continued from Page 1

the embryo (8,9). The fallopian tube is characterized by high levels of the carboxylic acids pyruvate (0.32mM) and lactate (10.5mM), and by relatively low levels of glucose (0.5mM). In contrast the uterus is characterized by lower levels of pyruvate (0.10mM) and lactate (5.2mM) and higher levels of glucose (3.15mM). Therefore, the embryo is exposed to different levels of carbohydrates before and after compaction, as the embryo passes into the uterus. This is consistent with the embryo preferring pyruvate and lactate over glucose until the blastocyst stage (10).

Significantly, pre and post compaction, the embryo has very different physiologies, which in turn can account for the changes in carbohydrate utilization (11).

Pre Compaction

address the changes in physiology and metabolism.

This is part of the rationale in the development of sequential culture media.

Minimizing intracellular stress Reducing stress as a means of facilitating embryo development is a prerequisite for maintaining embryo viability. If an embryo is stressed in culture, the stress can manifest as one or more of the following: slow cleavage rate, reduced development to the blastocyst, altered blastocyst differentiation, altered blastocyst metabolism, altered gene expression and imprinting, reduced viability and, in the worse case scenario, the development of birth defects. Clearly in order to minimize the stress one must first identify the possible sources.

There are several sources of stress to the embryo within the laboratory. The following list is not meant to be exclusive, but does include sources of stress we have encountered.

1. Inappropriate media formulations.

2. Inappropriate media supplementation.

3. Problems in the culture system.

4. Technical issues.

5. Lack of appropriate QC and QA. Examples for each of the above are discussed below: 1. Inappropriate media formulations. A good example of this is media that lack amino acids. Given the wide variety of niches that amino acids fill in the embryo’s physiology, their omission from culture media will lead to intracellular stress. For example, collection and manipulation of oocytes and embryos in a medium lacking amino acids will lead to the complete efflux of the intracellular pool, resulting in a loss of intracellular buffering capacity. Certainly in the mouse it has been demonstrated that collection of zygotes in a medium lacking amino acids has a profound negative effect on subsequent development to the blastocyst (21,22).

2. Inappropriate media supplementation. The best example of this is the use of serum. The numerous pathologies that serum induces in the preimplantation embryo have been described exhaustively (22,23).

Post Compaction

Low biosynthetic activity

High biosynthetic activity

Low QO2

High QO2

Pyruvate preferred nutrient

Glucose preferred nutrient

Non-essential amino acids

Non-essential + Essential amino acids

Maternal genome

Embryonic genome

Individual cells

Transporting epithelium

1 cell type

2 distinct cell types: ICM & Trophectoderm

Differences in Embryo Physiology Pre and Post Compaction

Within the fluids of the female reproductive tract there are also significant amounts of amino acids.

The roles of amino acids are many fold and include biosynthetic precursors (12), sources of energy (13), regulators of energy metabolism (14), osmolytes (15), buffers of pHi (16), antioxidants (17) and chelators (18). In vitro the mammalian embryo appears to exhibit a biphasic requirement for amino acids as well as carbohydrates (19,20). Logically therefore, in order to meet the requirements of the embryo as it develops, one should employ different media formulations to

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Given the available data, it is ethically unacceptable to use serum in any culture media used within an IVF laboratory.

3. Problems in the culture system. Problems in the culture system can arise as artifacts induced by the static nature of the incubation conditions. For example, when we incubate amino acids at 37oC, they spontaneously deaminate to release ammonium (24).

Ammonium has shown itself to be highly detrimental to both embryo development in culture and to fetal development post transfer (25). The vast majority of ammonium comes from the amino acid glutamine. The toxicity of glutamine can be alleviated by substituting this amino acid with the stable dipeptide form, alanylglutamine, which is equally as effective as glutamine, but completely stable at 37oC (26).

4. Technical issues. Technical issues can represent variability in the laboratory, such as the number of incubation chambers employed. The number of chambers can have a significant effect on the ability of embryos to develop and maintain their viability.

An insufficient number of chambers will result in their inability to maintain the correct temperature and gas phase, resulting in intracellular stress. This can be rectified by the acquisition of new chambers, preferably chambers that can provide a reduced oxygen environment, as a high oxygen concentration (20%) is another source of stress to the embryo (27).

