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Assisted Reproductive Technology

Micromanipulation in Assisted Reproductive Technologies

Robert B. Shabanowitz, PhD
Within the last several years the use of micromanipulation has rapidly developed as an additional adjunct to available assisted reproductive technologies in humans (see 18 & 19 for reviews). The micromanipulation of mammalian eggs developed as a procedure to study the early events of fertilization, especially as a useful method to observe the effects of materials injected into unfertilized ova or zygotes. Using this technique, glass micropipettes, only several microns wide at their tips, are used to pick up and manipulate single embryos or spermatozoa. This micromanipulation is performed while viewing the gametes under a microscope. At present, the most widely accepted and proven benefit of micromanipulation is assisted fertilization for severe male factor infertility. However, micromanipulation may eventually be utilized for corrective manipulations of gross fertilization errors, as well as for diagnosis (and correction) of genetic diseases and chromosomal disorders.

I. Assisted Fertilization
The indications for assisted fertilization are predominately associated with male factor problems as a cause or component of infertility. Corrective intervention for male factor infertility has largely been an underdeveloped area, but with the advent of IVF and GIFT technologies, the male factor component has rapidly become an area of intensive investigation. Assisted fertilization must be considered with cases of severe oligospermia (<5 million/ml), severe asthenozoospermia, immotile spermatozoa, unexplained failed fertilization due to the inability of sperm to traverse the outer egg investments, and multiples of these factors.

Zona By-Pass
The zona pellucida is a specialized extracellular matrix that surrounds all mammalian eggs. The zona pellucida is responsible for protecting the early developing embryo, and is the site to which sperm first bind the egg, in a species specific manner. The zona pellucida is also responsible for eliciting an acrosome reaction in sperm, a prerequisite for successful fertilization. Sperm must penetrate the zona pellucida prior to fertilization of the egg, and sperm contain specific enzymes used to digest the zona. The zona pellucida is also responsible for development of a block to polyspermy which prevents further sperm binding and penetration of the zona, and therefore helps prevent fertilization of the egg by more than one spermatozoa. The zona pellucida thus presents a barrier which sperm must negotiate prior to reaching the egg plasma membrane, and defects in sperm morphology, motility or function can preclude successful penetration of this acellular structure. Making a small hole in the zona pellucida is a simple yet elegant strategy to circumvent the requirement of sperm binding and penetration of this egg vestment.

A. Zona Drilling 
Zona drilling was first developed by Gordon and Talansky in 1986 (20). In this procedure, a small hole is made in the zona pellucida using a micropipette filled with acidified medium, or with enzymes such as trypsin or pronase that can digest and dissolve the zona pellucida. The resultant hole in the zona pellucida allows facile entry of sperm into the perivitelline space where they can then fertilize the egg. Although zona drilling has proven useful in animal models, its benefit in humans has been less promising as it has been shown that the acidic solutions used to digest the zona pellucida also damage the egg (21).

B. Partial Zona Dissection
Partial zona dissection (PZD) is another method developed to by-pass the need for sperm to attach to and penetrate the zona pellucida. In this technique, developed by Cohen (22), a sharp tipped micropipette is used to mechanically perforate the zona pellucida and form a "fertilization slit". Success of this technique has been improved by placing the egg in a hyperosmotic sucrose solution before perforating the zona pellucida. The hyperosmotic environment causes the egg to shrink, thereby temporarily creating a larger perivitelline space. This reduces the chances of damaging the oolemma when the micropipette pierces the zona pellucida. This technique has been shown to increase fertilization rates up to 50% in patients who have had prior poor or failed fertilization, and pregnancies have ensued using this procedure(23). PZD is not as effective, however, for asthenozoospermia, combined semen problems or immunologic infertility.

II. Microinsemination/Microfertilization
Micromanipulation can also be used to introduce sperm, either singly or in multiples, directly into the perivitelline space (sperm transfer) or directly into the egg cytoplasm (sperm microinjection). These techniques can be used when spermatozoon function is so poor that even a hole in the zona pellucida is insufficient.

A. Sperm Transfer
Sperm transfer involves the direct placement of sperm into the perivitelline space. This method can be used to place either single or multiple sperm in direct contact with the egg's plasma membrane. Just as in PZD, sucrose is used to osmotically shrink the egg prior to transfer. In order for spermatozoa to fuse with the oolemma, they must first be acrosome reacted. Human sperm require longer incubations in capacitating medium and are more variable in the percentage of acrosome reacted forms present than most other species. Therefore, to improve the fertilization efficiency of sperm transfer, 3-5 sperm are typically transferred to increase the probability of providing at least one sperm capable of fertilizing the egg. However, total sperm numbers must also be limited to prevent the incidence of polyspermy, which has been shown to increase with transfer of from 10-20 sperm (24). Whereas motile sperm are required for successful zona pellucida drilling and PZD, direct sperm transfer to the perivitelline space provides an attractive method for the treatment of severe oligospermia, as well as asthenozoospermia. Pregnancies from sperm microinsemination have been reported by Ng et al. (25), and human fertilization by microinsemination into the perivitelline space has even been successful using immotile sperm from a patient suffering from immotile cilia syndrome (26). Methods to improve the capacitation and acrosome reacting ability of human sperm will help the continued success of sperm transfer techniques.

