Hostname: page-component-669899f699-tpknm Total loading time: 0 Render date: 2025-05-01T00:56:31.380Z Has data issue: false hasContentIssue false
  • English
  • Français

Micromanipulation

Published online by Cambridge University Press:  02 March 2009

Simon Fishel ,
Judy Timson ,
Steven Green ,
Jenny Hall ,
Ken Dowell  and
Lucas Klentzeris
Simon Fishel*
Affiliation:
Department of Obstetrics and Gynaecology, University Hospital, Queen's Medical Centre, Nottingham, UK
Judy Timson
Affiliation:
Department of Obstetrics and Gynaecology, University Hospital, Queen's Medical Centre, Nottingham, UK
Steven Green
Affiliation:
Department of Obstetrics and Gynaecology, University Hospital, Queen's Medical Centre, Nottingham, UK
Jenny Hall
Affiliation:
Department of Obstetrics and Gynaecology, University Hospital, Queen's Medical Centre, Nottingham, UK
Ken Dowell
Affiliation:
Department of Obstetrics and Gynaecology, University Hospital, Queen's Medical Centre, Nottingham, UK
Lucas Klentzeris
Affiliation:
Department of Obstetrics and Gynaecology, University Hospital, Queen's Medical Centre, Nottingham, UK
*
Simon Fishel, NURTURE (Nottingham University Research & Treatment Unit in Reproduction), Department of Obstetrics & Gynaecology, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH, UK.
Get access Rights & Permissions [Opens in a new window]

Extract

Micromanipulation technology entered the forum of human conception in vitro in the late 1980s. It was erroneously perceived as a new technology – a purely mechanical approach to bypass failures of conception in vitro, the aetiology of which were unknown. In fact, it is the modification of technology developed since the beginning of this century, and its logical utilization for conception in vitro, preconception (polar body) biopsy and preimplantation (blastomere and trophectoderm) biopsy in the realm of human infertility and genetic disorders.

Type
Articles
Information
Reproductive Medicine Review , Volume 2 , Issue 3 , October 1993 , pp. 199 - 222
Copyright
Copyright © Cambridge University Press 1993

