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Ultrastructure of preimplantation genetic diagnosis-derived human blastocysts grown in a coculture system after vitrification
PREIMPLANTATION GENETIC DIAGNOSIS Ultrastructure of preimplantation genetic diagnosisderived human blastocysts grown in a coculture system after vitrification María-José Escribá, Ph.D.,a Carmen Escobedo-Lucea, Ph.D.,b Amparo Mercader, Ph.D.,a María-José de los Santos, Ph.D.,a,b Antonio Pellicer, M.D.,a,b,c and José Remohí, M.D.a,b,c a Instituto Universitario IVI; b IVI Foundation; and c Department of Pediatrics, Obstetrics and Gynecology, University School of Medicine, University of Valencia, Valencia, Spain
Objective: To evaluate ultrastructural features of preimplantation genetic diagnosis (PGD) blastocysts before and after vitrification. Design: Descriptive study of both vitrified and fresh hatching blastocysts. Setting: PGD program at the Instituto Universitario, Instituto Valenciano de Infertilidad. Patient(s): Patients undergoing PGD donated their abnormal embryos for research (n ⫽ 26). Intervention(s): Biopsied embryos were cultured in the presence of human endometrial cells until day 6. Sixteen blastocysts were vitrified. A total of 11 high-scored hatching blastocysts, 6 warmed and 5 fresh, were fixed for ultrastructure. Main Outcome Measure(s): The cytoskeleton structure, type of intercellular junctions, and basic intracellular organelles in trophoectoderm cells and the inner cell mass were analyzed. Result(s): Ten of 16 blastocysts (62%) survived the warming process. Six of these showed no signs of cell degeneration and light microscopy revealed similar ultrastructural characteristics to those of controls. However, in trophoectoderm cells from both fresh and cryopreserved blastocysts, a reduced number of tight junctions and the presence of degradation bodies were detected. Conclusion(s): The particular ultrastructural features observed in PGD-derived blastocysts could be related to embryo manipulation and culture conditions. Vitrification does not seem to alter blastocysts, as those that survive hatching do not display detectable cellular alterations when observed through electron microscopy. (Fertil Steril威 2006;86:664 –71. ©2006 by American Society for Reproductive Medicine.) Key Words: Blastocyst, human, PGD, ultrastructure, vitrification.
Zygotes and cleavage-stage embryos have traditionally been cryopreserved using low concentrations of cryoprotectant and considerably slow cooling rates so as to dehydrate the cell during cooling and prevent intracellular crystallization (1, 2). Although a 60%–70% embryo survival rate can be achieved with this method (3, 4), not all embryos respond well to slow freezing procedures. This is the case of cleavage-stage embryos that have undergone biopsy for preimplantation genetic diagnosis (PGD) purposes. These embryos have a lower capacity for recovery after cryopreservation than non-biopsied embryos (5, 6).
applied with different degrees of success. Human blastocysts behave differently from cleavage-stage embryos when in contact with cryoprotectants. They are less permeable to both cryoprotectants and water and shrink more slowly when placed in a cryoprotectant solution (9). These characteristics make blastocysts more vulnerable to damage by intracellular ice. Although initial trials with blastocyst cryopreservation using slow cooling methods were discouraging, present freezing methods have been rewarded with noticeable improvements in the blastocyst survival rate (10 –13).
Some modifications to conventional cryopreservation methods have been shown to improve the survival rate of biopsied cleavage-stage embryos (7, 8), whereas alternative approaches, such as freezing at further stages of development, can also be
Vitrification procedures combine fast, easy-to-use, and cheap technology; however, the high concentration of cryoprotectants used may compromise the later development of embryos, including their ability to implant, due to injuries or anomalies that may only be detected at an ultrastructural level (14 –18).
Received November 7, 2005; revised and accepted January 27, 2006. Reprint requests: María-José Escribá, Ph.D., Instituto Universitario IVI, Instituto Valenciano de Infertilidad, Plaza Policía Local, 3, 46015 Valencia, Spain (FAX: 34 963-05-09-99; E-mail: [email protected]).
We performed a descriptive study of the fine structure of both fresh, vitrified, and warmed human PGD-derived blastocysts grown in the presence of endometrial epithelial cells to determine whether our vitrification method causes damage
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Fertility and Sterility姞 Vol. 86, No. 3, September 2006 Copyright ©2006 American Society for Reproductive Medicine, Published by Elsevier Inc.
