micromanipulation of gametes for in vitro assisted fertilization

MOLECULAR REPRODUCTION AND DEVELOPMENT 28199-207 (1991)

Review Article

Micromanipulation of Gametes for In Vitro Assisted Fertilization A. IRITANI Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan

INTRODUCTION The main purpose of early investigations into sperm

injection conducted between 1965 and 1980 was to in- vestigate the early events of fertilization, such as membrane fusion between homologous and heterologous gametes, activation of the oocyte cytoplasm, and formation of male and female pronuclei. Very few papers were published in the field of gamete manipulation in rodents or in nonmammalian species.

In accordance with the rapid advancement of studies on in vitro fertilization (IVF) in mammals and of the development of many human IVF-ET programs, atten- tion focused upon difficult cases in which the binding of gametes was not achieved by conventional IVF, due to various reasons, especially infertility stemming from male factors. Since 1980, fundamental studies on the micromanipulation of gametes in nonhuman mammalian species have increased dramatically, resulting in basic knowledge and techniques (see Fig l), which may be useful in overcoming gamete binding problems, in- cluding those encountered in human infertility. Micro- manipulation of gametes has been a valuable technique from both the physiological and clinical view points.

EARLY GAMETE MICROMANIPULATION FOR PHYSIOLOGICAL STUDIES OF GAMETE

INTERACTION (TABLE 1) Hiramoto (1962) injected living sea urchin sperma-

tozoa into an unfertilized sea urchin egg, but the sperm nucleus did not decondense in the egg cytoplasm. When the egg was inseminated after sperm injection, the injected sperm nuclei then participated in the mitotic process, suggesting that activation of the egg cytoplasm is essential for decondensation of the sperm nucleus. Graham (1966) and Burn (1974) injected living frog spermatozoa into unfertilized frog eggs and found that the sperm nuclei transformed into pronuclei, suggesting that the frog egg unlike that of the sea urchin is easily activated by pricking with a microneedle (Briggs and King, 1952).

Uehara and Yanagimachi (1976,1977) applied the technique of sperm injection to determine whether spermatozoa or sperm nuclei injected into the hamster egg cytoplasm can develop into male pronuclei. They also examined whether spermatozoa, from various other species, would develop into pronuclei when injected into hamster oocytes. Isolated hamster nuclei developed into pronuclei when injected into hamster oocytes. It was also found that the nuclei of fresh, freeze-dried and frozen-thawed human spermatozoa were able to develop into pronuclei in hamster oocytes, suggesting that the cytoplasmic factors controlling the transformation of sperm nuclei into male pronuclei are not species specific (rat vs. mouse, Thadani, 1980; frog vs. human, Ohsumi et al., 1986; hamster vs. mice, rabbit vs. fish, Naish et al., 1987). Thadani (1979) investigated early events of hetero-specific fertilization between the mouse and the rat. He found that zona-free rat oocytes can be fertilized by mouse sperm and form both male and female pronuclei. He also examined whether sperm and oocyte membrane fusion is required for normal development, by injecting sperm directly into the cytoplasm. The injection of mouse sperm into rat oocytes resulted in a high proportion of activation and formation of both pronuclei. Capacitated and uncapacitated sperm reacted alike when injected into oocytes. The percentages of rat oocytes with normal pronuclei following injection of capacitated and uncapacitated mouse sperm were 65% and 86%, respectively, suggesting that sperm capacitation may not be necessary when spermatozoa are directly injected into cytoplasm. However, oocyte activation is necessary for male pronucleus formation. Thadani suggested that 5%

Received March 8, 1990; accepted June 20, 1990. Address reprint requests to Dr. A. Iritani, Department of Animal Science, College of Agriculture, Kyoto University, Kyoto 606, Japan.

0 1991 WILEY-LISS, INC.