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

Article purchase

Temporarily unavailable

References

1Ng, S-C, Bongso, TA, Ratnam, SS et al. Pregnancy after transfer of multiple sperm under the zona. Lancet 1988; ii: 790.CrossRefGoogle Scholar
2Fishel, SB, Antinori, S, Jackson, P et al. Twin birth after subzonal insemination. Lancet 1990; ii: 722.CrossRefGoogle Scholar
3Schmidt, HD. Minute structure of the hepatic lobules particularly with reference to the relationship between capiliary blood vessels and the hepatic cells. Am J Med Sci 1895; 37: 13–40.Google Scholar
4Peterfi, T. Das mikrurgische Verfahren. Naturwissenschaften 1923; 11: 81–87.CrossRefGoogle Scholar
5Taylor, AJ, Farber, D. Fatal effects of the removal of the micronucleus in euplotes. University of California Publications in Zoology 1924; 26: 131–35.Google Scholar
6Chambers, R, Fell, HB. Micro-operations on cells in tissue cultures. Proc R Soc Land 1931; 109: 381–92.Google Scholar
7Hammerling, J. Nucleo-cytoplasmic relationships in the development of Acetabularia. Int Rev Cytol 1953; 2: 475–98.CrossRefGoogle Scholar
8Lin, TP. Microinjection of mouse eggs. Science 1966; 151: 33–37.CrossRefGoogle ScholarPubMed
9Lin, TP. Micropipetting cytoplasm from the mouse eggs. Nature 1967; 216: 162–63.CrossRefGoogle Scholar
10Lin, TP. D-L-Methionine (Sulphur-35) for labelling unfertilized mouse eggs in transplantation. Nature 1956; 178: 1175–76.CrossRefGoogle Scholar
11Tarkowski, AK. Mouse chimereas developed from fused eggs. Nature 1961; 190: 857–60.CrossRefGoogle ScholarPubMed
12Moore, NW, Adams, CE, Rosen, LEA. Developmental potential of single blastomeres of the rabbit. J Reprod Fertil 1968; 17: 527–31.CrossRefGoogle ScholarPubMed
13Mintz, B. Allophenic mice of multi-embryo origin. In: Daniel, JC ed. Methods in mammalian embryology. San Francisco: Freeman, 1971: 186–214.Google Scholar
14Fetters, RM, Mettus, RV. Survival to term of chimaeric morulae produced by aggregation of 5–9 embryos in the mouse, Mus Musclus. Theriogenology 1984; 22: 167–74.CrossRefGoogle Scholar
15Gardner, RL, Edwards, RG. Control of the sex ratio at full term in the rabbit by transferring sexed blastocysts. Nature 1968; 218: 346–48.CrossRefGoogle ScholarPubMed
16Willadsen, SM. Developmental capacity of blastomeres from 4-cell and 8-cell sheep embryos. J Embryol Exp Morphol 1981; 65: 165–72.Google Scholar
17Markert, CL. Fertilization of mammalian eggs by sperm injection. J Exp Zool 1983; 228: 195–201.CrossRefGoogle ScholarPubMed
18Iritani, A, Hosoi, Y. Microfertilization by various methods in mammalian species. In: Joshinaga, K, Mori, T eds. Development of pre-implantation embryos and their environment. New York: Allan R Liss, 1989: 145–49.Google Scholar
19Keefer, CL, Younis, AI, Brackett, BG. Cleavage development of bovine oocytes fertilized by sperm injection. Mol Reprod Dev 1990; 25: 281–85.CrossRefGoogle ScholarPubMed
20Gordon, JW, Talansky, BE. Assisted fertilization by zona drilling, a mouse model for correction of oligospermia. J Exp Zool 1986; 239: 347–54.CrossRefGoogle Scholar
21Yang, X, Chen, J, Chen, Y, Foote, RH. Survival of rabbit eggs shrunken to aid in sperm micro-injection. Theriogenology 1988; 29: 336 (Abstract).CrossRefGoogle Scholar
22Lacham, O, Trounson, A, Holden, C, Mann, J, Sathananthan, H. Fertilization and development of mouse eggs injected under the zona pellucida with single spermatozoa treated to induce the acrosome reaction. Gamete Res 1989; 23: 233–43.CrossRefGoogle ScholarPubMed
23Metka, M, Haromy, T, Huber, J, Schurz, B. Artificial insemination using a micromanipulator. Fertilitat 1985; 1: 41–47.Google Scholar
24Laws-King, A, Trounson, A, Sathananthan, H, Kola, I. Fertilization of human oocytes by micro-injection of a single spermatozoon under the zona pellucida. Fertil Steril 1987; 48: 637–42.CrossRefGoogle Scholar