5. Lack of appropriate QC and QA. It is a sad fact that the IVF community is so small in relative terms, that the manufacturers of the contact supplies we use, such as culture dishes, tubes and pipettes, do not make their products with our needs in mind. This is manifest when one attempts to QC each lot of supplies that enters the program. The reality is that 25% of contact supplies tested in our program, using a 1-cell mouse embryo assay performed in a simple medium lacking amino acids, fails.

The requirements of this test are that the embryos have to pass the assay at several stages of development, including ≥ 60 % compaction at 9am on day 3, ≥ 50 % blastocyst at 4pm on day 4, ≥ 80 % expanded blastocyst at 9am on day 5 with a total cell number ≥ 50 (where day 1 is the pronulceate stage, and when embryos are placed into culture at 10am). Of the 25% of contact supplies that fail, not all are outright lethal, but do cause retarded embryo

development, i.e. they may actually fail the test on day 3 or 4, but look relatively good on day 5, so in effect the sample is suboptimal. Therefore, if an end point mouse embryo assay is used (e.g. percent blastocyst development on day 5), then we can introduce suboptimal contact supplies etc., into our laboratories.

Regrettably, the companies that manufacture such contact supplies are reticent to let outside laboratories test their products for use in human ART. They simply do not want to risk having to deal with such regulatory bodies as the FDA etc, for such a small market. (Once they have their products tested for ART, then they have to comply with the requirements of the FDA for medical devices.) We have offered our services gratis to various suppliers so they could then inform our community that they have safe contact supplies. To date our services have been declined. This highlights the problems we face being such a small fish in a very big biomedical pond.

The Development of Current Culture Media The approach taken in our laboratory over the past 15 years has been an amalgamation of the above Continued on Page 18

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two sections, i.e. to learn from the environment to which embryos are exposed in vivo, while at the same time studying the physiology and metabolism of the embryo in culture in order to determine what causes intracellular stress to the embryo. By being able to identify and monitor such stress we have been able to develop stage-specific culture media that substantially reduce culture-induced trauma. [The development and characterization of such sequential media have been published in detail elsewhere (27-31).] In other words we have taken both a “back to nature style” by looking at clues from the female reproductive tract, but have also studied the physiology of the embryo and used such information to create the least stressful environment to facilitate development.

This approach is fundamentally different to that taken by Biggers et al., who developed mouse embryo culture media using the simplex optimization procedure. This method used a computer program to generate simple culture media formulations (free of

amino acids, vitamins etc) based upon the response of mouse embryos in culture (32,33). Once a specific medium was formulated, tested and blastocyst development analyzed, the computer program would then generate several more media formulations for use in the next series of cultures. Blastocyst cell numbers, embryo physiology or viability were not parameters analyzed or used in the development of such media. This procedure was performed several times to generate media that supported high rates of blastocyst development of embryos derived from the oocytes of outbred mice (CF1) crossed with the sperm of an F1 hybrid male. Such media were subsequently modified by another laboratory to include amino acids (KSOMAA). This last phase of medium development was based upon previous studies on the mouse embryo (24) and did not involve the simplex procedure.

Recently this single medium formulation, KSOMAA, has been used to produce human blastocysts in culture (34,35).

Figure 1. A holistic analysis of human IVF.

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It is of no surprise that a single culture medium can support human blastocyst development, as several studies (11,27,36,37) have used a single medium formulation for this purpose. For example, although Bolton et al. (38) obtained a very respectable 40% blastocyst development from human pronuclear embryos using Earle’s supplemented with pyruvate and 10% maternal serum, the resultant implantation and pregnancy rate was only 7%. The real question remains; are blastocysts cultured in a single medium formulation more viable than those obtained through the use of sequential culture media? This question in the human remains to be answered in prospective, randomized clinical trials. However, there are data from two different animal models in which KSOMAA has been compared directly to sequential media (37). In these studies the efficacy of KSOMAA was compared to the sequential media G1/G2 in their

ability to support the development of the mouse and cow embryos in culture. In the cow, resultant cell numbers of the blastocysts were significantly higher when embryos were cultured in sequential media G1/G2 compared to those embryos cultured in KSOMAA (37). In the mouse model, sequential media supported significantly higher rates of blastocyst development. Furthermore, similar to the data on the cow, the blastocysts cultured in sequential media had significantly more cells than those cultured in KSOMAA (22,39). Of greatest importance, those blastocysts in the sequential media had significantly better inner cell mass development, which in turn reflected their increased viability over those embryos cultured in KSOMAA. Holistic Analysis of IVF From the above discussion it is evident that in Continued on Page 21

Figure 1. This figure serves to illustrate the complex and interdependent nature of human IVF treatment. For example, the stimulation regimen used not only impacts on oocyte quality (and hence embryo physiology and viability (72), but can also affect subsequent endometrial receptivity (73-75).