200 IRITANI

TABLE 1. Early Works of Fertilization by Microinjection of Homologous and Heterologous Spermatozoon Into Cytoplasm

Oocyte Sperm Results References Sea urchin Sea urchin Decondense" Hiramoto, 1962 Frog Frog Pro-N' Graham, 1966; Burn, 1974 Hamster Hamster, human Pro-N (69%,72%) Uehara and Yanagimachi, 1976 Hamster Hamster(cauda,caput epid.,testicular) Pro-N (69%,50%,72%) Uehara and Yanagimachi, 1977 Rat Mouse, deer mouse Pro-N (6748%) Thadani, 1979, 1980 Hamster Hamster (nuclei) Nuclei retained in cytoplasm (50%) Perreault and Zirkin, 1982 Mouse Mouse (intact,defect, immotile) Decondense, Pro-N Markert and Petters, 1977

Markert, 1983; Gomibuchi et al., 1988

Frog Human (nucleus) Decondense Ohsumi et al., 1986 Hamster Hamster Decondense (29%) Westhusin and Kraemer, 1986 Hamster Hamster, mouse, rabbit, fish, hepatocyte Pro-N Naish et al., 1987 Hamster Hamster, mouse, bull, human Pro-N Perreault et al., 1988 Hamster Three rodents (nucleus) Decondense (14-16%) Clarke and Johnson, 1988

"Decondense, Decondensation of sperm head. 'Pro-N, Transformation of sperm head into pronucleus.

Ram (nucleus) Decondense (38%)

polyvinyl pyloridone (PVP) in the sperm injection medium may have caused the activation. The data from their experiments indicate that sperm interactions with the oocyte cytoplasm are less species specific than interactions at the surface of the oocyte, and that the normal surface interactions of sperm and oocyte in membrane fusion are not essential for the initiation of development. Similar work was conducted by Markert (1983). Sperm heads, detached from tails by sonication, can be injected into unfertilized mouse oocytes to induce normal fertilization events. Immotile and grossly defective mouse sperm injected into the oocyte exhib- ited the same reactions found in fertilization with healthy sperm (Markert, 1983). When hamster, mouse, rabbit, and fish sperm nuclei, or hamster hepatocyte nuclei, were microinjected into hamster oocytes, the nuclei of each type transformed into pronuclei and started DNA synthesis (Naish et al., 1987). It is con- cluded from these results that the inteactions normally required for sperm to penetrate and fertilize oocytes are not biologically necessary and can be circumvented by direct injection of spermatozoa.

PRODUCTION OF OFFSPRING BY MICROFERTILIZATION IN RABBIT

AND CATTLE Cytoplasmic injection of a single spermatozoon di-

rectly into cytoplasm has contributed greatly to investigations of the early events and biological mechanisms of fertilization, such as sperm capacitation and the acrosome reaction, membrane fusion of both gametes, activation of oocytes, and cytoplasmic factors involved in transformation of sperm nuclei into pronuclei.

Embryos obtained by such injection have been transferred to recipient animals (Table 2). One of the main

purposes of applying this technique for animal production may be an efficient use of genetically important, but biologically inferior, male gametes from domestic and wild animals. In our laboratory a single uncapacitated rabbit sperm was injected into the cytoplasm of a series of superovulated rabbit oocytes. Sixty-three percent of the oocytes (52183) survived and forty-six (2462) developed to the pronuclear stage. Half of the fertilized zygotes cleaved to the 2-4-cell stage 24 h after microinjection (Hosoi et al., 1988). Keefer (1989) reported that injection of capacitated sperm yielded a significantly higher number of pronuclear eggs than did injection of uncapacitated sperm. In cattle, microinjection of in vitro capacitated bovine spermatozoa into the cytoplasm of in vitro matured bovine oocytes has been reported (Younis et al., 1989; Keefer et al., 1990). When in vitro capacitated, frozen-thawed bull spermatozoa were injected into cytoplasm only 2% (21 101) of the injected oocytes cleaved, but when spermatozoa were injected into oocytes activated by 5 minutes incubation in calcium ionophore, decondensation of sperm nuclei occurred in 37% of the injected oocytes, and 28% (6121) cleaved to the 2-4-cell stage 48 h after injection. These results indicate that bovine oocytes are not sufficiently stimulated by the injection procedure itself to complete meiosis, but calcium ionophore caused activation and normal cleavage following fertilization by sperm injection. Goto et al. (1990) matured bovine follicular oocytes in culture, activated them by incubation in the calcium ionophore, (A231871, for 10 minutes, and then a single spermatozoon was injected into their cytoplasm. Cleavage rate up to the 6-12-cell stage was 15%, and a calf was born after transfer of a blastocyst derived from microfertilization. In case of cytoplasmic injection, mechanical injury to the cy-

GAMETE MICROMANIPULATION FOR IVF 201

TABLE 2. Fertilization by Microinjection of Normal Spermatozoa Into Oocyte Cytoplasm Aiming Production of Offspring in the Rabbit and Cattle