25Lanzendorf, E, Maloney, MK, Veeck, LL, Slusser, J, Hodgen, GD, Rosenwaks, Z. A preclinical evaluation of human spermatozoa into human oocytes. Fertil Steril 1988; 49: 835–42.CrossRefGoogle ScholarPubMed
26Gordon, JW, Grunfeld, J, Garrisi, GJ, Talansky, BE, Richards, C, Laufer, N. Fertilization of human oocytes by sperm from infertile males after zona pellucida drilling. Fertil Steril 1988; 50: 68–73.CrossRefGoogle ScholarPubMed
27Cohen, J, Malter, M, Fehilly, C et al. Implantation of embryos after partial opening of oocyte zona pellucida to facilitate sperm penetration. Lancet 1988; ii: 162.CrossRefGoogle Scholar
28Cohen, J, Eisner, C, Kort, H et al. Impairment of the hatching process following IVF in the human and improvement of implantation by assisting hatching using micromanipulation. Human Reprod 1990; 5: 7–13.CrossRefGoogle ScholarPubMed
29Ng, S-C, Sathananthan, AH, Edirisinghe, WR et al. Fertilization of a human egg with sperm from a patient with immotile cilia syndrome: case report. In: Ratnam, SS, Teoh, ES, Anandakumar, C eds. Advances in fertility and sterility, Volume 4. Lancaster, UK: Parthenon Publishing, 1987: 716.Google Scholar
30Malter, HE, Cohen, J. Embryonic development after microsurgical repair of polyspermic human zygotes. Fertile Steril 1989; 52: 373–80.CrossRefGoogle ScholarPubMed
31Gordon, JW, Grunfeld, L, Garrisi, GJ, Navat, D, Laufer, N. Successful microsurgical removal of pronuclei from tripronuclear hamster zygotes. Fertil Steril 1989; 52: 367–72.CrossRefGoogle Scholar
32Rawlins, RG, Binor, Z, Radwaska, E, Dmowski, WP. Microsurgical enucleation of tripronuclear human zygotes. Fertil Steril 1988; 50: 266–72.CrossRefGoogle ScholarPubMed
33Malter, HE, Cohen, J. Partial zona dissection of human oocytes: a non-traumatic method using micromanipulation assists zona pellucida penetration. Fertil Steril 1989; 51: 139–48.CrossRefGoogle Scholar
34Cohen, J, Alikani, M, Adler, A et al. Microsurgical fertilization procedures: the absence of stringent criteria for patient selection. J Assist Reprod Genet 1992; 9: 197–206.CrossRefGoogle ScholarPubMed
35Cohen, J, Alikani, M, Maker, HE, Adler, A, Talansky, BE, Rosenwaks, Z. Partial zona dissection or subzonal insertion: microsurgical fertilization alternatives based on evaluation of sperm and embryo morphology. Fertil Steril 1991; 56: 696–706.CrossRefGoogle ScholarPubMed
36Malter, HE, Alikani, M, Grifo, J, Talanksy, BE, Rosenwaks, Z. Microsurgical fertilization and zona pellucida micromanipulation. In: Fishel, SB, Symonds, EM eds. Gamete and embryo micromanipulation in human reproduction. Sevenoaks, UK: Edward Arnold, 1993: 101–18.Google Scholar
37Mashiach, S. Micro-assisted reproduction: is it the treatment of choice after fertilisation failure? In: Fishel, SB, Symonds, EM eds. Gamete and embryo micromanipulation in human reproduction. Sevenoaks, UK: Edward Arnold, 1993: 133–37.Google Scholar
38Fishel, SB, Timson, J. Sperm-egg manipulation for the treatment of male infertility. In: Templeton, AA, Drife, JO eds. Infertility. London: Springer-Verlag, 1992: 115–132.CrossRefGoogle Scholar
39Fishel, SB, Timson, J, Antinori, S, Lisi, F, Rinaldi, L. Subzonal insemination and zona breaching techniques for assisted fertilisation. In: Fishel, SB, Symonds, EM eds. Gamete and embryo micromanipulation in human reproduction. Sevenoaks, UK: Edward Arnold, 1993: 79–97.Google Scholar
40Fishel, S, Timson, J, Lisi, F, Rinaldi, L. The evaluation of 225 patients undergoing subzonal insemination for the procurement of fertilization in vitro. Fertil Steril 1992; 57: 840–49.CrossRefGoogle ScholarPubMed
41Hall, J, Fishel, SB, Timson, J. Evaluation of sperm morphology as a prognosticator for SUZI or IVF. (Unpublished observations.)Google Scholar
