Fluorescein diacetate assessment of embryo viability after ultrarapid freezing of human multipronucleate embryos

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Maximillian Gilmore
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1 FERTILITY AND STERILITY Copyright The American Fertility Society l'rintmi on oeid-free paper in U.S.A. Fluorescein diacetate assessment of embryo viability after ultrarapid freezing of human multipronucleate embryos Vincenzo Noto, M.D. Rudi Campo, M.D. Patricia Roziers, B.S. Stephan Gordts, M.D.* Medical Center for Fertility Diagnostics and In Vitro Fertilization and Embryo Transfer, Leuven, Belgium Objective: To assess embryo viability after ultrarapid freezing-thawing. Design: We studied the fluorescence pattern of 35 ultrarapidly frozen-thawed multipronucleate human embryos exposed to fluorescein diacetate. Setting: All the embryos were obtained from the Medical Center for Fertility Diagnostics and In Vitro Fertilization and Embryo Transfer at Leuven (Belgium), a private care center. Patients, Participants: None. Interventions: None. Main Outcome Measure: The fluorescence pattern was evaluated at room temperature after a 1-minute incubation in fluorescein diacetate solution, 4 hours and 24 hours after thawing. Results: Healthy human multipronucleate embryos, when exposed to fluorescein diacetate, accumulated intracellular fluorescein and fluoresced brightly under ultraviolet light. On the other hand, embryos presenting submicroscopic cell membranes damage caused by different processes (e.g., high or low temperatures) lost the ability to accumulate intracellular fluorescein. All the ultrarapidly frozen embryos with normal looking blastomeres fluoresced brightly after a short exposure to fluorescein diacetate. Conclusions: Our experiments indicate an intact cell membrane permeability and an integrity of the intracytoplasmatic esterase enzyme activity of human embryos ultrarapidly frozen. Fertil Steril 55:1171, 1991 Ultrarapid freezing of mammalian embryos using high concentrations of dimethyl sulfoxide (DMSO) as cryoprotectant was first described by Trounson et a1. in This freezing method seemed to be a very useful and interesting alternative to the more complicated, expensive, and time-consuming slow freezing techniques. Other papers have been published on this promising field and very encouraging results in terms of survival and cleavage rates have been presented; in 1988 Trounson et a1. 2 and Trounson and Sjoblom 3 published two different reports on the application Received August 13, 1990; revised and accepted February 7, * Reprint requests: Stephan Gordts, M.D., Medical Center for Fertility Diagnostics and In Vitro Fertilization and Embryo Transfer, Tiensevest 168, B-3000 Leuven, Belgium. of this freezing procedure to human fertilized oocytes at very early stages of in vitro development. The first pregnancy resulting from this method of freezing on human embryos was reported and published in 1990 by our group.4 Until now in our center, two patients delivered healthy babies. To understand if an embryo or a single blastomere is still viable after cryostorage, different viability assays are available. In our study, an invasive method has been used on human multipronucleate embryos to assess survival after ultrarapid freezing. This method, related to the invasive measurement of fluorescein diacetate uptake by living cells, may provide a valuable functional criterion to improve the freezing technique. The use of fluorescein diacetate for cell lines cultured in vitro was introduced for the first time by Rotman and Papermaster. 5 Living mammalian cells Noto et ai. Fluorescein test of quick frozen embryos 1171

in vitro accumulate intracellular fluorescein when exposed to fluorescein diacetate because, being nonpolar, it can readily pass into the cell where it is hydrolyzed by esterases to yield fluorescein.

