D.K. Gardner

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Although  considerable progress has been made in our ability to culture the human  embryo, there has been relatively little research on the role and  composition of the embryo transfer medium. The media used for embryo  culture and transfer are almost pure aqueous solutions supplemented with  protein, usually at 5 to 10%. This protein is usually in the form of  serum albumin or whole serum. However, such media even when supplemented  with protein bear little similarity to the fluids of the female  reproductive tract, which are more viscous in nature. In an attempt to  increase the viscosity of the transfer medium Menezo et al., (1989) used  1% placental collagen. However, there was no increase in implantation  rates as a result of its use. Although albumin is the most abundant  macromolecule in the female reproductive tract, glycosaminoglycans  (GAGS) are found at high concentrations in the fluid of the female  reproductive tract of several mammalian species (Zorn et al., 1995). One  of these GAGS, hyaluronan, a linear polysaccharide of alternating  D-glucuronic acid and N-acetyl-D-glucosamine residues, increases in  concentration at the time of implantation in the mouse embryo (Carson et  al., 1987). Furthermore, human embryos have the surface receptor for  hyaluronan, CD44, throughout development from the oocyte to the  blastocyst stage (Campbell et al., 1995). In a study on mouse embryo  development and transfer Gardner et al., (1999) determined that  hyaluronan could substitute for serum albumin in culture media. However,  it was found that at the time of transfer the presence of hyaluronan in  the medium resulted in a significant increase in implantation rates.  This benefit did not depend on the presence of any macromolecule in the  culture medium. Embryos cultured to the blastocyst stage in the absence  of any protein/macromolecule benefited from the presence of hyaluronan  in the transfer medium. There are several possible means by which  hyaluronan could facilitate implantation; hyaluronan has been shown to  increase cell-cell adhesion and cell-matrix adhesion and so may function  during the initial stages of apposition and attachment of the  blastocyst and endometrium. However, even though the human embryo may  specifically bind hyaluronan, it is plausible that the beneficial  effects of hyaluronan may be manifest through other indirect routes. For  example, hyaluronan can promote angiogenesis by both its degradation  products and by interaction with EGF. The latter point is of particular  interest given the stimulatory effect of EGF on implantation in the  mouse. Alternatively, the beneficial effect of hyaluronan in the  transfer medium may be a physical phenomenon, by facilitating rapid  diffusion of the contents of the transfer medium (the embryo) with the  fluid of the uterus. As uterine fluid is a viscous solution, the  transfer of a relatively aqueous solution, such as culture medium with  albumin or serum, to the uterine lumen will result in the slow dispersal  of the medium and embryo with the luminal contents. In conclusion, the  polysaccharide hyaluronan may prove to be effective in facilitating  improvements in embryo transfer. As hyaluronan can be obtained from  bioengineered bacteria in an endotoxin-free form, transfer media can be  standardized and biological variation eliminated.


CAMPBELL S, SWANN HR, APLIN JD, ET AL. CD44 is expressed throughout pre-implantation human embryo development. Hum. Reprod. 10, 425-430, 1995.

CARSON DD, DUTT A. AND TANG J-P. Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function. Dev. Biol. 120, 228-235, 1987.

GARDNER DK, LANE M AND RODRIGUEZ-MARTINEZ H. Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronate for embryo culture and transfer in the mouse. Hum. Reprod. 14, 1999.

MENEZO Y, ARNAL F, HUMEAU C, ET AL. Increased viscosity in transfer medium does not improve the pregnancy rates after embryo transfer. Fertil. Steril. 52, 680-682, 1989.

ZORN TMT, PINHAL MAS, NADER HB, ET AL. Biosynthesis of glycosaminoglycans in the endometrium during the initial stages of pregnancy of the mouse. Cell. Mol. Biol. 41, 97-106, 1995.