42Fishel, S. Gamete and embryo micromanipulation in human reproduction: perspicacity or opacity? In: Fishel, SB, Symonds, EM eds. Gamete and embryo micromanipulation in human reproduction. Sevenoaks, UK: Edward Arnold, 1993: 193–203.Google Scholar
43Payne, D, McLaughlin, KJ, Depypere, HT, Kirby, CA, Warnes, GM, Matthews, CD. Experience with zona drilling and zona cutting to improve fertilization rates of human oocytes in vitro. Human Reprod 1991; 6: 423–31.CrossRefGoogle ScholarPubMed
44Schmutzler, AG, Al-Hasani, S, Casper, AN, Viabold, A, Diedrich, K, Krebs, D. Assisted fertilization: SZI and PZD with mostly severe and very severe teratozoospermia. Abstract from VIIIth Meeting of ESHRE. Human Reprod 1992; Abstract 256: 115.Google Scholar
45Palermo, G, Joris, H, Devroey, P, Van Steirteghem, AC. Induction of acrosome reaction in human spermatozoa used for subzonal insemination. Human Reprod 1992; 7: 248–54.CrossRefGoogle ScholarPubMed
46Van de Leur, JJCM, Zeilmaker, GH. Microinsemination compared with IVF in the same patients. Abstract from VIIIth Meeting of ESHRE. Human Reprod 1992; Abstract 252: 137–38.Google Scholar
47Jean, M, Barriere, P, Sagot, P, L'Hermite, A, Lopes, P. Utility of zona pellucida drilling in cases of severe semen alterations in man. Fertil Steril 1992; 57: 591–96.CrossRefGoogle ScholarPubMed
48Tucker, MJ, Bishop, FM, Cohen, J, Wiker, SR, Wright, V. Routine application of partial zona dissection for male factor infertility. Human Reprod 1991; 6: 676–81.CrossRefGoogle ScholarPubMed
49Ng, S-C, Bongso, A, Ratnam, SS. Micro-injection of human oocytes: the technique for severe oligoasthenoteratozoospermia. Fertil Steril 1991; 56: 1117–23.CrossRefGoogle Scholar
50Fishel, SB, Lymberopolous, G, Timson, J. Increasing insemination concentration for IVF/microdrop IVF with spermatozoa from infertile males. (Unpublished observations.)Google Scholar
51Palermo, G, Joris, H, Devroey, P, Van Steirteghem, AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17–18.CrossRefGoogle ScholarPubMed
52Iritani, A. Micromanipulation of gametes for in vitro assisted fertilization. Mol Reprod Dev 1991; 28: 199–207.CrossRefGoogle ScholarPubMed
53Veeck, LL, Oehninger, S, Acosta, AA, Muasher, SJ. Sperm microinjection in a clinical in vitro fertilization program. Proceedings of the 45th Annual Meeting of the American Fertility Society, 13–16 November 1989, San Francisco. Birmingham, Alabama: American Fertility Society, 1990.Google Scholar
54Kola, I, Lacham, O, Jensen, RPS, Turner, N, Trounson, A. Chromosomal analysis of human oocytes fertilised by micro-injection of spermatozoa into the perivitelline space. Human Reprod 1990; 5: 575–77.CrossRefGoogle Scholar
55Aitken, RJ. The zona-free hamster oocyte penetration test and the diagnosis of male infertility. Int J Androl 1986; 6: 1–199.Google Scholar
56Ng, S-C, Liow, L, Bongso, TA et al. Micromanipulation and possible new approaches to help the severe male-factor patient. J Assist Reprod Genet 1992; 9: 191–96.CrossRefGoogle ScholarPubMed
57Fuscaldo, G, Sovieszczuk, D, Trounson, AO. Improved fertilization rates following micro-injection of human spermatozoa pretreated with 2'-deoxyadenosine and pentoxifylline: VIIth World Congress IVF and Assisted Procreation, Paris. Human Reprod 1991; 110: 145–46 [Abstract].Google Scholar
58Suarez, SS, Wolf, DP, Mei, ZEL. Induction of the acrosome reaction in human spermatozoa by a fraction of human follicular fluid. Gamete Res 1986; 14: 107–21.CrossRefGoogle Scholar
59Tesarik, J. Comparison of acrosome reaction-inducing activities of human cumulus oophorus, follicular fluid and ionophore A23187 in human sperm populations of proven fertilizing ability in vitro. J Reprod Fertil 1985; 74: 383–88.CrossRefGoogle ScholarPubMed
60Bleil, JD, Wassarman, PM. Sperm-egg interactions in the mouse: sequence of events and induction of the acrosome reaction by a zona pellucida glycoprotein. Dev Biol 1983; 95: 317–24.CrossRefGoogle ScholarPubMed