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that is detectable at the ultrastructural level but not with conventional light microscopy. MATERIALS AND METHODS Embryo Generation, Culture Conditions, and PGD Analysis Embryos were obtained from women participating in our PGD program. Ovarian stimulation was performed using GnRH analogues (GnRH-a) and FSH in either long or short treatment protocols (19). Four hours after ovum retrieval, intracytoplasmic sperm injection (ICSI) was performed as previously described (20). Fertilized embryos were kept individually in culture from days 2–5, in the presence of epithelial endometrial cells (21, 22). Embryo biopsy was performed on day 3 of development. To summarize, after zona drilling with Tyrode=s acid, one or two blastomeres were removed per embryo and chromosomes 13, 16, 18, 21, 22, X, and Y were analyzed by means of fluorescent in situ hybridization (23). On day 5, the chromosomally normal blastocysts with the highest morphological scores were selected for embryo transfer. Twenty-six abnormal embryos were admitted into the study once the informed and signed consent of the progenitors was obtained. Vitrification and Warming Procedures Blastocyst vitrification was performed following our previously reported procedure (24, 25), based on the method of Yokota and co-workers (26), with some minor modifications. In short, 16 day-5, abnormal blastocysts were placed for 2 minutes in 1 mL of Dulbecco phosphate-buffered saline solution supplemented with 20% (v/v) serum (hereafter sPBS). Embryos were incubated in 20% (v/v) ethylene glycol for 4 minutes and then in 25% (v/v) ethylene glycol and dimethyl sulfoxide (DMSO) in s-PBS. Each blastocyst was loaded into a 0.25-mL French straw and then identified, sealed, and plunged into liquid nitrogen within ⱕ45 seconds. Samples were kept in liquid nitrogen for ⱖ1 month. For warming, each straw was immersed into a 38°C water bath for 5–10 seconds. The straw content was expelled into a 0.75 M sucrose solution in s-PBS. Dilution of cryoprotectants was performed by six-step incubations for 1 minute each in decreasing sucrose solutions. Finally, recovered embryos were kept at 37°C in 1 mL of co-culture medium (CCM, Scandinavian IVF, Göteborg, Sweden) in 5% CO2 and 95% relative humidity. Approximately 12 hours after warming, blastocysts were examined under conventional light microscopy at ⫻400 magnification to assess their morphological appearance and state of blastocoel expansion. A blastocyst was considered to have survived when both inner cell mass (ICM) and trophoectoderm were intact and reexpansion of the blastocoel was observed. Fertility and Sterility姞
Electron Microscopy Initial blastocyst assignation to each group was at random. The control group was comprised by five fresh abnormal blastocysts that on day 6 of development were at the hatching stage and scored high on morphology (5 of 10 cultured). On the other hand, the vitrified group consisted of 6 vitrified and warmed hatching blastocysts of 16 initially cryopreserved. Thus, a total of 11 day-6 human blastocysts were included in the fine microscopic study. The group size difference was due to the expected loses as a result of vitrification, earlier estimated at 40% (24, 25). On day 6 of embryo development, both chromosomally abnormal fresh blastocysts and surviving vitrified and warmed hatching blastocysts were retrieved from the culture media and repeatedly washed in a 0.1 M phosphate buffer (pH 7.4) solution. After this step, samples were fixed in 3% glutaraldehyde solution in phosphate buffer for 30 minutes at 37°C and then postfixed in 2% OsO4 in phosphate buffer. Blastocysts were block-stained with 2% uranyl acetate (Electron Microscopy Science, Hatfield, PA). Dehydration was performed using a graded series of ethanol and infiltration in propylene oxide (Lab Baker, Deventry, Holland). Embryos were then kept in araldite overnight. The next day, blastocysts were individually flat-embedded in araldite (Durkupan, Fluka, Sigma-Aldrich, Madrid, Spain) and blocks were allowed to polymerize at 70°C for ⱕ5 days. Semithin (0.5 m) and ultrathin (70 nm) sections were cut with glass and diamond knives using a Reichert Jung Ultracut ultramicrotome (Reichert A.G., Vienna, Austria) and placed on formvar-coated 50-mesh copper grids. Ultrastructural observation was carried out under a 100-kV Jem 1010 Jeol 500 electron microscope (Jeol, Akishima, Tokyo, Japan). RESULTS Using transmission electron microscopy, four of the five “fresh” PGD-derived hatching blastocysts were classified as being of excellent or good quality, defined by the presence of mitosis, absence of picnosis or vacuoles, and a clear distinction of the two main cell lineages, trophoectoderm and ICM. Sixteen abnormal blastocysts at different developmental stages were vitrified. Seven blastocysts (six at the hatching stage and one already hatched) survived warming and were scored in terms of good or excellent quality. Three of the intact blastocysts were of reasonable quality (two hatching and one hatched) and six degenerated (10/ 16; 62% survival rate). The fine microscopic study was only performed on the six surviving high-scoring hatching blastocysts, which would normally have been selected for transfer based purely on their morphological score. 665
FIGURE 1 Morphological feature of trophoectodermal (TP) cells of a fresh PGD-derived hatching blastocyst. Note the nucleus (Nu) containing a large nucleolus (No), abundant tubular mitochondria (mt), and external microvilli (mv). Bar ⫽ 2,000 nm.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
The ultrastructural features observed in these six cryopreserved blastocysts were comparable to those in fresh control blastocysts. The cubic-plane epithelium of the trophoectoderm was formed by polarized cells showing abundant superficial microvilli on their external surface and a smooth face on their blastocoelic side (Fig. 1). At the cytoplasmic level, blastodermal cells are characterized by the presence of glucogen electrodense vesicles and cytokeratin filament throughout the cytoplasm, usually encroached on the plasmatic membrane and participating in specialized epithelial junctions. These are formed by apical tight junctions and basal desmosome in homologous trophoectoderm-to-trophoectoderm junctions (Fig. 2), and by hemidesmosomes when heterologous 666
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trophoectoderm-to-ICM junctions exist. Among the intracytoplasmic organules, the abundant population of large mitochondria should be highlighted. These organelles are elongated in shape and have defined transverse cristae with no electrodense material, and are usually located near microfilaments. We also detected lipidic droplets, ribosomes, Golgi complexes, and cisterns of rough endoplasmic reticulum, which were sometimes found to be dilated, as previously described (27–30). At the nuclear level, blastodermal cells showed an organizer nucleus surrounded by a continuous, smooth, double membrane with a reticular nucleolus (Fig. 1). Cells of the ICM have a cubic shape, with some specialized intercellular gap junctions (Fig. 3).