B. Sperm Microinjection
Microinjection involves the direct placement of sperm into the egg cytoplasm. This is accomplished by piercing both the zona pellucida and egg plasma membrane with a transfer pipette no wider than a red blood cell. Sperm microinjection was first reported by Hiramoto in 1962, using sea urchin eggs (27), and the technique was first described in a mammalian species (mouse) by Lin in 1966 (28), who microinjected bovine gamma globulin into mouse embryos and followed their development after recovery and transplantation to females. As might be expected, however, this procedure can result in a high rate of egg injury. With the proper technique and training, this procedure is extremely effective, especially for the treatment of severe male factor infertility (37). The procedure requires extensive training and is very time consuming. However, it is rapidly becoming the most effective and successful methods of sperm transfer.

III. Corrective Manipulations of Gross Fertilization Errors

A. Pronuclear Reduction
Penetration of an egg by more than one spermatozoon results in a polyspermic egg. Normally, the egg can prevent this by a plasma membrane associated fast block to polyspermy, as well as a more slowly developing slow block to polyspermy believed to be due to the biochemical modification of the zona pellucida elicited by cortical granule enzymes, which are released into the perivitelline space immediately subsequent to fertilization (29). Suboptimal cortical granule exocytosis, as in immature metaphase I eggs or older eggs can therefore result in higher rates of polyspermy. Although the continued development of polyspermic eggs is severely compromised, these eggs can continue to develop and divide. However, in order to prevent potential obstetrical risks, one would not intentionally transfer such aneuploid embryos.

Excess male pronuclei can be removed by micromanipulation, thereby correcting an originally aneuploid condition. This process is accomplished by temporarily arresting the aneuploid embryo at the pronuclear stage with cytoskeletal inhibitors, and then specifically removing excess male pronuclei by microsuction. Rawlins et al. (30) have recently reported preliminary attempts to correct tripronuclear human eggs using this method. Male pronuclei were identified by their larger size and attendant sperm tail piece. Enucleation was successful in all embryos, and syngamy occurred in one, although subsequent cleavage was not observed. The removal of redundant sperm pronuclei, however, may not become a useful clinical procedure because of the initial low incidence of polyspermy (<5% in most IVF programs), low embryo survival rates, and the fact that most patients will have several normally fertilized eggs available. An additional drawback is the inadvertent removal of the female rather than male pronucleus; this could result in the development of androgenomes. Furthermore, it is has been reported that up to 40% of tripronuclear zygotes, the most common form of aneuploidy seen, may actually contain one pseudopronucleus which, in fact, contains no chromatin (31). These eggs are normal diploids, and therefore cleave and develop normally.

IV. Diagnosis - Correction

A. Blastomere Separation - Blastocyst Biopsy
The ability to remove individual blastomeres or a small sample of trophectoderm from early growing embryos offers some of the most fascinating prospects for the benefit of advanced micromanipulative techniques. The removal of blastomeres (32) must usually be performed at an early embryonic stage, typically at four to eight cells. A similar technique, called blastocyst or trophectoderm biopsy (33) involves removal of mural trophoblast cells. The individual blastomeres of very early embryos remain totipotent, and removal of portions of the early embryo do not compromise the continued growth and development of a viable organism. Individual cells can be removed by creating a small crack in the zona pellucida, and the early blastomeres, which are not firmly attached to one another can then be removed from the zona pellucida. Once removed, a variety of manipulations are possible. Individual blastomeres can be transplanted to an empty "donor" zona pellucida and can thereby provide a means of producing monozygotic offspring. Although this technique is common in the production of monozygotic farm animals to increase the yield of genetically superior animals, this is not a likely scenario in humans. However, if these blastomeres can successfully be frozen-thawed, they could provide a bank of additional "embryos" available for transfer should pregnancy fail to occur on the first occasion.

Using specific DNA probes and the ability to amplify DNA signals using the polymerase chain reaction (PCR), individual cells can also be utilized for the diagnosis of chromosomal abnormalities. Determination of sex could also be performed for identification of sex chromosome-linked genetic diseases. Separated cells could be frozen and stored pending results of such screening, thus developing methods for preimplantation diagnosis. If diagnosis could be completed within a few days, freezing may not even be required since embryos could continue to grow in culture and embryo transfer of blastocysts would only be performed after test results were available. These techniques have already been used in animal models to identify x-linked traits (32). The use of such advanced techniques in humans, however, remains speculative. Considerably more research will be required to evaluate the ability of such manipulated embryos to continue to survive, develop and produce normal offspring.