2 in vitro accumulate intracellular fluorescein when exposed to fluorescein diacetate because, being nonpolar, it can readily pass into the cell where it is hydrolyzed by esterases to yield fluorescein. Because fluorescein is polar, it cannot cross back through the cell membrane and therefore accumulates intracellularly. Thus, a living cell becomes highly fluorescent when exposed shortly to fluorescein diacetate because of the integrity of the cell membrane and of the esterase enzyme activity.6 Human Embryos MATERIALS AND METHODS In this study, 35 human multipronucleate embryos (derived from polyspermic zygotes) at different stages of development (from I-cell to very early cleavage stages) were cryopreserved. These embryos, obtained after superovulation induction as a treatment for in vitro fertilization (IVF), represented only 9% of all the embryos obtained in vitro in our center. As a positive internal control for our experiments, 10 nonfrozen multipronucleate embryos were used. Twelve nonviable multipronucleate embryos were used as a negative control: 6 killed by heating for 30 minutes in a waterbath set at 56 C and 6 showing clearly signs of cellular degeneration after quick freezing, as assessed by light microscopy. Five multipronucleate cleaved embryos were exposed to the freezing/thawing solutions without cold storage in liquid nitrogen to determine an eventual cell toxicity because of the high concentrations of cryoprotectant used. Culture conditions and embryo grading before and after ultrarapid freezing/thawing were already described. 4 Freezing Procedure The freezing procedure too has already been described in our previous report, 4 but some details were modified. In brief, a DMSO (Union Chimique BeIge, Leuven, Belgium) concentration of 3 mol/l was used instead of 3.5 mol/l in combination with 0.25 mol/l sucrose (Sigma, St. Louis, MO) in embryo transfer (ET) freezing medium (Gibco Bethesda Research Laboratories, Paisley, Scotland) containing 4 mg/ml bovine serum albumin (BSA). The exposure time of the embryos to this cryoprotective solution before storage in liquid nitrogen was extended to exactly 3 minutes. Thawing Procedure Also for the thawing we modified the procedure. After a storage period of a few days, the straws con- taining the multipronucleate embryos were rapidly removed from liquid nitrogen and held for 10 seconds at room temperature (RT) before being plunged into a waterbath at 37 C. The content of the straws was emptied in ET freezing medium containing mol/l sucrose and left in this solution for 7 minutes before being washed in Earle's medium (Earle's balanced salt solution, Gibco Bethesda Research Laboratories) and cultured at 37 C for further development. Fluorescein Diacetate Assay To perform the viability assay using fluorescein diacetate (Sigma), a stock solution of 5 mg fluorescein diacetate/ml acetone was prepared and stored at -20 C. The day of the viability test a solution of 0.5 JLL fluorescein diacetate stock/ml modified Dulbecco's phosphate-buffered saline [PBS, Gibco Bethesda Research Laboratories] containing 4 mg BSA/mL was prepared and immediately used (final concentration 2.5 JLg fluorescein diacetate/ml). Embryos cultured in Earle's medium containing 15% heat-inactivated wife serum were washed several times in modified Dulbecco's PBS to remove the phenol red from the extracellular compartment because under ultraviolet light phenol red can become spontaneously fluorescent. The embryos, 4 hours and 24 hours after thawing, were incubated in modified Dulbecco's PBS containing fluorescein diacetate for exactly 1 minute at RT and washed again in modified Dulbecco's PBS without fluorescein diacetate for at least 1 minute to remove intercellular fluorescein diacetate. The embryos were subsequently exposed to the ultraviolet light before photographic record. All the observations under ultraviolet light were carried out using an inverted microscope (Fluovert-FS, Leitz GmBH, Wetzlar, Germany) set on phase-contrast and incident-light fluorescence optics (Leitz combination of filters that contained an excitation filter BP , splitting mirror, and absorption filter LP 515). All photographs were recorded on 400 ASA films. Different exposure times were used, but 5 seconds seemed to be enough to trigger the camera. RESULTS Nonfrozen Normal Looking Human Multipronucleate Embryos Ten human multipronucleate embryos (2- to 4- cell stage) were exposed to fluorescein diacetate so Noto et al. Fluorescein test of quick frozen embryos Fertility and Sterility

Figure 1 Three human multipronucleate embryos after exposure to fluorescein diacetate (positive internal control).

Figure 2 Five human multipronucleate embryos after cryostorage and exposure to fluorescein diacetate.

Fluorescence was only present inside the blastomeres and never in the perivitelline space (Fig. 1). Differences in fluorescence intensity among healthy blastomeres were never observed.

experiment was the understanding ofthe eventually present intrinsic cytotoxicity of the compounds used at very high concentration to quick freeze-thaw.