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FIGURE 2 Electron micrograph showing the ultrastructural morphology of vitrified and warmed hatching human blastocysts. Detail of specialized cell-to-cell junction between trophoectodermal (TP) cells, formed by apical tight junctions (black arrowhead) and desmosomes (white arrowhead) associated with cytokeratin filaments (ck). Note subcellular characteristics of TP cells such as the mitochondrial (mt) location near to the cytokeratin filaments and the smooth surface of the nucleus (Nu). Bar ⫽ 1,000 nm.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
At the cytoplasmic level, the abundance of ribosomes, absence of keratohyalin filaments, and the small size and reduced number of tubular mitochondrion were noticeable. A characteristic of these cells is the large ratio of nucleus to cytoplasm. The nucleus is surrounded by a continuous double membrane with numerous finger-shaped invaginations and usually contains more than one nucleolus of a large magnitude with a reticulate ultrastructure and granular content, all of which corresponds to the cytoplasmic abundance of ribosomes. In the fifth fresh hatching control blastocyst, we observed, in some trophoectodermal cells, fewer electrodense mitochondria containing fewer cristae and of a smaller size, and that were sometimes swollen and occasionally enveloped by lysosomes and vesicles derived from the reticulum. The final digestion was evident due to the empty cytoplasmic areas with membrane. In two of the six vitrified blastocysts, we also noticed the occasional presence of the mitochondria–lysosome complexes described earlier in the trophoblastic cells of one of the control embryos (Fig. 4). Fertility and Sterility姞
DISCUSSION Vitrification techniques have been successfully applied on the different developmental stages of the embryo (7, 9, 12, 26, 31–36). In this study, fine structure analysis of fresh and vitrified and warmed human PGD-derived blastocysts was performed to investigate whether our vitrification method modifies the structure of cytoskeleton and/or other cytoplasmic organelles. Although the ultrastructural characteristics of human blastocysts have previously been reported (27–30), this is, to our knowledge, the first report on the fine structure of vitrified and warmed human blastocysts derived from embryos that have been subjected to biopsy and cultured in the presence of human epithelial endometrial cells. During cryopreservation, embryos can suffer several types of injury (37). Although intracellular ice is the major cause of structural injury in slow freezing procedures (38), in the case of vitrification, the high cooling rates associated with high concentrations of cryoprotectants may induce osmotic stress and chemical toxicity in the cells (14, 15). Such injuries are not always observed under light microscopic observation (16 –18). 667
FIGURE 3 Fresh (noncryopreserved) PGD-derived human blastocysts. Electron micrograph of specialized gap junction (arrowhead) between adjacent cells of the ICM. Note the large nucleus (Nu) vs. the cytoplasm volume. Original magnification, ⫻50,000.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
Our vitrification protocol, compared to others, uses higher cryoprotectant concentration, rendering comparable survival rates (62%) (39, 40). Moreover, the ultrastructural characteristics of our vitrified and warmed human blastocysts were similar to those of control embryos, which indicates that our vitrification procedure, based on the equimolar combination of ethylene glycol and DMSO as cryoprotectants, preserves the embryonic structures and cellular junctions. On day 6 of embryo development, in both cryopreserved and hatching control blastocysts, each of the cellular lineages was clearly distinguishable, and their fine structure almost coincided with that of prior observations (27–30, 41). Concerning the nature of cell junctions, we report specific discrepancies with earlier ultrastructural descriptions of human blastocysts. Whereas the epithelial type, formed by apical tight junctions and desmosomes associated with microfilaments (27, 30) or tonofilaments (41), is the most frequently described junction between trophoblast cells, it was a feature only occasionally observed in our blastocysts. Instead, desmosomes anchored to cytokeratin filaments were the most commonly observed cell junction in our study. With respect to ICM cells, we observed no specialized cell junctions between them, but rather isolated gap junctions. 668
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Contrary to our findings, Sathananthan et al. (27) described the presence of desmosomes and gap junctions between ICM and trophoblast cells and between ICM cells. This peculiarity could be due to the different circumstances of the blastocysts. First, Sathananthan and co-workers studied the ultrastructure of normal day-6 blastocysts whereas our study analyzed chromosomally abnormal PGD-derived embryos. Second, our embryos underwent cellular biopsy on day 3. Several studies have related the morphological characteristics of individual blastocysts to the total number of cells (42). Blastomere loss at earlier stages of development, as well as physical removal or damage of ⬍50% of the original blastomeres before culture have been reported to be associated with lower number of cells in resultant blastocysts (42–51), possibly causing changes in the distribution of cellular junctions in both the trophoectoderm cells and the ICM. Third, the practice of making a hole in the zona pellucida (ZP; zona drilling) to remove one or two blastomeres may also partially explain such discrepancies. During expansion, the blastocyst volume increases twofold to threefold with a subsequent thinning of the ZP. The trophoblast epithelium stretches due to fluid intake, thereby increasing the hydrostatic pressure within the blastocoel, forming a continuous robust epithe-
