Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca 2 + fluxes and oocyte cytoplasmic dislocation*

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1 r FERTILITY AND STERILITY Copyright ~ 1995 American Society for Reproductive Medicine Printed on acid free paper in U. S. A. Key elements of a highly efficient intracytoplasmic sperm injection technique: Ca 2 + fluxes and oocyte cytoplasmic dislocation* Jan Tesarik, M.D., Ph.D.t Mario Sousa, M.D., Ph.D.* American Hospital of Paris, Neuilly sur Seine, and Institut National de la Sante et de la Recherche Medicale (lnserm), Clamart, France Objective: To analyze the mechanism by which modifications of the intracytoplasmic sperm injection (ICSI) technique influence success rates. Design: Prospective clinical study supplemented with an experimental analysis ofca 2 + fluxes provoked by the injection procedure. Setting: Private hospital and public research center. Patients: Patients treated by IVF and ICSI. Interventions: Intracytoplasmic sperm injection. Main Outcome Measures: Fertilization and pregnancy rates and intracellular free Ca 2 + concentration. Results: The inclusion of vigorous aspiration of oocyte cytoplasm improved outcomes oficsi. In a series of 100 consecutive cases treated with this technique, the fertilization and pregnancy rates were 87% of total metaphase II oocytes injected and 52% of total treatment cycles, respectively. Enhanced Ca 2 + influx into the injected oocytes and dislocation of the oocyte cytoplasm, including the development of a focus of persistent Ca 2 + discharge around the injected sperm head, were the main characteristics of this highly successful technique. Conclusions: Vigorous aspiration of oocyte cytoplasm may facilitate fertilization after ICSI by increasing the oocyte Ca 2 + load at the time of injection, by establishing a more intimate contact of the injected sperm head with oocyte intracellular Ca 2 + stores, or by a conjunction of these mechanisms. Fertil Steril 1995;64:770-6 Key Words: Intracytoplasmic sperm injection, fertilization, oocyte activation, Ca 2 + fluxes, confocal microscopy Received November 9, 1994; revised and accepted April 24, * Supported in part by grant 7173 from the Junta de Andalucia, Sevilla, Spain (to J.T.) and by a grant from the Calouste Gulbenkian Foundation, Porto, Portugal (to M.S.). Support for work with the confocal microscopy system was also obtained from the Institut Paris-Sud sur les Cytokines, Paris, France. t Reprint requests: Jan Tesarik, M.D., Ph.D., Center for Reproductive Biology and Medicine, American Hospital of Paris, 63 Boulevard Victor Hugo, Neuilly sur Seine, France (FAX: ). * On postdoctoral fellowship from Laboratory of Cell Biology, Institute of Biomedical Sciences, University ofoporto, Porto, Portugal. The technique of microsurgical sperm transfer into the oocyte cytoplasm (intracytoplasmic sperm injection [les!]) has been developed in the animal model in the mid-70s (1) and recently has been applied with success in humans (2). According to latest evaluations (3, 4), lesl is the most efficient method of microsurgically assisted fertilization actually available to be used with ejaculated, epididymal, or testicular sperm. Nonetheless, success rates reported from different centers are somewhat inconsistent, mainly because of differences in the fertilization rate. In our laboratory, we used the human aged oocyte model (5) to evaluate the impact of different modifications of the injection technique on fertilization rates after lesl (Tesarik J, unpublished data). These experiments showed that the percentage of fertilization can be doubled when the sperm injection is preceded by vigorous aspiration of part of oocyte cytoplasm into the microinjection needle as compared with the technique using simple injection or involving only gentle cytoplasmic aspiration. In search for the mechanism of this beneficial effect, 770 Tesarik and Sousa Intracytoplasmic sperm injection Fertility and Sterility