Oocyte Sperm Results References

Rabbit Rabbit (intact, epididymal) Fert. rate (53%) Iritani et al., 1988 Cleavage rate (30%)

Rabbit Rabbit (intact, epididymal) Fert. rate (60%) Hosoi et al., 1988 Offspring (2/72;2.8%) Iritani, 1988, 1990

Rabbit Rabbit (intact, nucleus, 18 h preincubated) Cleavage rate (25%) Keefer et al., 1989 (Preincubated, intact) Foetal development

Iritani and Hosoi, 1989

Bovine Bovine (capacitated) Control oocyte cleavage rate (2%) Younis et al., 1989 (activated) Preincubated oocyte cleaved (28%), Keefer et al., 1990

Bovine Bovine (killed by freeze-thawing) 6-12 cell (15%) Goto et al., 1990 (activated) Morula-Bla. (10%)

A calf born

toskelton and organelles in the cytoplasm may be caused by the injection needle and biological contami- nants may be introduced. These unfavorable conditions may cause a loss of embryos after transfer of the microfertilized embryos. Nevertheless, Hosoi et al. obtained delivery of young after transfer of microfertilized rabbit embrys into the oviducts of a pseudopregnant doe in 1985 at The Johns Hopkins Medical School (unpublished). Recently we transferred 72 2-4-cell rabbit microfertilized embryos into the oviducts of eight recipient does and obtained two offspring. However, the fertilization and cleavage rates were high (Table 2).

MICROFERTILIZATION BY VARIOUS METHODS

Most of the research on microfertilization just de- scribed was conducted by cytoplasmic sperm injection. With this method satisfactory fertilization rates may be obtained. However, the developmental rate of transferred embryos is quite low, probably due to some del- eterious effect on the egg cytoplasm. Furthermore, cytoplasmic microinjection bypasses all natural barriers to sperm penetration and removes all biological selec- tivity from the process, such as sperm binding to the zona pellucida and fusion of the sperm with the vitelline membrane (Gordon and Laufer, 1988). This may be the key reason why many clinicians prefer other methods.

Since 1986 when the paper by Gordon and Talansky appeared reporting the zona drilling method for the microfertilization of mice, two additional methods, perivitelline injection and zona dissection, have been widely applied. As shown in Figure 1, frequently applied techniques in the micromanipulation of gametes to assist fertilization are 1) injection of spermatozoa directly into cytoplasm (cytoplasmic injection), 2) injection of spermatozoa under the zona pellucida (perivitelline injection), 3) opening the zona pellucida (partial

CYTOPLASMIC INJECTION - - - - - - -

* Monospermic Q- ’\ ’Zona pellucida ( * Intact ‘Perivitelline space

PERIVITELLINE INJECTION _-------

* Polyspermic ? * Acrosome-reacted

i ’ I Ooplasm M-II plate

ZONA DISSECTION (ZONA OPENING) __-------

1 s t polar body ‘

ZONA DRILLING

* Polyspermic ? * Acrosome-reacted

--------

Fig. 1. In vitro fertilization using various methods of gamete micromanipulation.

zona dissection, PZD), and 4) digesting a hole in the zona pellucida (zona drilling).

Cytoplasmic Injection The advantages are as follows: Any sperm type

(sperm head, immotile sperm, dead sperm, and grossly

202 IRITANI

TABLE 3. Microfertilization in Mice in Relation to Clinical Application for Human Infertility*

Sperm type Methods Results References Intact ZD (acid) Fertilized Gordon and Talansky, 1986 Intact, AR, PVI No fertilization Barg et al., 1987

Intact PVI Fert. rate (25%) Mann, 1988

Intact ZD Overcome immunological Conover and Gwatkin, 1988 block of penetration Ahmad et al., 1989

Intact PZD, ZD High fert. rate at low sperm conc. Depypere et al., 1988 Intact, AR PVI (single sperm) Fert. rate (30-40%), high cleavage rate Lacham et al., 1989 Intact, ZD Fert. rate; control (52%) Talansky and Gordon, 1988

Intact PZD Blast. rate; control (23%) Odawara and Lopata, 1989

Intact, AR PVI (single sperm) Pro-N (5%) Mettler et al., 1988 Sterile TG male, ZD (acid) Fert. rate (71%) Gordon, 1988

*ZD, zona-drilling; PZD, partial zona dissection; PVI, perivitelline injection; AR, acrosome reacted.

immobilized

Off spring (13%)

( 106/ml) ZD (78%)

(103-1o4/m1) PZD (51%)

IVF failure Offspring (29183; 35%)

defective sperm) can be injected, and both capacitation and the acrosome reaction appear to be unnecessary. Mechanical pricking of the oocytes and such reagents as polyvinylpyrolidone (PVP), when included in the injection medium, appear to be effective for oocyte activation (Markert, 1983; Iritani and Hosoi, 1989).