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FIGURE 4 Electron microscopy of vitrified and warmed hatching blastocyst. (A) Trophoblast (Tp) cell showing an autophagic vacuole (v) containing cellular debris, probably mitochondrion (mt). (B) Degradation body (*) formed by empty cytoplasmic areas with no content of residual material but surrounded by a membrane. Original magnification, ⫻20,000.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
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lium with specialized cell junctions, characteristic of closely integrated tissues (27, 30, 41). This process could differ in biopsied embryos in which creating an artificial hole in the ZP for blastomere removal would permit blastocysts to hatch out of the zona with ease during expansion. Therefore, it would be reasonable to assume that assisted hatched blastocysts may not yet have developed or may never develop the ultrastructural mechanism observed in the regular mechanical process of hatching (30). In fact, we did not observe trophoblastic plump cells or “zona-breaker” cells in the PGD-derived blastocysts, which are supposedly involved in the process of embryo hatching (30). Whether these features are characteristic of earlier developmental stages such as expansion, or of a particular mechanism of hatching or timing in human PGD-derived blastocysts, remains to be elucidated. These discrepancies could also be due to the presence of endometrial epithelial cells during blastocyst formation and growth in vitro. Uterine epithelial cells are the first site of contact between maternal and fetal tissue, therefore it is possible that a signal from those cells induces trophoectoderm cells to become depolarized, affecting the subsequent endometrial attachment. Thus, it is possible that the frequency and distribution of the tight junctions are influenced by culture conditions. In the present study, some trophoblast cells of highscoring analyzed blastocysts (27%; 2/6 cryopreserved and 1/5 control) presented autophagic vacuoles with cellular debris (probably mitochondrion), which was the result of lysosomal digestion. Moreover, we have also observed empty cytoplasmic areas without content and surrounded by a membrane, which have previously been interpreted as degradation bodies (18, 30). The origin of these features is uncertain, but it has been suggested that they exclude apoptotic, abnormal cells and cytoplasmic fragments from the embryo proper (30, 41, 52), or participate in the ubiquitinmediated degradation of paternal mitochondria carried by the spermatozoa during fertilization (53–55). Because mitochondrial elimination was only observed in the trophoblast cells, the hypothesis is supported if, in fact, paternal mitochondria are unequally sequestered to a region of the embryo that does not contribute to the ICM (56). Alternatively, because this fine structure analysis was performed on chromosomally abnormal embryos, it is possible that the presence of such autophagic vacuoles would be related to these abnormalities, in particular to monosomies. Whereas autophagic vacuoles were not observed in 45,X0 blastocysts, they were detected in the trophoectodermal cells of autosomal monosomic embryos. Whether this fact is related to the implantation failure earlier described in these embryos remains to be determined. To summarize, the lack of difference in terms of ultrastructure between survived vitrified and control blastocysts encourages us to use our vitrification procedure for clinical purposes. The differences observed between PGD-derived 670
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human blastocysts grown in the presence of epithelial endometrial cells compared to previous reports need to be further investigated. Acknowledgments: The authors especially thank José-Manuel García-Verdugo, Ph.D., for his intellectual support in the area of cellular morphology and Mario Soriano-Navarro, Ph.D., for his technical support. The authors also thank all the embryologists of the IVF and PGD departments as well as the gynecologists of IVI Valencia.
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38. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to ⫺196 degrees and ⫺269 degrees C. Science 1972;178:411– 4. 39. Mukaida T, Nakamura S, Tomiyama T, Wada S, Oka C, Kasai M, et al. Vitrification of human blastocysts using cryoloops: clinical outcome of 223 cycles. Hum Reprod 2003;18:384 –91. 40. Huang CC, Lee TH, Chen SU, Chen HH, Cheng TC, Liu CH, et al. Successful pregnancy following blastocyst cryopreservation using super-cooling ultra-rapid vitrification. Hum Reprod 2005;20:122– 8. 41. Sathananthan H, Gunasheela S, Menezes J. Critical evaluation of human blastocysts for assisted reproduction techniques and embryonic stem cell biotechnology. Reprod BioMed Online 2003;7:219 –27. 42. Fong CY, Bongso A. Comparison of human blastulation rates and total cell number in sequential culture media with and without co-culture. Hum Reprod 1999;14:774 – 81. 43. Hardy K, Hooper MA, Handyside AH, Rutherford AJ, Winston RM, Leese HJ. Non-invasive measurement of glucose and pyruvate uptake by individual human oocytes and preimplantation embryos. Hum Reprod 1989;4:188 –91. 44. Somers GR, Trounson AO, Wilton LJ. Allocation of cells to the inner cell mass and trophectoderm of 3/4 mouse embryos. Reprod Fertil Dev 1990;2:51–9. 