V. Nuclear Substitution/Cloning
The first report of nuclear substitution or cloning into enucleated eggs (frog) was reported by Briggs and King in 1952 (34). This technique requires the removal of the nucleus from one cell (cytoplast), followed by the transfer of a nucleus, surrounded by a small portion of cytoplasm (karyoplast) derived from a second cell into the previously enucleated cell. The fusion of cytoplast and karyoplast can be accomplished by direct microinjection, or by using Sendai virus to promote fusion (35). Nuclear substitution would allow the continued propagation of a specific genetic composition. The success of nuclear substitution is limited to totipotent nuclei. The full developmental potential of isolated nuclei is species dependant, and in most mammals, this potential is reduced very early in embryonic development. Nuclei from differentiated cells are unable to support the development of a new organism after transfer to enucleated eggs. It is doubtful that this technology will ever become useful in humans, although it may be useful for the preservation of rare species or breeds. However, this technique continues to be of importance in the study of the individual roles of nucleus and cytoplasm in development and differentiation.

VI. Transgenic Animals
The ability to transfer specific DNA into eggs or early embryos is well established (for review see 36). Cloned genes can be transferred to eggs or early embryos by direct microinjection or by viral vectors. A percentage of cells may then incorporate this material into their own DNA repertoire, and the cloned genes may then be expressed in the adults of these genetically altered animals. Transgenic mice have become a powerful tool to study the activity of cloned genes. This technology may someday enable correction of certain genetic abnormalities after identification by preimplantation diagnosis.

The advanced reproductive technologies discussed encompass many years of basic research in the areas of fertilization and early development. Several of these technologies have been easily adapted, and proven successful in the treatment of certain human reproductive impairments. Continuing research in animal models, as well as controlled studies in humans, however, will be required to bring all these technologies to the available repertoire of the reproductive infertility specialist.

18. Ng S-C, Bongso A, Sathananthan H, Ratnam SS: Micromanipulation: its relevance to human in vitro fertilization. Fert. Steril. 53,203, 1990.

19. Iritani A: Current status of biotechnological studies in mammalian reproduction. Fert. Steril. 50:543, 1988.

20. Gordon JW, Talansky BE: Assisted fertilization by zona drilling: a mouse model for correction of oligospermia. J. Exp. Zool. 239:347, 1986.

21. Ng S-C, Bongso A, Chang S-I, Sathananthan H, Ratnam S: Transfer of human sperm into the perivitelline space of human oocytes after zona-drilling or zona-puncture. Fert. Steril. 52:73, 1989.

22. Malter HE, Cohen J: Partial zona dissection of the human oocyte: a nontraumatic method using micromanipulation to assist zona pellucida penetration. Fert. Steril. 51:139, 1989.

23. Cohen J, Malter H, Fehilly C, Wright G, Elsner C, Kort H, Massey J: Implantation of embryos after partial opening of oocyte zona pellucida to facilitate sperm penetration. The Lancet, July 16: 162, 1988.

24. Lassalle B, Courtot AM, Testart J: In vitro fertilization of hamster and human oocytes by microinjection of human sperm. Gamete Res 16:69, 1987.

25. Ng S-C, Bongso A, Ratnam SS, Sathananthan H, Chan CLK, Wong PC, Hagglund L, Anandakumar C, Wong YC, Goh VHH: Pregnancy after transfer of sperm under zona. The Lancet, October 1: 790, 1988.

26. Bongso TA, Sathananthan AH, Wong PC, Ratnam SS, Ng SC, Anandakumar C, Ganatra S: Human fertilization by micro-injection of immotile spermatozoa. Human Reprod. 4:175, 1989.

27. Hiramoto Y: Microinjection of the live spermatozoa into sea urchin eggs. Exp. Cell Res. 27,416: 1962.

28. Lin TP: Microinjection of mouse eggs. Science 151:333, 1966.

29. Shabanowitz RB, O'Rand MG: Characterization of the human zona pellucida from fertilized and unfertilized eggs. J. Reprod. Fert. 82:151, 1988.

30. Rawlins RG, Binor Z, Radwanska E, Dmowski WP: Microsurgical enucleation of tripronuclear human zygotes. Fert. Steril. 50:266, 1988.

31. Van Blerkom J, Bell H, Henry G: The occurrence, recognition and developmental fate of pseudo-multipronuclear eggs after in-vitro fertilization of human oocytes. Human Reprod. 2:217, 1987.

32. Monk M, Handyside AH: Sexing of preimplantation mouse embryos by measurement of X- linked gene dosage in a single blastomere. J. Reprod. Fert. 82:365, 1988.

33. Summers PM, Campbell JM, Miller MW: Normal in-vivo development of marmoset monkey embryos after trophectoderm biopsy. Human Reprod. 3:389, 1988.

34. Briggs R, King TJ: Transplantation of living nuclei from blastula cells into enucleated frog eggs. Proc. Natl. Acad. Sci. USA 38:455, 1952.

35. McGrath J, Solter D: Nuclear transplantation in the mouse embryo by microsurgery and cell fusion. Science 220:1300, 1983.

36. Palmiter RD, Brinster RL: Transgenic mice. Cell 41:343, 1985.

37. Van Steirteghem et al.: High fertilization and implantation rates after intracytoplasmic sperm injection. Human Reprod. 8:1061, 1993.

For more information about treatment options:
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