3 Figure 1 Three human multipronucleate embryos after exposure to fluorescein diacetate (positive internal control). The blastomeres are brightly fluorescent, and no differences in terms of fluorescence intensity are observed among the cells. Figure 2 Five human multipronucleate embryos after cryostorage and exposure to fluorescein diacetate. The embryo on the right side, morphologically nonsurviving as assessed by light microscopy, shows no sign of fluorescence. lution at RT (normal control). All the embryos became brightly fluorescent. Fluorescence was only present inside the blastomeres and never in the perivitelline space (Fig. 1). Differences in fluorescence intensity among healthy blastomeres were never observed. After 15 minutes, fluorescence was still present inside the blastomeres with the same intensity, whereas at RT complete fading was observed only 1 hour after fluorescen diacetate exposure. experiment was the understanding ofthe eventually present intrinsic cytotoxicity of the compounds used at very high concentration to quick freeze-thaw. As expected, no differences in terms of fluorescence were found between treated (but nonfrozen) embryos and the control ones. Nonviable Human Multipronucleate Embryos Twelve human multipronucleate embryos, 6 killed by heating and 6 morphologically degenerating after cryostorage, were taken as a model of nonviable embryos. In fact, in all those embryos no fluorescence was found after exposure to fluorescein diacetate, even when increasing the incubation time or incubating the embryos at 37 C. Furthermore, using an exposure time > 5 seconds no reliable fluorescent blastomeres were obtained to be compared to the highly fluorescent ones of the healthy embryos. This kind of on-off answer to fluorescein diacetate allowed us to use for the same experiment cryostored embryos and healthy ones together (Fig. 2), just to allow an easy comparison and to avoid the need to determine very subtle changes in fluorescence using sophisticated and expensive microspectrophotometers. Human Multipronucleate Cleaved Embryos After Quick Freezing-Thawing Procedure Thirty-five multipronucleate cleaved embryos were exposed to fluorescein diacetate after cryostorage at -196 C. A survival rate of 60% was obtained. only considered embryos presenting after thawing at lea,st 50% of their original blastomeres intact. All the embryos with normal looking blastomeres fluoresced brightly after exposure to fluorescein diacetate, indicating an intact cell membrane permeability and an integrity of the intracytoplas- We Human Multipronucleate Embryos Exposed to the Cryoprotectant Without Freezing In a short series (n = 5), we exposed multipronucleated cleaved embryos to cryoprotective solution for 3 minutes and transferred them in thawing solution, omitting the freezing step. The aim of this Figure 3 Five human multipronucleate embryos after ultrarapid freezing/thawing and subsequent exposure to fluorescein diacetate. Morphologically surviving blastomeres are brightly fluorescent with the same intensity observed for embryos exposed to fluorescein diacetate but nonfrozen. Noto et al. Fluorescein test of quick frozen embryos 1173

4 matic esterase enzyme activity (Fig. 3). The morphologically degenerating embryos (n = 14) showed no detectable fluorescence after exposure to fluorescein diacetate, even using prolonged incubation times. DISCUSSION Different tests to assess cell viability after cold storage of human embryos have been described in the literature, but only a few can be used in practice for IVF aims. They can be classified according to Pegg 7 in four groups. The first group (physical integrity) includes the microscopic observation (light or electron microscopy) of the frozen-stored embryos. The gross appearance of the blastomeres and the integrity of the zona pellucida are normally used; however, a normal morphological appearance can never prove that a cell will function, nor can appearance be quantified in the manner required of a viability assay. Electron microscopy gives, of course, more information, but it needs the destruction of the blastomeres. More subtle aspects of physical integrity may be useful. For instance, dye exclusion tests are a widely used type of staining assay. They are based on the assumption that live cells will exclude certain dyes, like trypan blue, nigrosin, or fast green, whereas dead cells will be stained. Thus, the dye exclusion tests are intended as a measure of the plasma membrane integrity, but unfortunately, the results in the assay of cryoinjury are sometimes difficult to interpret The second group of viability assays includes the study of the mitotic activity because reproduction is one ascertained aspect of living structures. Of course, dividing cells need a very high level of functional integrity, but application to human embryos is limited by a certain number of difficulties. First of all, when deoxyribonucleic acid synthesis is used as a parameter for mitotic activity, the measure of the incorporation of labeled precursors such as [3H] thymidine in the embryos is needed, meaning the complete destruction of themy On the other hand, if the mitotic index to assess embryo viability after cryostorage is used, another kind of problem should be taken into account: if human embryos are cultured in vitro not all of them will cleave and reach very advanced embryo stages (like morula or blastocyst stage), independent ofthe culture conditions. This phenomenon, well known for other animal species as the two-cell block, is responsible for the very low percentage of human embryos reaching blastocyst stage «30%) and for the difficulties en- countered when trying to extrapolate results obtained on animal models to human species. On the other hand, there is some evidence that the rate of cleavage may well predict viability of the embryosthe more rapidly growing embryos are more likely to establish a viable pregnancy than the slower cleaving ones. However, Plachot et al.12 found that viability, as expressed by simple morphological assessment of dividing embryos, is not correlated with developmental capacity, except for totally degenerated embryos. They calculated that in patients who became pregnant only 42% of the replaced embryos were viable. The third group of viability assays (metabolic activity) includes the study of the uptake of metabolites, the production of catabolites, and the study of intracellular enzymatic reactions and membrane transport. In this case, metabolic functions always depend on intact enzyme systems, whereas structural integrity is rarely required. Different types of metabolic tests are known, but the need of a welltrained team and, especially, of expensive laboratory instruments, limit the use of these tests in an IVF laboratory. Probably the metabolic activity that has most commonly been measured on human embryos is the uptake of glucose and pyruvate.13 Furthermore, bioassays using culture media have demonstrated that the so-called early pregnancy associates thrombocytopaenia is because of an embryo-derived platelet-activating factor (P AF).14 Implantation did not occur when embryo-derived P AF was not produced. This work can be done in a noninvasive way, allowing the embryos to be replaced into the uterine environment. Of course, such an approach does not raise considerable ethical concerns. The use of fluorescein diacetate, as demonstrated by Mohr and Trounson,6 will not only detect inadequacies in culture conditions before they are evident morphologically, but it is also a parameter of the integrity of the cell membrane and the esterase enzyme activity. The ultimate group is the most important: the in vivo function, meaning the ability of a gamete, in the case of cryopreserved spermatozoa, to fertilize or in the case of cryopreserved embryos the ability of an embryo to implant and develop when transferred to the natural environment represented by the maternal uterus. Naturally, the production of a clinical pregnancy leading to the birth of a normal baby is the only indicator of embryonic viability, but such an approach to the viability assessment is essentially retrospective.1 5 This study, carried out using human multipronucleate embryos, has shown that surviving embryos 1174 Noto et al. Fluorescein test of quick frozen embryos Fertility and Sterility