Furthermore, the health and dietary status of the patient can have a profound effect on the subsequent developmental capacity of the oocyte and embryo (76,77). The dietary status of patients attending IVF is typically not considered as a compounding variable, but growing data would indicate otherwise.

In the schematic, the laboratory has been broken down into its core components, only one of which is the culture system. The culture system has in turn been broken down to its components, only one of which is the culture media. Therefore, it would appear rather simplistic to assume that by changing only one part of the culture system (i.e. culture media), that one is going to mimic the results of a given laboratory or clinic.

A major determinant of the success of a laboratory and culture system is the level of quality control and quality assurance in place. For example, one should never assume that anything coming into the laboratory that has not been pre-tested with a relevant bioassay (e.g. mouse embryo assay), is safe merely because a

previous lot has performed satisfactorily. Only a small percentage of the contact supplies and tissue culture ware used in IVF comes suitably tested. Therefore it is essential to assume that everything entering the IVF laboratory without a suitable pretest is embryo toxic until proven otherwise.

In our program the 1-cell mouse embryo assay (MEA) is employed to prescreen every lot of tissue culture ware that enters the program, i.e. plastics that are approved for tissue culture. Around 25% of all such material fails the 1-cell MEA (in a simple medium lacking protein after the first 24h). Therefore, if one does not perform QC to this level, 1 in 4 of all contact supplies used clinically will be embryo toxic. In reality many programs cannot allocate the resources required for this level of QC; and when embryo quality is compromised in the laboratory, it is the media that are held responsible, when in fact the lab ware is more often the culprit. For complete details of the conditions for embryo recovery, culture and MEA that are used in our laboratories, the reader may consult the following texts (27,78,79).

‘Modified from an article in Reproductive BioMedicine Online by Gardner DK and Lane M. Towards a single embryo transfer. RBM Online 2003;6:470-481. With permission from Reproductive Healthcare Ltd.’

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Continued from Page 19

order to optimize embryo development, one has to do more than consider the culture media in isolation.

Rather, one must take a more holistic look at the IVF cycle. Figure 1 is an attempt to present some of the key factors that impact the outcome of a given IVF cycle.

Only by examining all of the factors present in Figure 1 can one begin to optimize the treatment of an IVF cycle.

used glucose uptake and lactate production to determine glycolytic activity in individual day-5 mouse blastocysts prior to transfer. Blastocysts were classified as viable or non-viable according to their rate of glucose uptake and lactate production. Mouse blastocysts of equal dimensions and morphology subsequently had their metabolism quantitated non-invasively and then classified as either viable or non-viable. It was found that those blastocysts which exhibited a pattern of glycolytic utilization similar to that of embryos developed in vivo had a developmental potential of 80%, while those blastocysts which exhibited an excessive lactate production (i.e. aberrant glycolytic activity), had a developmental potential of only 6%. Interestingly, when a retrospective analysis of glucose uptakes was performed on the blastocysts transferred in this study, those blastocysts classified as viable had a significantly higher rate of glucose uptake than those blastocysts classified as non-viable. Therefore, it would appear that both the rate of nutrient uptake and its subsequent fate are important determinants of embryo viability.

Similarly, Van den Bergh et al. (49) showed that, in patients who conceived following blastocyst transfer, their embryos had a higher glucose uptake than those blastocysts which failed to establish a pregnancy. Similar to the data reported on the mouse (48), viable human blastocysts had a significantly lower glycolytic activity than those embryos which did not establish a pregnancy.

More recently, two studies have determined the relationship between human embryo nutrition and subsequent development in vitro. Gardner et al. (50) determined that glucose consumption on day 4 by human embryos was twice as high in those embryos that went on to form blastocysts. Furthermore, it was determined that blastocyst quality affected glucose uptake. Poor quality blastocysts consumed significantly less glucose than top scoring embryos.