Perivitelline Injection This technique circumvents the process of sperm

penetration through the zona pellucida, so that sperm with extremely poor motility and even immotile sperm can be introduced. A short exposure of the oocytes to the hypertonic solution is recommended to minimize oocyte injury. The sperm to be injected should be capacitated and acrosome reacted. With this method selection of biologically normal sperm is achieved to some extent by the process of membrane fusion. However, polyspermic fertilization may occur when multiple sperm are injected.

Zona Drilling (ZD) A small hole is produced in the zona pellucida by

digestion with a stream of acid Tyrode’s or acid PBS solution (pH 2-3) flowing from the tip of the micropi- pet. Another method is to pierce the zona pellucida with a fine needle after prior softening with chymotrypsin. For this procedure the sperm must be capacitated, acrosome reacted and progressively motile. ZD can be utilized for oligospermia with weak sperm motility. The disadvantage is that this procedure cannot be applied to immotile spermatozoa. The oocytes are also injured to a certain extent.

Partial Zona Dissection (PZD) PZD has been also called zona opening or zona cut-

ting. The oocyte is held by a pipette and a portion of the

zona pellucida is cut with a fine glass needle or metal microblade. An advantage of this procedure over cytoplasmic injection is that one potential sperm selection barrier, the vitelline membrane, is present as in ZD. In this procedure the sperm must be capacitated and acrosome reacted for fusion with the vitelline membrane. A disadvantage of this method is that an abnormal embryo hatching process has been observed after embryo transfer (Talansky and Gordon, 1988; Malter and Cohen, 1989~). If at least some motility is present PZD may prove to be a useful approach for human infertility therapy.

MICROFERTILIZATION IN MICE AIMING AT CLINICAL APPLICATION FOR

HUMAN INFERTILITY A considerable number of research reports on micro-

fertilization using such methods as zona drilling (ZD), perivitelline injection (PVI), and partial zona dissection (PZD) have been conducted and have provided valuable information for clinical application of these techniques (Table 3).

Conover and Gwatkin (1988) showed that fertilization occurred in ZD oocytes even when penetration through the zona was blocked immunologically by a monoclonal antibody against zona pellucida sperm re- ceptor glycoprotein. Recently Ahmad et al. (1989) reported that ZD does not overcome the infertility of sperm from infertile tw5/tW7l males. However, sperm from T/tw5’w71 males, which carries only one t complex leading to a reduction in sperm transport and infertility by normal in vitro fertilization, were found to fertilize 32% of T/t mature oocytes when ZD was carried out with acid Tyrode’s solution, and 78% of the fertilized eggs cleaved to the 2-cell stage. Spermatozoa from t/t mice have very poor progressive motility between


TABLE 4. Quality Check of Human Spermatozoa Using Hamster Oocytes

Sperm type Methods Results References Frozen-thawed, Perivitelline injection Penetration rate; control (8%) Lassalle and Testart, 1988

Ionophore treated (2-12 sperm) frozen-thawed (51%) ionophore (28%)

sperm decondensed (90%) and pronucleated

Round head, acrosomeless Cytoplasmic injection Both control and abnormal Lanzendorf et al., 1988a,b