61Tompkins, FT, Houghton, JA. The rapid induction of the acrosome reaction of human spermatozoa by electropermeabilisation. Fertil Steril 1988; 50: 329–36.CrossRefGoogle Scholar
62Green, S, Fishel, SB, Timson, J, Hall, J. The induction of the acrosome reaction in human sperm populations undergoing micromanipulation. (Unpublished observations.)Google Scholar
63Ng, S-C, Bongso, TA, Liow, SL, Edirisinghe, R, Tok, V, Ratnam, SS. Controversies in micro-injection. J Assist Reprod Genet 1992; 9: 186–89.CrossRefGoogle ScholarPubMed
64Martin-DeLeon, PA, Shaver, EL, Gammal, EB. Chromosome abnormalities in rabbit blastocytes resulting from spermatozoa aged in the male tract. Fertil Steril 1973; 24: 212–19.CrossRefGoogle Scholar
65Martin, RH, Ko, E, Rademaker, A. Human sperm chromosome compliments after micro-injection of hamster eggs. J Reprod Fertil 1988; 84: 179–86.CrossRefGoogle Scholar
66Sathananthan, AH, Kola, I, Osborne, A et al. Centrioles in the beginning of human development. Proc Natl Acad Sci USA 1991; 88: 4806–10.CrossRefGoogle ScholarPubMed
67Goto, K, Kinoshita, A, Takuma, Y, Ogawa, K. Fertilization of bovine oocytes by the injection of immobilised, killed spermatozoa. Vet Rec 1990; 127: 517–20.Google ScholarPubMed
68Cohen, J, Eisner, C, Kort, H et al. Impairment of the hatching process following IVF in a human and improvement of implantation by assisting hatching using micromanipulation. Human Reprod 1990; 5: 7–13.CrossRefGoogle Scholar
69Cohen, J. Assisted hatching of human embryos. J In Vitro Fert Embyro Transf 1991; 8: 179–90.CrossRefGoogle ScholarPubMed
70Cohen, J, Alikani, M, Trowbridge, J, Rosenwaks, Z. Implantation enhancement by selective assisted hatching using zona drilling of human embryos with poor prognosis. Human Reprod 1992; 7: 685–91.CrossRefGoogle ScholarPubMed
71Malter, HE, Cohen, J. Blastocyst formation and hatching in vitro following zona drilling of mouse and human embryos. Gamete Res 1989; 24: 67–80.CrossRefGoogle ScholarPubMed
72Cohen, J, Feldberg, D. Effects of the size and number of zone pellucida openings on hatching and tropho-blast outgrowth in the mouse embryo. Mol Reprod Dev 1991; 30: 70–78.CrossRefGoogle Scholar
73Dale, B, Gualitierei, R, Talevi, R, Tosti, E, Santella, L, Elder, K. Intercellular communication in the early human embryo. Mol Reprod Dev 1991; 29: 22–28.CrossRefGoogle ScholarPubMed
74Verlinksy, Y, Ginsburg, N, Lifchez, A, Valle, J, Moise, J, Strom, CM. Analysis of the first polar body: preconception genetic diagnosis. Human Reprod 1990; 5: 826–69.Google Scholar
75Handyside, AH. Pre-implantation diagnosis strategies for embryo biopsy and genetic analysis. In: Fishel, SB, Symonds, EM eds. Gamete and embryo micromanipulation in human reproduction. Sevenoaks, UK: Edward Arnold, 1993: 173–82.Google Scholar
76Fishel, SB. Prelude to pre-implantation diagnosis in the human. In: Fishel, SB, Symonds, EM eds. Gamete and embryo micromanipulation in human reproduction. Sevenoaks, UK: Edward Arnold, 1993: 141–54.Google Scholar
77Navidi, W, Arnheim, N. Using PCR in preimplantation genetic disease diagnosis. Human Reprod 1991; 6: 836–49.CrossRefGoogle ScholarPubMed
78Verlinsky, Y, Rechitsky, S, Evsikov, S et al. Preconception and pre-implantation diagnosis for cystic fibrosis. Prenat Diagn 1992; 12: 103–10.CrossRefGoogle Scholar
79Handyside, AH, Lesko, J, Winston, RML, Hughes, MR. Birth of a normal girl following pre-implantation diagnostic testing of cystic fibrosis. N Engl J Med 1992; 327: 905–909.CrossRefGoogle Scholar
80Lesko, J, Snabes, M, Handyside, A, Hughes, M. Amplification of the cystic fibrosis delta-F508 mutation from single cells; applications towards genetic diagnosis of the pre-implantation embryo. Am J Hum Genet 1991; 49: 223.Google Scholar