Significantly, within a cohort of human blastocysts from the same patient with the same alpha-numeric score, i.e. 4AA (27), there exists a significant spread of metabolic activities. Therefore, assessing metabolic activity and metabolic normality may prove to be a feasible way to determine embryonic

In vivo rates of embryo development in vitro Historically embryos cultured in vitro lag behind their in vivo developed counterparts (40,41). However, with the development of sequential media, based upon the premise of meeting the changing requirements of the embryo and minimizing trauma, in vivo rates of embryo development can now be attained in vitro in the mouse (22,42). This is a significant development for the laboratory, for now we have a culture system capable of producing blastocysts at the same time and with the same cell number and allocation to the inner cell mass as embryos developed in the female reproductive tract. This helps to explain why the implantation rates of human blastocysts developed in our laboratory approximate those of blastocysts developed in vivo (43).

Selection of Viable Embryos Since the inception of IVF there have been several attempts to correlate the morphology of pronucleate and cleavage-stage embryos with their development to the blastocyst stage and with their subsequent viability.

The approaches taken to assess embryos on successive days of development have been recently reviewed (44).

With such scoring systems implantation rates as high as 28% for pronucleate embryos (45), 48% for day 3 embryos (46) and 70% for blastocysts transferred on day 5 (47) have been reported for selected groups of patients.

However, at best, attempts to select embryos based upon morphology are akin to one walking in to the physician for an annual check up, and the doctor looks at you and says “Well you look fine!”, but does not conduct any further investigation. Therefore, tests that can assess the physiology and metabolism and even the genetics of the embryos developed are highly desirable.

In a prospective study, Lane and Gardner (48)

Continued on Page 22

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“health”. In a study on amino acids, Houghton et al., (51) determined that alanine release into the surrounding medium on day 2 and day 3 was highest in those embryos that did not form blastocysts.

It has also been shown that human blastocysts produce ammonium, indicating that they are actively transaminating amino acids (50). This leads one to propose that using the non-invasive assessment of carbohydrate and/or amino acid metabolism may be of use in selecting embryos for single embryo transfer.

Furthermore, with the advent of polar body and blastomere biopsy, one is now able to analyze the karyotype of the embryo (52,53). This will hopefully lead to the ability to select for both normal karyotype and physiology.

Cryopreservation It is evident that if we move to reduce the numbers of embryos transferred, then we will have more embryos to cryopreserve. Therefore, the ability to optimize cryopreservation of embryos is a prerequisite for moving to single embryo transfer.

In recent years there have been significant advances in the area of cryobiology for storage of animal oocytes and embryos. There has been a steady move towards vitrification as the procedure of choice for animal embryos (54). Additionally, the use of vitrification has enabled the successful cryopreservation of embryos from species that had previously not been able to be cryopreserved, such as the pig (55). Most significantly have been the improvements in the ability to cryopreserve animal oocytes using ultra-rapid vitrification procedures such as open-pulled straw and loop vitrification.

These successes in the field of animal ART have lead to interest in vitrification to improve the success of human oocyte and blastocyst cryopreservation.

However, to date there is relatively little information as to the role of vitrification in human ART. Slow freezing of blastocysts The first report of human blastocyst freezing dates back to 1985 (56). In this study blastocysts were cryopreserved using a slow-freezing protocol with the cryoprotectant glycerol added in 6 steps of increasing concentration. However, subsequent larger trials of blastocyst freezing reported disappointing

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rates of survival (around 50% and 60%), low pregnancy rates (9% and 7%) and high rates of fetal loss (32%)(57). Such low rates could also be due to the rather poor culture systems employed in these studies. Menezo et al., (58) reported a doubling of the pregnancy rate by modifying the protocol to a 2 step exposure to glycerol. A further report of 500 cycles of cryopreservation of blastocysts grown using co-culture with Vero cells using this 2 step protocol reported survival rates of around 80% and an ongoing pregnancy rate of 19% and an implantation rate of 13% (59). The implantation rate of frozen blastocysts in this study was around half of that reported for the transfer of fresh blastocysts. Most clinics freezing human blastocysts are using a modification of this Menezo protocol.