5 exposed shortly to high DMSO concentrations and quickly immersed in liquid nitrogen are able to survive the tremendous shock caused by the dramatic shrinkage occurring during the incubation in freezing solution under severe hyperosmotic conditions. On the other hand, the thawing conditions, representing maybe the most critical moment during the whole freezing procedure, do not impair embryo survival. Furthermore, already achieved human pregnancies after ultrarapid freezing confirm the efficacy of this freezing system. In our opinion, only the still unsatisfactory pregnancy rate (PR) obtained using this technique remains the most disappointing aspect. Other researches in our IVF laboratory are now undertaken, trying to improve the freezing protocol and the PRo REFERENCES 1. Trounson A, Peura A, Kirby C: Ultrarapid freezing: a new low-cost and effective method of embryo cryopreservation. Fertil Steril 48:843, Trounson A, Peura A, Freemann L, Kirby C: Ultrarapid freezing of early cleavage stage human embryos and eightcell mouse embryos. Fertil Steril 49:822, Trounson A, Sjoblom P: Cleavage and development of human embryos in vitro after ultrarapid freezing and thawing. Fertil Steril 50:373, Gordts S, Roziers P, Campo R, Noto V: Survival and pregnancy outcome after ultrarapid freezing of human embryos. Fertil Steril 53:469, Rotman B, Papermaster BW: Membrane properties ofliving mammalian cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci USA 55:134, Mohr LR, Trounson AO: The use of fluorescein diacetate to assess embryo viability in the mouse. J Reprod Ferti158:189, Pegg DE: Viability assays for preserved cells, tissues, and organs. Cryobiology 26:212, Dolan MF: Viability assay-a critique. Fed Proc 15(Suppl): 275, Green AE, Silver RK, Krug M, Coriell LL: Preservation of cell cultures by freezing in liquid nitrogen vapor. Proc Soc Exp BioI Med 116:462, Pegg DE: In vitro assessment of cell viability in human bone marrow preserved at -79 C. J Appl Physiol 19:123, Anderson GB, Foote RH: Effects of low temperature upon subsequent nucleic acid and protein synthesis of rabbit embryos. Exp Cell Res 90:73, Plachot M, Mandelbaum J, Junca A M, Cohen J, Salat-Baroux J, Da Lage C: Morphologic and cytologic study of human embryos obtained by in-vitro fertilization. In Future Aspects in Human In Vitro Fertilization, Edited by W Feichtinger, P Kemeter. Berlin, Springer-Verlag, 1987, p Hardy K, Hooper MAK, Handyside AH, Rutherford AJ, Winston RML, Leese HJ: Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod 4:188, O'Neill C, Saunders DM: Assessment of embryo quality. Lancet 2:1035, Whittingham DG: Viability assays for mammalian ova. Cryobiology 15:245, 1978 N oto et ai. Fluorescein test of quick frozen embryos 1175