81Handyside, AH, Pattinson, JK, Penketh, RJA, Delhanty, JDA, Winston, RML, Tuddenham, EDO. Biopsy of human pre-implantation embryos and sexing by DNA amplification. Lancet 1989; i: 347–49.CrossRefGoogle Scholar
82Kontogianni, EH, Hardy, K, Handyside, A. Co-amplification of X- and Y-specific sequences for sexing preimplantation human embryos. In: Verlinsky, Y, Kulier, A eds. Preimplantation genetics. New York: Plenum Press, 1991: 139–45.CrossRefGoogle Scholar
83Handyside, AH, Delhanty, JDA. Cleavage stage biopsy of human embryos and diagnosis of X-linked recessive disease. In: Edwards, RG ed. Preimplantation diagnosis of human genetic disease. Cambridge: Cambridge University Press, 1993 (in press).Google Scholar
84Strom, CM, Rechitsky, S, Verlinsky, Y. Reliability of gender determination using the polymerase chain reaction (PCR) for single cells. J In Vitro Pert Embryo Transf 1991; 8: 225–29.CrossRefGoogle ScholarPubMed
85Holding, C, Monk, M. Diagnosis of beta-thallasemia by DNA amplification in single blastomeres from mouse pre-implantation embryos. Lancet 1989; ii: 532–35.CrossRefGoogle Scholar
86Li, H, Cui, X, Arnheim, N. Direct electrophoretic detection of the allelic state of the single DNA molecules in human sperm by using the polymerase chain reaction. Proc Natl Acad Sci USA 1990; 87: 4580–84.CrossRefGoogle ScholarPubMed
87Gomez, CM, Muggleton-Harris, AL, Whittingham, D, Hood, L, Readhead, C. Rapid pre-implantation detection of mutant (shiverer) and normal alleoles of the mouse myeline basic protein gene allowing selective implantation and birth of live young. Proc Natl Acad Sci USA 1990; 87: 4481–84.CrossRefGoogle Scholar
88Pickering, SJ, McConnell, JM, Johnson, MH, Braudie, PR. Reliability of detection by polymerase chain reaction of the sickle cell-containing region of the β-globin gene in single human blastomeres. Hum Reprod 1992; 7: 630–36.CrossRefGoogle ScholarPubMed
89Dokras, A, Sargeant, IL, Ross, C, Gardner, RL, Barlow, DH. Trophectoderm biopsy in human blastocysts. Hum Reprod 1990; 5: 821–25.CrossRefGoogle ScholarPubMed
90Fishel, SB, Cohen, J, Fehily, C, Purdy, JM, Walters, E, Edwards, RG. Factors influencing human embryonic development in vitro. N Y Acad Sci 1985; 442: 342–56.CrossRefGoogle ScholarPubMed
91Hardy, K, Handyside, AH, Winston, RML. Human blastocysts; cell number, death and allocation during later pre-implantation development in vitro. Development 1989; 107: 597–604.CrossRefGoogle Scholar
92Dawson, KJ, Rutherford, AJ, Winston, NJ, Subak-Sharpe, R, Winston, RML. Human blastocyst transfer, is it a feasible proposition? Hum Reprod 1988; 145(suppl): 44–45.Google Scholar
93Bolton, VN, Wren, ME, Parsons, JH. Pregnancies after in vitro fertilization and transfer of human blastocysts. Fertil Steril 1991; 55: 830–32.CrossRefGoogle ScholarPubMed
94Handyside, AH, Kontogianni, EH, Hardy, K, Winston, RML. Pregnancies from human pre-implantation embryos sexed by Y-specific DNA amplification. Nature 1990; 344: 768–70.CrossRefGoogle Scholar
95Wilton, L, Shaw, JM, Trounson, AO. Successful singlecell biopsy and cryopreservation of pre-implantation mouse embryos. Fertil Steril 1989; 51: 513–17.CrossRefGoogle Scholar
96Griffin, DK, Wilton, L, Handyside, AH, Winston, RML, Delhanty, JDA. Dual fluorescent in situ hybridisation for simultaneous detection of X and Y chromosome-specific probes for the sexing of human pre-implantation embryonic nuclei. Hum Genet 1992; 89: 18–22.CrossRefGoogle Scholar
97Cohen, J, Malter, HE, Talansky, BE, Grifo, J. Micromanipulation of human gametes and embryos. New York: Raven Press, 1992.Google ScholarPubMed
98Fishel, S, Timson, J, Lisi, F, Jacobson, M, Rinaldi, L, Gobetz, L. Micro-assisted fertilization (MAP) in patients who have failed subzonal insemination (SUZI). 1993 (unpublished observations).CrossRefGoogle Scholar