Using the criterion for freezing that day 5 or day 6 blastocysts should be expanded with a well defined ICM and trophectoderm, and a modified Menezo slow freeze protocol, there are now reports of ongoing pregnancy and implantation rates of around 50% and 30% respectively (60,61).

Vitrification of blastocysts The literature documents the successful vitrification of blastocysts from rodents such as the mouse (62,63) and domestic animals such as the cow (64,65). There are also a limited number of studies on the vitrification of the cleavage stage human embryo (66-68). Recently there have been several reports of ongoing pregnancies after ultra-rapid vitrification of human blastocysts (69,70). The reported implantation rates for vitrification of blastocysts are around 25%.

Vitrification of human oocytes and embryos is in the very early stages of development. Vitrification has dramatically improved the success of cryopreservation of embryos, but especially oocytes in the field of animal ART. Therefore, with continued research it is likely that the vitrification procedures that have improved success rates in animal embryos will also do so in the human, although vitrification cannot be considered a routine procedure for humans as yet.

Conclusions With the advances in culture technology and the development of more detailed embryo scoring systems (and possibly non-invasive viability assays), together Continued on Page 24

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Continued from Page 23

References

with an increased ability to successfully cryopreserve embryos, single embryo transfer will become a reality for a growing number of patients. To attain this goal, it is essential to consider the IVF cycle in its entirety, by taking a holistic approach. Some may like to call this “Zen and the art of ART”.

To determine the feasibility of single embryo transfer we have performed a prospective randomized trial of one versus two blastocyst transfers in patients with good ovarian reserve (71). The transfer of a single blastocyst resulted in an implantation and ongoing pregnancy rate of 60.9% with no twins. The transfer of two blastocysts resulted in an implantation rate of 56% and an ongoing pregnancy rate of 76%, with a 47.4% incidence of twins. Using the selection criteria for this study, approximately three quarters of our clinic’s current patients would be eligible for single blastocyst transfer, no small percentage. ■ Dr. David K. Gardner, Ph.D., Scientific Director, Colorado Center for Reproductive Medicine, Englewood, CO. [Email: dgardner@colocrm.com]

1. Lipitz S, Frenkel Y, Watts C, Ben Rafael Z, Barkai G, and Reichman B. High-order multifetal gestation--management and outcome. Obstet Gynecol 1990;76:215-8.

2. Gardner MO, Goldenberg RL, Cliver SP, Tucker JM, Nelson KG, and Copper RL. The origin and outcome of preterm twin pregnancies. Obstet Gynecol 1995;85:553-7.

3. Adashi EY, Barri PN, Berkowitz R, Braude P, Bryan E, Carr J, Cohen J, Collins J, Devroey P, Frydman R, Gardner D, Germond M, Gerris J, Gianaroli L, Hamberger L, Howles C, Jones H, Jr., Lunenfeld B, Pope A, Reynolds M, Rosenwaks Z, Shieve LA, Serour GI, Shenfield F, Templeton A, Van Steirteghem A, Veeck L, and Ulla-Britt W. Infertility therapy-associated multiple pregnancies (births): an ongoing epidemic. Reprod Biomed Online 2003;7:515-42.

4. Evans MI, Dommergues M, Wapner RJ, Lynch L, Dumez Y, Goldberg JD, Zador IE, Nicolaides KH, Johnson MP, Golbus MS. Efficacy of transabdominal multifetal pregnancy reduction: collaborative experience among the world’s largest centers.

Obstet Gynecol 1993;82:61-6.

5. Cusick W and Gleicher N. Multiple conceptions, implantation, and prematurity. Assisted Reproductive Reviews 1995;246.

6. Kanhai HH, de Haan M, van Zanten LA, GeerinckVercammen C, van der Ploeg HM, and Gravenhorst JB. Followup of pregnancies, infants, and families after multifetal pregnancy reduction. Fertil Steril 1994;62:955-9.

7. Goldfarb JM, Austin C, Lisbona H, Peskin B, and Clapp M. Cost-effectiveness of in vitro fertilization. Obstet Gynecol 1996;87:18-21.

8. Gardner DK, Lane M, Calderon I, and Leeton J. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril 1996;65:349-53.

9. Gardner DK, Lane M, and Schoolcraft WB. Physiology and culture of the human blastocyst. J Reprod Immunol 2002;55:85-100.