Sonicated, incubated Cytoplasmic injection Incubation in TEST-Yolk Martin et al., 1988 in TEST-Yolk provided more analyzable

sperm karyotypes

normal and Tlt spermatozoa. Failure to bind to the zona pellucida was not a cause of fertilization failure, since they observed that many sperm of both Tlt and tlt bound to oocytes, suggesting that tlt sperm may be sterile due to physiological or biochemical defects. Depypere et al. (1988) applied a ZD procedure in par- allel with zona dissection. The fertilization rate of zona-drilled mouse oocytes inseminated with 2 x lo4 spe rdml was much higher (73%) than with intact oocytes (5%). The polyspermic fertilization rate was only 7% in the ZD oocytes. Lacham et al. (1989) microinjected a single sperm under the zona pellucida. In this case, the fertilization rate was not correlated with the proportion of acrosome reacted spermatozoa (54 to 81%), which was determined after the capacitation treatment. In case of single sperm injection, there would be a possibility of injecting uncapacitated spermatozoon. It is recommended to inject more than two spermatozoa to increase fertilization rates, since it is difficult to select a single fully acrosome reacted spermatozoon. Odawara and Lopata (1989) found that opening the zona of oocytes did not influence the fertilization rate with lo5 sperdml , but when the sperm concentration was decreased to 104/ml, the zona- opened oocytes showed a significantly higher fertilization rate (63%) and blastocyst formation (51%) than intact controls, 38% and 23%, respectively, confirming the early reports of Gordon and Talansky (1986) and Conover and Gwatkin (1988). The polyspermy rate for zona-opened oocytes was 3%, and for the control oocytes was 5%. Mouse oocytes were zona-drilled and exposed to an extremely low concentration of sperm suspension from sterile transgenic mice (Gordon, 1988). As shown in Table 3, a high fertilization rate was obtained compared with intact oocytes and 29 of the 83 transferred embryos developed into young mice. There are few papers on micromanipulation in the rat, probably due to the vulnerability of oocytes and embryos to manipulation in this species. Vanderhyden et al. (1988) reported that ZD of oocytes by acid Tyrode’s solution increased the penetration rates of in vitro matured oocytes, but the formation of normal pronuclei was decreased.

QUALITY CHECK OF HUMAN SPERMATOZOA (TABLE 4)

Lassalle and Testart (1988) investigated the effects of various pretreatments of human sperm, such as freeze-thawing and calcium ionophore exposure, before injection into the perivitelline space of hamster oocytes. The penetration rates for each treatment (51% and 71%, respectively) were higher than with untreated sperm (8%). The highest penetration rate was obtained with calcium ionophore-treated or freeze- thawing, 57% and 71%, respectively. When only one sperm was injected the penetration rates for ionophore treated, frozen-thawed, and untreated sperm were 19%, 28%, and 0%, respectively. When 2-12 sperm were injected the rates rose to 74%, 50%, and 50%, respectively. The fertilization rate with 5-12 sperm was twice that with 2-4 sperm.

Some cases of severe teratozoospermia may exhibit round-headed (acrosomeless) spermatozoa, which cannot bind to and penetrate zona-free hamster oocytes. Lanzendorf et al. (1988) attempted to determine by microinjection if such abnormal sperm are capable of decondensation and pronuclear formation. The sperm of three such infertile men were injected into the cytoplasm of zona-intact hamster oocytes. As shown in Ta- ble 4, 90% of the injected abnormal spermatozoa were capable of decondensation or pronuclear formation, suggesting that if the inability to penetrate an oocyte is bypassed, the sperm from these infertile men may at least participate in the early events of fertilization.

Microfertilization will probably be applied mainly for male factor infertility, when genetically abnormal spermatozoa may often be expected. In such cases, chromosomal analysis of male gametes should be carried out before clinical use of sperm at least in cases of severe abnormalities. Martin et al. (1988) developed a technique for analyzing chromosomal abnormality. They reported that sperm incubation in TEST-Yolk buffer (Brandriff et al., 1985) before microinjection provided more analyzable karyotypes, with a significantly lower frequency of chromosomal abnormality than was the case with unincubated sperm.

204 IRITANI

TABLE 5. Clinical Application of Fertilization by Micromanipulation of Gametes

Sperm type Methods Results References 6 IVF failure group Cytoplasmic injection Pro-N rate; Lanzendorf et al., 1988 5 IVF pregnant group

Normal, capacitated; Perivitelline single sperm 42% W19) oocytes were Laws-King e t al., 1987

Male infertile Zona-drilling (acid Tyrode, Fert. rate; Gordon e t al., 1988

IVF failure group (0%) pregnant group (58%)

coiled or twin tailed injection fertilized

chymotrypsin) control (25%) drilled (32%)

IVF failed, Oligospermia Perivitelline injection Pregnancy obtained Ng et al., 1988 Ng et al., 1989

Malter et al., 1989

Zona-drilling (acid PBS)