45. Geber S, Winston RM, Handyside AH. Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis. Hum Reprod 1995;1: 1492– 6. 46. Dumoulin JC, Bras M, Coonen E, Dreesen J, Geraedts JP, Evers JL. Effect of Ca2⫹/Mg2⫹-free medium on the biopsy procedure for preimplantation genetic diagnosis and further development of human embryos. Hum Reprod 1998;13:2880 –3. 47. Dumoulin JC, Coonen E, Bras M, van Wissen LC, Ignoul-Vanvuchelen R, Bergers-Jansen JM, et al. Comparison of in-vitro development of embryos originating from either conventional in-vitro fertilization or intracytoplasmic sperm injection. Hum Reprod 2000;15:402–9. 48. Devreker F, Hardy K, Van den Bergh M, Vannin AS, Emiliani S, Englert Y. Amino acids promote human blastocyst development in vitro. Hum Reprod 2001;16:749 –56. 49. Dumoulin JM, Coonen E, Bras M, Bergers-Janssen JM, IgnoulVanvuchelen RC, van Wissen LC, et al. Embryo development and chromosomal anomalies after ICSI: effect of the injection procedure. Hum Reprod 2001;16:306 –12. 50. Archer J, Gook DA, Edgar DH. Blastocyst formation and cell numbers in human frozen-thawed embryos following extended culture. Hum Reprod 2003;18:1669 –73. 51. Dumoulin JC, Derhaag JG, Bras M, Van Montfoort AP, Kester AD, Evers JL, et al. Growth rate of human preimplantation embryos is sex dependent after ICSI but not after IVF. Hum Reprod 2005;20:484 –91. 52. Levy RR, Cordonier H, Czyba JC, Guerin JF. Apoptosis in preimplantation mammalian embryo and genetics. Ital J Anat Embryol 2001;106: 101– 8. 53. Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly C, Schatten G. Ubiquitinated sperm mitochondria, selective proteolysis, and the regulation of mitochondrial inheritance in mammalian embryos. Biol Reprod 2000;63:582–90. 54. Sutovsky P, Motlik J, Neuber E, Pavlok A, Schatten G, Palecek J, et al. Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts. Cloning Stem Cells 2001;3: 157– 61. 55. Sutovsky P. Ubiquitin-dependent proteolysis in mammalian spermatogenesis, fertilization, and sperm quality control: killing three birds with one stone. Microsc Res Tech 2003;61:88 –102. 56. Birky CW Jr. Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution. Proc Natl Acad Sci USA 1995;92: 11331– 8.
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Objective: To evaluate ultrastructural features of preimplantation genetic diagnosis (PGD) blastocysts before and after vitrification. Design: Descriptive study of both vitrified and fresh hatching blastocysts. Setting: PGD program at the Instituto Universitario, Instituto Valenciano de Infertilidad. Patient(s): Patients undergoing PGD donated their abnormal embryos for research (n ⫽ 26). Intervention(s): Biopsied embryos were cultured in the presence of human endometrial cells until day 6. Sixteen blastocysts were vitrified. A total of 11 high-scored hatching blastocysts, 6 warmed and 5 fresh, were fixed for ultrastructure. Main Outcome Measure(s): The cytoskeleton structure, type of intercellular junctions, and basic intracellular organelles in trophoectoderm cells and the inner cell mass were analyzed. Result(s): Ten of 16 blastocysts (62%) survived the warming process. Six of these showed no signs of cell degeneration and light microscopy revealed similar ultrastructural characteristics to those of controls. However, in trophoectoderm cells from both fresh and cryopreserved blastocysts, a reduced number of tight junctions and the presence of degradation bodies were detected. Conclusion(s): The particular ultrastructural features observed in PGD-derived blastocysts could be related to embryo manipulation and culture conditions. Vitrification does not seem to alter blastocysts, as those that survive hatching do not display detectable cellular alterations when observed through electron microscopy. (Fertil Steril威 2006;86:664 –71. ©2006 by American Society for Reproductive Medicine.) Key Words: Blastocyst, human, PGD, ultrastructure, vitrification.
Zygotes and cleavage-stage embryos have traditionally been cryopreserved using low concentrations of cryoprotectant and considerably slow cooling rates so as to dehydrate the cell during cooling and prevent intracellular crystallization (1, 2). Although a 60%–70% embryo survival rate can be achieved with this method (3, 4), not all embryos respond well to slow freezing procedures. This is the case of cleavage-stage embryos that have undergone biopsy for preimplantation genetic diagnosis (PGD) purposes. These embryos have a lower capacity for recovery after cryopreservation than non-biopsied embryos (5, 6).
applied with different degrees of success. Human blastocysts behave differently from cleavage-stage embryos when in contact with cryoprotectants. They are less permeable to both cryoprotectants and water and shrink more slowly when placed in a cryoprotectant solution (9). These characteristics make blastocysts more vulnerable to damage by intracellular ice. Although initial trials with blastocyst cryopreservation using slow cooling methods were discouraging, present freezing methods have been rewarded with noticeable improvements in the blastocyst survival rate (10 –13).
Some modifications to conventional cryopreservation methods have been shown to improve the survival rate of biopsied cleavage-stage embryos (7, 8), whereas alternative approaches, such as freezing at further stages of development, can also be
Vitrification procedures combine fast, easy-to-use, and cheap technology; however, the high concentration of cryoprotectants used may compromise the later development of embryos, including their ability to implant, due to injuries or anomalies that may only be detected at an ultrastructural level (14 –18).