10. Biggers JD and Stern S. Metabolism of the preimplantation mammalian embryo. Adv Reprod Physiol 1973;6:1-59.

11. Gardner DK, Pool TB, and Lane M. Embryo nutrition and energy metabolism and its relationship to embryo growth, differentiation, and viability. Semin Reprod Med 2000;18:205-18.

12. Crosby IM, Gandolfi F, and Moor RM. Control of protein synthesis during early cleavage of sheep embryos. J Reprod Fertil 1988;82:769-75.

13. Rieger D, Loskutoff NM, and Betteridge KJ. Developmentally related changes in the metabolism of glucose and glutamine by cattle embryos produced and co-cultured in vitro. J Reprod Fertil 1992;95:585-95.

14. Gardner DK and Lane M. The 2-cell block in CF1 mouse embryos is associated with an increase in glycolysis and a decrease in tricarboxylic acid (TCA) cycle activity: alleviation of the 2-cell block is assocated with the restoration of in vivo metabolic pathway activities. Biol Reprod 1993;48 Suppl 1:152.

DOES EVERYONE AT YOUR FERTILITY CENTER RECEIVE THE CLINICAL EMBRYOLOGIST? New recipient or new address NAME: ________________________________________ ADDRESS: _____________________________________ _______________________________________________ CITY: _________________________________________ STATE:___________ ZIP: ________________________ Copy & send to: Embryologistsʼ Newsletter 115 N.W. 84th Way, Coral Springs, FL 33071

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15. Van Winkle LJ, Haghighat N, and Campione AL. Glycine protects preimplantation mouse conceptuses from a detrimental effect on development of the inorganic ions in oviductal fluid. J Exp Zool 1990;253:215-9.

16. Edwards LJ, Williams DA, and Gardner DK. Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH. Hum Reprod 1998;13:3441-8.

17. Liu Z and Foote RH. Development of bovine embryos in KSOM with added superoxide dismutase and taurine and with five and twenty percent O2. Biol Reprod 1995;53:786-90.

18. Lindenbaum A. A survey of naturally occurring chelating ligands. Adv Exp Med Biol 1973;40:67-77.

19. Lane M and Gardner DK. Differential regulation of mouse embryo development and viability by amino acids. J Reprod Fertil 1997;109:153-64.

20. Steeves TE and Gardner DK. Temporal and differential effects of amino acids on bovine embryo development in culture.

Biol Reprod 1999;61:731-40.

21. Gardner DK and Lane M. Alleviation of the ‘2-cell block’ and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod 1996;11:2703-12.

22. Gardner DK and Lane M. Towards a single embryo transfer. RBM Online 2003;6:470-81.

23. Gardner DK. Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biol Int 1994;18:1163-79.

24. Gardner DK and Lane M. Amino acids and ammonium regulate mouse embryo development in culture. Biol Reprod 1993;48:377-85.

25. Lane M and Gardner DK. Increase in postimplantation development of cultured mouse embryos by amino acids and induction of fetal retardation and exencephaly by ammonium ions. J Reprod Fertil 1994;102:305-12.

26. Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J, and Hesla J. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 1998;13:3434-40.

27. Gardner DK and Lane M. Embryo culture systems. eds.

Trounson, A. O. and Gardner, D. K. Handbook of In Vitro Fertilization, Second Edition. 205-264. 1999. Boca Raton, CRC Press.

28. Gardner DK. and Lane M. Embryo culture systems. eds. Gardner, D. K. and Trounson, A. O. Handbook of In Vitro Fertilization. 85-114. 1993. Boca Raton, CRC Press.

29. Gardner DK. Culture of mammalian embryos in the absence of serum and somatic cells. Cell Biol Int 1994;18:1163-79.

30. Gardner DK and Lane M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum Reprod Update 1997;3:367-82.

31. Pool TB, Atiee S.H., and Martin JE. Oocyte and embryo culture: basic concepts and recent advances. Infertility and Reproductive Medicine Clinics of North America 1998;9:181-203.

32. Lawitts JA and Biggers JD. Optimization of mouse embryo culture media using simplex methods. J Reprod Fertil 1991;91:543-56.