Articles

The First World Congress On: Controversies in Obstetrics, Gynecology & InfertilityPrague, Czech Republic - 1999

Protein-Free Medium for Embryo Transfer

The routine transfer  of cleavage stage embryos to the patient’s uterus on day 2 or 3 has two  inherent drawbacks; firstly the embryo does not reside in the uterus at  such time, and secondly, up to the 8-cell stage one is observing a  cleaving oocyte, in that the majority of embryonic genes have not been  activated. Therefore it is not possible to assess true embryonic  developmental potential of the conceptus at this time. Consequently the  transfer of cleavage stage embryos has been associated with low  implantation rates (10-30%). A further problem associated with the  transfer of cleavage stage embryo is that in order to attain acceptable  pregnancy rates, multiple embryos are routinely transferred to the  patient, which in turn frequently culminates in multiple gestations. In  the USA, where the national average for the number of embryos  transferred is 4, high order multiple gestations are regularly reported.  The issue of high order multiple gestations is of concern to all who  practice IVF, and has enormous, medical, social and financial  implications (Gardner and Schoolcraft, 1998). 

Increasing embryo implantation rates  should not only reduce the need to transfer multiple embryos, but  should also increase the overall efficacy of an IVF cycle. Over the past  decade a resurgence of interest in embryo physiology has led to the  formulation of sequential culture media designed to cater for the  changing requirements of the embryo as it develops and differentiates  (Gardner and Lane, 1997). Such media were specifically designed based on  data regarding the physiology of the maternal reproductive tract and  embryo. In a prospective randomized trial a significantly higher  implantation rate for blastocysts transferred on day 5 (50%), compared  to cleavage stage embryos transferred on day 3 (30%), was confirmed in  patients with a good response to gonadotropins (Gardner et al., 1998).  Subsequently, it has been determined that the quality of the blastocyst  formed has a direct impact on pregnancy outcome (Schoolcraft et al.,  1999). When a patient has at least one blastocyst with a well developed  inner cell mass and trophectoderm, implantation rates of 65% can be  achieved. Therefore it is evident that blastocyst culture and transfer  can be used to effectively eliminate high order multiple gestations, and  perhaps equally as importantly, will lead to the introduction of single  embryo transfer, while establishing pregnancy rates of 50% or greater  in certain groups of patients. In a retrospective analysis of blastocyst  transfer for all patients entering an IVF program, Marek et al., (1999)  determined that for all age groups, extended culture and transfer was  associated with an increase in implantation and pregnancy rates, while  observing a significant decrease in the number of embryos transferred.  An important point about this report is that less than 7% of oocyte  retrievals resulted in no embryo transfers, compared to 3% no transfers  for day 3. Therefore, concerns regarding no transfer after extended  culture appears unjustified. To conclude, blastocyst transfer can be  used to effectively eliminate high order multiple gestations. Indeed,  single blastocyst transfers should now be considered as a routine  procedure. In the general patient population, preliminary results  indicate that blastocyst transfer does increase the overall efficacy of  IVF.

GARDNER DK AND LANE M. Culture and selection of viable blastocysts: a feasible proposition for human IVF? Hum. Reprod. Update 3, 367-382, 1997.

GARDNER DK, SCHOOLCRAFT WB, WAGLEY L, ET AL. A prospective randomized trial of blastocyst culture and transfer in in vitro fertilization. Hum. Reprod.13, 3434-3440, 1998.

GARDNER DK AND SCHOOLCRAFT WB. Elimination of high order multiple gestations by blastocyst culture and transfer. In Female Infertility Therapy: Current Practice. Eds. Z Shoham, C Howles and H Jacobs. Martin Dunnitz, London, pp. 267-74, 1998.

MAREK D, LANGLEY M, GARDNER DK, ET AL. Introduction of blastocyst culture and transfer for all patients in an IVF program. Fertil. Steril. December, 1999.

SCHOOLCRAFT WB, GARDNER DK, LANE M, ET AL. Blastocyst culture and transfer: Analysis of results and parameters affecting outcome in two IVF programs. Fertil. Steril. October, 1999.

The routine transfer of cleavage stage embryos to the patient’s uterus on day 2 or 3 has two inherent drawbacks; firstly the embryo does not reside in the uterus at such time, and secondly, up to the 8-cell stage one is observing a cleaving oocyte, in that the majority of embryonic genes have not been activated.