Received November 7, 2005; revised and accepted January 27, 2006. Reprint requests: María-José Escribá, Ph.D., Instituto Universitario IVI, Instituto Valenciano de Infertilidad, Plaza Policía Local, 3, 46015 Valencia, Spain (FAX: 34 963-05-09-99; E-mail: [email protected]).
We performed a descriptive study of the fine structure of both fresh, vitrified, and warmed human PGD-derived blastocysts grown in the presence of endometrial epithelial cells to determine whether our vitrification method causes damage
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that is detectable at the ultrastructural level but not with conventional light microscopy. MATERIALS AND METHODS Embryo Generation, Culture Conditions, and PGD Analysis Embryos were obtained from women participating in our PGD program. Ovarian stimulation was performed using GnRH analogues (GnRH-a) and FSH in either long or short treatment protocols (19). Four hours after ovum retrieval, intracytoplasmic sperm injection (ICSI) was performed as previously described (20). Fertilized embryos were kept individually in culture from days 2–5, in the presence of epithelial endometrial cells (21, 22). Embryo biopsy was performed on day 3 of development. To summarize, after zona drilling with Tyrode=s acid, one or two blastomeres were removed per embryo and chromosomes 13, 16, 18, 21, 22, X, and Y were analyzed by means of fluorescent in situ hybridization (23). On day 5, the chromosomally normal blastocysts with the highest morphological scores were selected for embryo transfer. Twenty-six abnormal embryos were admitted into the study once the informed and signed consent of the progenitors was obtained. Vitrification and Warming Procedures Blastocyst vitrification was performed following our previously reported procedure (24, 25), based on the method of Yokota and co-workers (26), with some minor modifications. In short, 16 day-5, abnormal blastocysts were placed for 2 minutes in 1 mL of Dulbecco phosphate-buffered saline solution supplemented with 20% (v/v) serum (hereafter sPBS). Embryos were incubated in 20% (v/v) ethylene glycol for 4 minutes and then in 25% (v/v) ethylene glycol and dimethyl sulfoxide (DMSO) in s-PBS. Each blastocyst was loaded into a 0.25-mL French straw and then identified, sealed, and plunged into liquid nitrogen within ⱕ45 seconds. Samples were kept in liquid nitrogen for ⱖ1 month. For warming, each straw was immersed into a 38°C water bath for 5–10 seconds. The straw content was expelled into a 0.75 M sucrose solution in s-PBS. Dilution of cryoprotectants was performed by six-step incubations for 1 minute each in decreasing sucrose solutions. Finally, recovered embryos were kept at 37°C in 1 mL of co-culture medium (CCM, Scandinavian IVF, Göteborg, Sweden) in 5% CO2 and 95% relative humidity. Approximately 12 hours after warming, blastocysts were examined under conventional light microscopy at ⫻400 magnification to assess their morphological appearance and state of blastocoel expansion. A blastocyst was considered to have survived when both inner cell mass (ICM) and trophoectoderm were intact and reexpansion of the blastocoel was observed. Fertility and Sterility姞
Electron Microscopy Initial blastocyst assignation to each group was at random. The control group was comprised by five fresh abnormal blastocysts that on day 6 of development were at the hatching stage and scored high on morphology (5 of 10 cultured). On the other hand, the vitrified group consisted of 6 vitrified and warmed hatching blastocysts of 16 initially cryopreserved. Thus, a total of 11 day-6 human blastocysts were included in the fine microscopic study. The group size difference was due to the expected loses as a result of vitrification, earlier estimated at 40% (24, 25). On day 6 of embryo development, both chromosomally abnormal fresh blastocysts and surviving vitrified and warmed hatching blastocysts were retrieved from the culture media and repeatedly washed in a 0.1 M phosphate buffer (pH 7.4) solution. After this step, samples were fixed in 3% glutaraldehyde solution in phosphate buffer for 30 minutes at 37°C and then postfixed in 2% OsO4 in phosphate buffer. Blastocysts were block-stained with 2% uranyl acetate (Electron Microscopy Science, Hatfield, PA). Dehydration was performed using a graded series of ethanol and infiltration in propylene oxide (Lab Baker, Deventry, Holland). Embryos were then kept in araldite overnight. The next day, blastocysts were individually flat-embedded in araldite (Durkupan, Fluka, Sigma-Aldrich, Madrid, Spain) and blocks were allowed to polymerize at 70°C for ⱕ5 days. Semithin (0.5 m) and ultrathin (70 nm) sections were cut with glass and diamond knives using a Reichert Jung Ultracut ultramicrotome (Reichert A.G., Vienna, Austria) and placed on formvar-coated 50-mesh copper grids. Ultrastructural observation was carried out under a 100-kV Jem 1010 Jeol 500 electron microscope (Jeol, Akishima, Tokyo, Japan). RESULTS Using transmission electron microscopy, four of the five “fresh” PGD-derived hatching blastocysts were classified as being of excellent or good quality, defined by the presence of mitosis, absence of picnosis or vacuoles, and a clear distinction of the two main cell lineages, trophoectoderm and ICM. Sixteen abnormal blastocysts at different developmental stages were vitrified. Seven blastocysts (six at the hatching stage and one already hatched) survived warming and were scored in terms of good or excellent quality. Three of the intact blastocysts were of reasonable quality (two hatching and one hatched) and six degenerated (10/ 16; 62% survival rate). The fine microscopic study was only performed on the six surviving high-scoring hatching blastocysts, which would normally have been selected for transfer based purely on their morphological score. 665