Reinseminated to one day old

Low fert. rate (lo%),

45% (52/115) of PZD oocytes meiosis arrest

Normal, capacitated sperm PZD oocytes fertilized; monospermy

Eleven male factor couples PZD, Cleavage rate; cont. (33%) Malter and Cohen, 1989 rate (52%)

Zona-drilling (acid Tyrode) PZD (68%) Two twin (PZD) and one twin

(cont.) pregnancies

CLINICAL APPLICATION OF GAMETE MICROMANIPULATION

Microfertilization by Various Methods in Cases of Human Male Factor Infertility

Many clinical applications of microfertilization have been reported (Table 5). The fertilization rate of IVF in cases of male infertility is significantly lower than the fertilization rate of other IVF patient groups (Lanzen- dorf et al., 1988b). Microinjection of a spermatozoon directly into cytoplasm was applied to such male factor infertility patients, even those with extremely poor semen quality. Lanzendorf et al. found that oocytes from 5 of 11 patients microinjected with human spermatozoa demonstrated successful male pronuclear formation (58%). Laws-King et al. (1987) microinjected a single spermatozoon into the perivitelline space. Spermatozoa were capacitated by exposure to calcium-depleted medium containing strontium chloride for 20-24 h (Mor- timer et al., 1986). Eight of the nineteen (42%) oocytes manipulated were fertilized. Fertilization occurred with spermatozoa from donors with semen profiles in the normal fertile range and those with semen of low quality. Gordon et al. (1988) applied IVF by ZD to infertile couples, who had failed to achieve fertilization in prior conventional IVF. Zona drilling was by application of acid Tyrode’s solution (pH 2.3) from a microneedle and by mechanical drilling after exposure of oocytes to Ham’s F10 medium containing alpha-chymotrypsin (0.5 mg/ml). In all, 63 oocytes were recovered from ten couples and manipulated. The fertilization rate of the zona-drilled oocytes (32%) was higher than that of the nondrilled oocytes (25%)) although probably not significantly. Five of the ten eggs were diploid and transferred, although pregnancy was not obtained

Since successful fertilization has been achieved over

a wide range of intervals in oocyte collection and insemination (Edwards et al., 1984; Trounson et al., 19821, several studies were conducted on the reinsemination of unfertilized 1-day-old human oocytes, which were judged to have failed IVF. Malter et al. (1989) applied the procedure of PZD to oocytes that had failed IVF. The oocytes were shrunk by incubation in a sucrose solution and then reinseminated. There was a trend toward a reduced fertilization rate (from 60% to 40%) and an increase in polyspermy as the time between sucrose exposure and reinsemination in sucrose- free medium increased. The incidence of polyspermy was higher with oocytes manipulated more than 25 h after recovery than with those manipulated 21-24 h after recovery, supporting the idea that the ability to block polyspermy is reduced in aged oocytes.

The procedures of PZD and ZD have been compared (Malter and Cohen, 1989a). The proportions of monospermic fertilization for PZD, ZD, and control oocytes were 26%, 17%, and 3%, respectively. Both the fertilization rate (79%) and the cleavage rate (68%) of PZD oocytes were almost twice those of the controls (53% and 33%). Transfer of two PZD and a single control embryos into two patients resulted in twin prenancies (Cohen et al., 1988). A third twin pregnancy was established after transfer of only micromanipulated embryos. Recently a patient delivered healthy dizygotic twins. In addition to the above mentioned three preg nancies, three more were subsequently established (Malter and Cohen, 1989a).

Developmental Abnormality of the Micromanipulated Embryos

Investigations of the developmental normality of embryos derived from micromanipulated oocytes, espe-