33. Lawitts JA and Biggers JD. Culture of preimplantation embryos. Methods Enzymol 1993;225:153-64.

34. Biggers JD and Racowsky C. The development of fertilized human ova to the blastocyst stage in KSOMAA medium: is a two-step protocol necessary? Reprod BioMedicine Online 2002;5:133-40.

35. Wiemer K, Anderson AR, Kyslinger L, and Weikert ML. Embryonic development and pregnancies following sequential culture in human tubal fluid and modified simplex optimized medium containing amino acids. Reprod BioMedicine Online 2002;5: 36. Gardner DK and Lane M. Culture of viable human blastocysts in defined sequential serum-free media. Hum Reprod 1998;13 Suppl 3:148-59.

37. Gardner DK and Lane M. Development of viable mammalian embryos in vitro : Evolution of sequential media. eds. Cibelli, J., Lanza, R. P., Campbell, K. H. S., and West, M. D. Principles of Cloning. 187-213. 2002. New York, Academic Press.

38. Bolton VN, Wren ME, and Parsons JH. Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil Steril 1991;55:830-2.

39. Gardner DK and Lane M. Culture media for the human embryo. eds. Revelli A, Tur-Kaspa, I., Holte JG, and Massobrio M. Biotechnology of human reproduction. 181-199. 2002.

Parthenon Press.

40. Bowman P and McLaren A. Cleavage rate of mouse embryos in vivo and in vitro. J Embryol Exp Morphol 1970;24:203-7.

41. Harlow GM and Quinn P. Development of preimplantation mouse embryos in vivo and in vitro. Aust J Biol Sci 1982;35:187-93.

42. Reed L.C., Lane M, and Gardner, D. K. In vivo rates of mouse embryo development can be attained in vitro.

Theriogenology 59, 349. 2003.

43. Buster JE, Bustillo M, Rodi IA, Cohen SW, Hamilton M, Simon JA, Thorneycroft IH, and Marshall JR. Biologic and morphologic development of donated human ova recovered by nonsurgical uterine lavage. Am J Obstet Gynecol 1985;153:211-7.

44. Gardner DK and Sakkas D. Assessment of embryo viability: the ability to select a single embryo for transfer--a review. Placenta 2003;24 Suppl B:S5-12.

45. Scott LA and Smith S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum Reprod 1998;13:1003-13.

46. Gerris J, De Neubourg D, Mangelschots K, Van Royen E, Van de MM, and Valkenburg M. Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod 1999;14:2581-7.

47. Gardner DK, Lane M, Stevens J, Schlenker T, and Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril 2000;73:1155-8.

48. Lane M and Gardner DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum Continued on Page 26

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Reprod 1996;11:1975-8.

49. Van den Bergh, M., Devreker, F., Emiliani, S., and Englert, Y. Glycolytic activity: a possible tool for human blastocyst selection. RBM Online 3, Suppl 1, 8. 2001.

50. Gardner DK, Lane M, Stevens J, and Schoolcraft WB. Noninvasive assessment of human embryo nutrient consumption as a measure of developmental potential. Fertil Steril 2001;76:1175-80.

51. Houghton FD, Hawkhead JA, Humpherson PG, Hogg JE, Balen AH, Rutherford AJ, and Leese HJ. Non-invasive amino acid turnover predicts human embryo developmental capacity.

Hum Reprod 2002;17:999-1005.

52. Munne S and Wells D. Preimplantation genetic diagnosis.

Curr Opin Obstet Gynecol 2002;14:239-44.

53. Voullaire L, Slater H, Williamson R, and Wilton L. Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization. Hum Genet 2000;106:210-7.

54. Kasai M. Cryopreservation of mammalian embryos. Mol Biotechnol 1997;7:173-9.

55. Dobrinsky JR, Pursel VG, Long CR, and Johnson LA. Birth of piglets after transfer of embryos cryopreserved by cytoskeletal stabilization and vitrification. Biol Reprod 2000;62:564-70.

56. Cohen J, Simons RF, Fehilly CB, Fishel SB, Edwards RG, Hewitt J, Rowlant GF, Steptoe PC, and Webster JM. Birth after replacement of hatching blastocyst cryopreserved at expanded blastocyst stage [letter]. Lancet 1985;1:647.

57. Hartshorne GM, Elder K, Crow J, Dyson H, and Edwards RG. The influence of in-vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Hum Reprod 1991;6:136-41.