FIGURE 1 Morphological feature of trophoectodermal (TP) cells of a fresh PGD-derived hatching blastocyst. Note the nucleus (Nu) containing a large nucleolus (No), abundant tubular mitochondria (mt), and external microvilli (mv). Bar ⫽ 2,000 nm.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
The ultrastructural features observed in these six cryopreserved blastocysts were comparable to those in fresh control blastocysts. The cubic-plane epithelium of the trophoectoderm was formed by polarized cells showing abundant superficial microvilli on their external surface and a smooth face on their blastocoelic side (Fig. 1). At the cytoplasmic level, blastodermal cells are characterized by the presence of glucogen electrodense vesicles and cytokeratin filament throughout the cytoplasm, usually encroached on the plasmatic membrane and participating in specialized epithelial junctions. These are formed by apical tight junctions and basal desmosome in homologous trophoectoderm-to-trophoectoderm junctions (Fig. 2), and by hemidesmosomes when heterologous 666
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trophoectoderm-to-ICM junctions exist. Among the intracytoplasmic organules, the abundant population of large mitochondria should be highlighted. These organelles are elongated in shape and have defined transverse cristae with no electrodense material, and are usually located near microfilaments. We also detected lipidic droplets, ribosomes, Golgi complexes, and cisterns of rough endoplasmic reticulum, which were sometimes found to be dilated, as previously described (27–30). At the nuclear level, blastodermal cells showed an organizer nucleus surrounded by a continuous, smooth, double membrane with a reticular nucleolus (Fig. 1). Cells of the ICM have a cubic shape, with some specialized intercellular gap junctions (Fig. 3).
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FIGURE 2 Electron micrograph showing the ultrastructural morphology of vitrified and warmed hatching human blastocysts. Detail of specialized cell-to-cell junction between trophoectodermal (TP) cells, formed by apical tight junctions (black arrowhead) and desmosomes (white arrowhead) associated with cytokeratin filaments (ck). Note subcellular characteristics of TP cells such as the mitochondrial (mt) location near to the cytokeratin filaments and the smooth surface of the nucleus (Nu). Bar ⫽ 1,000 nm.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
At the cytoplasmic level, the abundance of ribosomes, absence of keratohyalin filaments, and the small size and reduced number of tubular mitochondrion were noticeable. A characteristic of these cells is the large ratio of nucleus to cytoplasm. The nucleus is surrounded by a continuous double membrane with numerous finger-shaped invaginations and usually contains more than one nucleolus of a large magnitude with a reticulate ultrastructure and granular content, all of which corresponds to the cytoplasmic abundance of ribosomes. In the fifth fresh hatching control blastocyst, we observed, in some trophoectodermal cells, fewer electrodense mitochondria containing fewer cristae and of a smaller size, and that were sometimes swollen and occasionally enveloped by lysosomes and vesicles derived from the reticulum. The final digestion was evident due to the empty cytoplasmic areas with membrane. In two of the six vitrified blastocysts, we also noticed the occasional presence of the mitochondria–lysosome complexes described earlier in the trophoblastic cells of one of the control embryos (Fig. 4). Fertility and Sterility姞
DISCUSSION Vitrification techniques have been successfully applied on the different developmental stages of the embryo (7, 9, 12, 26, 31–36). In this study, fine structure analysis of fresh and vitrified and warmed human PGD-derived blastocysts was performed to investigate whether our vitrification method modifies the structure of cytoskeleton and/or other cytoplasmic organelles. Although the ultrastructural characteristics of human blastocysts have previously been reported (27–30), this is, to our knowledge, the first report on the fine structure of vitrified and warmed human blastocysts derived from embryos that have been subjected to biopsy and cultured in the presence of human epithelial endometrial cells. During cryopreservation, embryos can suffer several types of injury (37). Although intracellular ice is the major cause of structural injury in slow freezing procedures (38), in the case of vitrification, the high cooling rates associated with high concentrations of cryoprotectants may induce osmotic stress and chemical toxicity in the cells (14, 15). Such injuries are not always observed under light microscopic observation (16 –18). 667
FIGURE 3 Fresh (noncryopreserved) PGD-derived human blastocysts. Electron micrograph of specialized gap junction (arrowhead) between adjacent cells of the ICM. Note the large nucleus (Nu) vs. the cytoplasm volume. Original magnification, ⫻50,000.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
Our vitrification protocol, compared to others, uses higher cryoprotectant concentration, rendering comparable survival rates (62%) (39, 40). Moreover, the ultrastructural characteristics of our vitrified and warmed human blastocysts were similar to those of control embryos, which indicates that our vitrification procedure, based on the equimolar combination of ethylene glycol and DMSO as cryoprotectants, preserves the embryonic structures and cellular junctions. On day 6 of embryo development, in both cryopreserved and hatching control blastocysts, each of the cellular lineages was clearly distinguishable, and their fine structure almost coincided with that of prior observations (27–30, 41). Concerning the nature of cell junctions, we report specific discrepancies with earlier ultrastructural descriptions of human blastocysts. Whereas the epithelial type, formed by apical tight junctions and desmosomes associated with microfilaments (27, 30) or tonofilaments (41), is the most frequently described junction between trophoblast cells, it was a feature only occasionally observed in our blastocysts. Instead, desmosomes anchored to cytokeratin filaments were the most commonly observed cell junction in our study. With respect to ICM cells, we observed no specialized cell junctions between them, but rather isolated gap junctions. 668