cially a t the stages of blastocyst expansion and hatching, are important for predicting pregnancy outcome. A few studies of the hatching process of embryos derived from micromanipulated zonae have been reported. Hatching of mouse blastocysts with manipulated zonae pellucidae usually occurred a day earlier when compared with that of control oocytes (Malter and Cohen, 1989~). Talansky and Gordon (1988) reported that some of the micromanipulated embryos lost their zonae entirely. In others the blastomeres were extruded a t earlier cleavage stages leading to blastomere separa- tion, aggregation of cleavage embryos to form giant embryos and formation of split blastocysts. The char- acteristic patterns found during cleavage suggested that although ZD provides a possibility of increasing the number of live births, some developmental abnormalities may result, leading to embryo loss, spontane- ous chimerism, and embryo splitting to produce monozygotic twins. Odawara and Lopata (1989) also reported that zona-opening promotes early hatching of blastocysts in mice, but that there is no significant dif- ference in birth rates between blastocysts with intact zonae (42%) and those with opened zonae (36%). Malter and Cohen (1989~) investigated abnormalities of human embryos derived from manipulated oocytes. Fifty percent of l-day-old human oocytes were fertilized following PZD and re-insemination. Forty-eight percent were monospermic, of which 87% (13/15) cleaved, six compacted, and four cavitated. Three of four blastocysts extruded through the PZD incision. The zona pellucidae did not thin and one blastocyst was bisected spontaneously as it was caught between the thick ridges of the PZD hole. These results indicate that the hatching process of embryos derived from the manipulated oocytes may be abnormal. Mouse embryos start hatching earlier. Extrusion occurs earlier and embryos may be trapped by the thick edge of the opened zona and bisected randomly. This may lead to artificial twinning or the formation of trophoblastic vesicles (Malter and Cohen, 1989~).

Microsurgical Repair of Polypronuclear Human Embryos

Assisted fertilization, subzonal insertion of multiple sperm, ZD, or PZD are all associated with the problem of polyspermic fertilization, although the polyspermic rate is generally reduced when manipulated oocytes are exposed to low concentrations of poor quality spermatozoa. Two recent papers have reported the microsurgical repair of polyspermic human embryos (Malter and Cohen, 1989b; Gordon et al., 1989). Correction of polyspermy through pronucleus extraction has been investigated in human pronuclear eggs fertilized by conventional IVF and ZD. Eighteen polyspermic zygotes from zona intact oocytes and seven from previously zona-drilled oocytes were obtained. These zygotes, which originally contained three to five pronuclei, survived and cleaved after removal of the excess pronuclei.

However, both survival and cleavage rates were higher from intact oocytes than from PZD oocytes. Criteria used for identification of male pronuclei were 1) pronucleus closely associated with the sperm tail 2) a comparatively greater pronuclear size, and 3) greater distance from the second polar body. Six of the eleven zygotes were incubated in cytochalasin-D for 30 minutes before manipulation, and five were manipulated without preincubation in cytoskeletal relaxing agents. When cytoskeletal relaxants were used, all six embryos survived after enucleation and cleaved (Gor- don et al., 1989). These results indicated that it is possible to microsurgically “epronuleate” from tripronuclear human zygotes and obtain further development. This technique could be effectively used for repair of polyspermic eggs derived from microfertilization and ordinary IVF. However, viable embryos were not obtained after correction of polyspermy using microsurgical extraction of tripronuclear human zygotes (Rawlins et al., 1988).

Possibility of Using Immature Prophase-I Oocytes Collected at a Conventional

IVF Program In the Norfolk human IVF-ET program immature

oocytes make-up approximately one fourth of all oocytes collected (Veeck, 1988). Despite nuclear maturation in vitro, fertilization and cleavage, those embryos derived from a prophase-I (P-I) oocyte rarely con- tribute to a viable pregnancy, suggesting that some cytoplasmic factors required for early cleavage events and subsequent embryonic development are formed during in vivo maturation (Sorensen et al., 1985; Kish- imoto, 1986). Transfusion of a small amount of ooplasm of in vivo matured oocytes into immature P-I oocytes appears to compensate for this deficiency (Flood et al., 1990). Thus transfused monkey oocytes, matured and transferred to the fallopian tube for fertilization, resulted in a pregnancy rate that was comparable with that of in vivo matured M-I1 oocytes. Such ooplasmic transfusion is a good example of the practical application of micromanipulation that may be clinically useful in a human IVF-ET program.

For efficient clinical application of micromanipulation a satisfactory animal model of assisted fertilization with naturally occurring, or artificially induced, abnormal spermatozoa should be established. Species differences in the susceptibility of oocytes to micromanipulation and ease of sperm replacement have been found. Therefore, selection of animal species having gametes that are comparatively easy to manipulate and similar to human oocytes in their characteristics would be highly desirable. Furthermore, minimizing gamete damage during the process of micromanipulation and in vitro development and technical improve- ment for reducing polyspermy are important aspects for future research.

206 IRITANI

ACKNOWLEDGMENTS The author is grateful to Dr. R. B. L. Gwatkin for his

reading and commenting on this manuscript.

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