Are Blastocysts the Answer?

With the routine use  of exogenous gonadotropins to stimulate the production of multiple  oocytes, clinics are faced with the daily decision of which of the  resulting embryos to replace in the mother and which to cryopreserve.  There is therefore an obvious clinical need to be able to select those  embryos most likely to implant after replacement in the uterus. Methods  of predicting and quantifying embryo viability have been discussed  extensively (Gardner and Leese, 1999). Parameters for consideration  include assessment of morphology, development in culture, production of  platelet activating factor, production of human chorionic gonadotropin,  enzyme leakage, oxygen uptake measurements, and nutrient uptake  measurements. The ability to predict whether an embryo will develop in  culture has been associated with rate of cleavage in animal studies  (McKiernan and Bavister, 1994). It was observed that the speed at which  an embryo developed was related to its ability to form a blastocyst.  Furthermore, morulae and blastocysts which developed from slowly  dividing embryos gave rise to lower fetal development after transfer  than those embryos which developed from more rapidly dividing cleavage  stages (McKiernan and Bavister, 1994). Similarly, Shoukir et al., (1998)  have observed that those embryos forming blastocyst earlier are the  more viable. (Scott and Smith, 1998) have determined that pronuclear  embryo morphology can be used to select embryos for transfer. A  potential criterion for the quantification of embryo viability is energy  metabolism, which can be readily measured non-invasively for individual  embryos in culture (Gardner and Leese, 1999). Lane and Gardner (1996)  used both glucose uptake and lactate production (to estimate glycolytic  activity) to select prospectively individual day-4 mouse blastocysts for  transfer. Prior to any transfers the hypothesis developed was that the  most viable embryos would be the ones with a glycolytic activity closest  to that observed for embryos developed in vivo, around 50%. Therefore  those embryos exhibiting excessive lactate production were proposed to  be non-viable i.e. they had lost control over their energy metabolism.  When morphologically identical blastocysts were selected at random for  transfer, resultant fetal development was 20% per blastocyst  transferred. However, when blastocysts were prospectively selected with a  glycolytic activity similar to in vivo developed blastocysts, the  resultant fetal development rate was 80%, a 4-fold increase over random  selection. In contrast, those blastocysts with an abnormally elevated  glycolytic activity had a fetal development rate of only 6%. Such data  therefore supports the hypothesis that it is possible to select embryos  for transfer using metabolic criteria. This hypothesis warrants further  investigation in clinics performing single blastocyst transfers.  Interestingly, the quality of human blastocysts, as assessed using the  development of the inner cell mass and trophectoderm, can be used  successfully to identify those embryos with an implantation rate of over  65%. Other markers of blastocyst function such as hCG and PAF  production prior to transfer also warrant further study. In conclusion,  there are several parameters that can be used in order to predict or  quantitate embryo viability. It is envisaged that not one but two or  more approaches will be most effective in identifying those embryos most  likely to give rise to a viable pregnancy, culminating in single embryo  transfers. 


GARDNER DK AND LEESE HJ. Assessment of embryo metabolism and viability. In Handbook of In Vitro Fertilization, Second Edition. Eds. A Trounson and DK Gardner. CRC Press, Boca Raton, pp 347-372, 1999.

LANE M AND GARDNER DK. Selection of viable mouse blastocysts prior to transfer using a metabolic criterion. Hum. Reprod. 11, 1975-1978, 1996.

MCKIERNAN SH AND BAVISTER BD. Timing of development is a critical parameter for predicting successful embryogenesis. Hum. Reprod. 9, 2123-2129, 1994.

SCOTT LA AND SMITH S. The successful use of pronuclear embryo transfers the day following oocyte retrieval. Hum. Reprod. 13, 1003-1013, 1998.

SHOUKIR Y, CHARDONNENS D, CAMPANA A, ET AL. The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Hum. Reprod. 13, 676-81, 1998.

Author | D.K. Gardner

Articles

Protein-Free Medium for Embryo Transfer

Are Blastocysts the Answer?

Markers of Viability