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Contrary to our findings, Sathananthan et al. (27) described the presence of desmosomes and gap junctions between ICM and trophoblast cells and between ICM cells. This peculiarity could be due to the different circumstances of the blastocysts. First, Sathananthan and co-workers studied the ultrastructure of normal day-6 blastocysts whereas our study analyzed chromosomally abnormal PGD-derived embryos. Second, our embryos underwent cellular biopsy on day 3. Several studies have related the morphological characteristics of individual blastocysts to the total number of cells (42). Blastomere loss at earlier stages of development, as well as physical removal or damage of ⬍50% of the original blastomeres before culture have been reported to be associated with lower number of cells in resultant blastocysts (42–51), possibly causing changes in the distribution of cellular junctions in both the trophoectoderm cells and the ICM. Third, the practice of making a hole in the zona pellucida (ZP; zona drilling) to remove one or two blastomeres may also partially explain such discrepancies. During expansion, the blastocyst volume increases twofold to threefold with a subsequent thinning of the ZP. The trophoblast epithelium stretches due to fluid intake, thereby increasing the hydrostatic pressure within the blastocoel, forming a continuous robust epithe-
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FIGURE 4 Electron microscopy of vitrified and warmed hatching blastocyst. (A) Trophoblast (Tp) cell showing an autophagic vacuole (v) containing cellular debris, probably mitochondrion (mt). (B) Degradation body (*) formed by empty cytoplasmic areas with no content of residual material but surrounded by a membrane. Original magnification, ⫻20,000.
Escribá. Fine analysis of vitrified PGD-derived human blastocysts. Fertil Steril 2006.
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lium with specialized cell junctions, characteristic of closely integrated tissues (27, 30, 41). This process could differ in biopsied embryos in which creating an artificial hole in the ZP for blastomere removal would permit blastocysts to hatch out of the zona with ease during expansion. Therefore, it would be reasonable to assume that assisted hatched blastocysts may not yet have developed or may never develop the ultrastructural mechanism observed in the regular mechanical process of hatching (30). In fact, we did not observe trophoblastic plump cells or “zona-breaker” cells in the PGD-derived blastocysts, which are supposedly involved in the process of embryo hatching (30). Whether these features are characteristic of earlier developmental stages such as expansion, or of a particular mechanism of hatching or timing in human PGD-derived blastocysts, remains to be elucidated. These discrepancies could also be due to the presence of endometrial epithelial cells during blastocyst formation and growth in vitro. Uterine epithelial cells are the first site of contact between maternal and fetal tissue, therefore it is possible that a signal from those cells induces trophoectoderm cells to become depolarized, affecting the subsequent endometrial attachment. Thus, it is possible that the frequency and distribution of the tight junctions are influenced by culture conditions. In the present study, some trophoblast cells of highscoring analyzed blastocysts (27%; 2/6 cryopreserved and 1/5 control) presented autophagic vacuoles with cellular debris (probably mitochondrion), which was the result of lysosomal digestion. Moreover, we have also observed empty cytoplasmic areas without content and surrounded by a membrane, which have previously been interpreted as degradation bodies (18, 30). The origin of these features is uncertain, but it has been suggested that they exclude apoptotic, abnormal cells and cytoplasmic fragments from the embryo proper (30, 41, 52), or participate in the ubiquitinmediated degradation of paternal mitochondria carried by the spermatozoa during fertilization (53–55). Because mitochondrial elimination was only observed in the trophoblast cells, the hypothesis is supported if, in fact, paternal mitochondria are unequally sequestered to a region of the embryo that does not contribute to the ICM (56). Alternatively, because this fine structure analysis was performed on chromosomally abnormal embryos, it is possible that the presence of such autophagic vacuoles would be related to these abnormalities, in particular to monosomies. Whereas autophagic vacuoles were not observed in 45,X0 blastocysts, they were detected in the trophoectodermal cells of autosomal monosomic embryos. Whether this fact is related to the implantation failure earlier described in these embryos remains to be determined. To summarize, the lack of difference in terms of ultrastructure between survived vitrified and control blastocysts encourages us to use our vitrification procedure for clinical purposes. The differences observed between PGD-derived 670
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human blastocysts grown in the presence of epithelial endometrial cells compared to previous reports need to be further investigated. Acknowledgments: The authors especially thank José-Manuel García-Verdugo, Ph.D., for his intellectual support in the area of cellular morphology and Mario Soriano-Navarro, Ph.D., for his technical support. The authors also thank all the embryologists of the IVF and PGD departments as well as the gynecologists of IVI Valencia.
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