58. Menezo Y, Nicollet B, Herbaut N, and Andre D. Freezing cocultured human blastocysts. Fertil Steril 1992;58:977-80.

59. Kaufman RA, Menezo Y, Hazout A, Nicollet B, DuMont M, and Servy EJ. Cocultured blastocyst cryopreservation: experience of more than 500 transfer cycles. Fertil Steril 1995;64:1125-9.

60. Gardner DK, Lane M, Stevens J, and Schoolcraft WB. Changing the start temperature and cooling rate in a slowfreezing protocol increases human blastocyst viability. Fertil Steril 2003;79:407-10.

61. Veeck L. Does the developmental stage at freeze impact on clinical results post-thaw? RBM Online 2003;6:367-74.

62. Lane M, Schoolcraft WB, and Gardner DK. Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertil Steril 1999;72:1073-8.

63. Zhu SE, Sakurai T, Edashige K, Machida T, and Kasai M. Cryopreservation of zona-hatched mouse blastocysts. J Reprod Fertil 1996;107:37-42.

64. Vajta G, Holm P, Greve T, and Callesen H. Survival and development of bovine blastocysts produced in vitro after assisted hatching, vitrification and in-straw direct rehydration. J Reprod Fertil 1997;111:65-70.

65. Donnay I, Auquier P, Kaidi S, Carolan C, Lonergan P, Mermillod P, and Massip A. Vitrification of in vitro produced bovine blastocysts: methodological studies and developmental

capacity. Anim Reprod Sci 1998;52:93-104.

66. Vanderzwalmen P, Delval A, and Chatziparasidou A. Pregnancies after vitrification of human day 5 embryos. Hum Reprod 1997;12:O-198.

67. Mukaida T, Wada S, Takahashi K, Pedro PB, An TZ, and Kasai M. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos. Hum Reprod 1998;13:2874-9.

68. Saito H, Ishida GM, Kaneko T, Kawachiya S, Ohta N, Takahashi T, Saito T, and Hiroi M. Application of vitrification to human embryo freezing. Gynecol Obstet Invest 2000;49:145-9.

69. Reed ML, Lane M, Gardner DK, Jensen NL, and Thompson J. Vitrification of human blastocysts using the cryoloop method: successful clinical application and birth of offspring. J Assist Reprod Genet 2002;19:304-6.

70. Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M, and Takahashi K. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003;18:384-91.

71. Gardner DK, Surrey E, Minjarez D, Leitz A, Stevens J, and Schoolcraft WB. Single blastocyst transfer: A prospective randomized trial. Fertil Steril 2004; 81: 551-5.

72. Hardy K, Robinson FM, Paraschos T, Wicks R, Franks S, and Winston RM. Normal development and metabolic activity of preimplantation embryos in vitro from patients with polycystic ovaries. Hum Reprod 1995;10:2125-35.

73. Simon C, Garcia Velasco JJ, Valbuena D, Peinado JA, Moreno C, Remohi J, and Pellicer A. Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil Steril 1998;70:234-9.

74. Van der Auwera I, Pijnenborg R, and Koninckx PR. The influence of in-vitro culture versus stimulated and untreated oviductal environment on mouse embryo development and implantation. Hum Reprod 1999;14:2570-4.

75. Ertzeid G and Storeng R. The impact of ovarian stimulation on implantation and fetal development in mice. Hum Reprod 2001;16:221-5.

76. Kwong WY, Wild AE, Roberts P, Willis AC, and Fleming TP. Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension. Development 2000;127:4195-202.

77. Gardner DK, Stilley K, and Lane M. High protein diet inhibits inner cell mass formation and increases apoptosis in mouse blastocysts developed in vivo by increasing the levels of ammonium in the reproductive tract. Reprod Fertil Dev 2004;16:190.

78. Lane M and Gardner DK. Preparation of gametes, in vitro maturation. eds. Gardner, D. K, Lane M, and Watson, A. A laboratory guide to the mammalian embryo. 24-40. 2004. New York, Oxford University Press.

79. Gardner DK and Lane M. Culture of the mammalian preimplantation embryo. eds. Gardner, D. K, Lane M, and Watson, A. A laboratory guide to the mammalian embryo. 4161. 2004. New York, Oxford University Press.

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