2 several possibilities have been envisaged. First of all, we believed that the enhanced aspiration of oocyte cytoplasm might decrease the risk of later expulsion ofthe injected spermatozoon from the oocyte. However, other data have shown that a failure of oocyte activation, rather than sperm expulsion, is the main cause of fertilization failure after ICSI when the vigorous aspiration step is not included (6, 7). Hence, the effect of oocyte cytoplasmic aspiration appears to be somehow related to events responsible for oocyte activation. It is well known that in humans, as in other mammalian species, Ca 2 + is the principal second messenger implied in the triggering of oocyte activation, both after IVF (8) and after ICSI (9). Changes in the intracellular free Ca 2 + concentration ([Ca 2 +]i) that occur in human oocytes as an immediate consequence of the ICSI procedure also were addressed (9). Moreover, spatial analysis ofca 2 + signals generated in human oocytes in response to fertilization (10) have suggested that different regions of the oocyte cytoplasm are populated by different types of intracellular Ca 2 + stores so that the transmission of the oocyte activating signal from the injected spermatozoon to corresponding effectors may be influenced by the type and actual physiological state of the oocyte Ca 2 + stores in the immediate vicinity of the injected spermatozoon. Obviously, all of these potential factors can be influenced by the oocyte cytoplasmic aspiration. This study was undertaken to verify whether an improvement of fertilization scores, similar to that seen with the aged oocyte model, also can be obtained by vigorous cytoplasmic aspiration in fresh oocytes, to examine the effect of the aspiration on Ca 2 + fluxes accompanying the injection procedure, and to evaluate the potential consequences of the oocyte cytoplasmic dislocation, resulting from the aspiration, for the local cytoplasmic microenvironment at the site of the sperm head deposition. Clinical Part MATERIALS AND METHODS In the clinical part of this study, patients attending our assisted reproduction program were assigned randomly to one of two groups. In the first group, ICSI was carried out with the use of a previously described technique (7, 9-11). Briefly, this technique was characterized by injection of a single spermatozoon together with the smallest amount possible of a solution consisting of 10% polyvinylpyrrolidone in Sperm Preparation Medium (Medicult, Copenhagen, Denmark). Because the penetration of the microinjection needle into the oocyte usually produced a deep invagination of the oocyte plasma membrane, a minimal amount of the oocyte cytoplasm was aspirated into the needle tip just to make sure that the subsequent injection actually would result in sperm deposition in the oocyte cytoplasm and not merely in the extended portion of the perivitelline space. During this aspiration, the backward movement of the sperm head within the microinjection needle was minimal and did not exceed 10 p,m. Given the needle internal diameter of 6 p,m, the calculated maximum volume of aspirated cytoplasm was 283 p,m 3 We use the term gentle aspiration technique to refer to this procedure. In the second group, oocytes were injected with the use of a modified technique, which is referred to as vigorous aspiration technique throughout this study. Unlike the gentle aspiration technique, this technique was characterized by a vigorous cytoplasmic aspiration preceding the sperm injection. The aspiration was started as soon as the tip of the microinjection needle had been pushed through the oocyte and approached as closely as possible the oocyte plasma membrane opposite to the puncture site. The volume of the aspirated cytoplasm was controlled by following the backward movement of the sperm head within the needle, and the aspiration was continued until the head retrograded for?: 100 p,m from its original position at the needle tip. Hence the calculated minimum volume of aspirated cytoplasm was 2,826 p,m 3, which is 10 times the maximum volume aspirated with the gentle technique. The aspirated cytoplasm then was reinjected into the oocyte together with the spermatozoon. With both the gentle aspiration technique and the vigorous aspiration technique, freshly ejaculated spermatozoa were treated with 1 mg/ml pentoxifylline (Torental; Hoechst, Puteaux, France) during the last 10 minutes ofthe semen liquefaction period followed by filtration through a discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient and washing in medium as described (12). The inclusion of the pentoxifylline preincubation step facilitated the isolation of motile spermatozoa for injection in cases of extremely severe oligoasthenozoospermia. All other methods used, including patient selection, ovarian stimulation, gamete handling, and preparation of micromanipulation instruments, were those described previously (7, 9-11). Only metaphase II oocytes were injected. Experimental Part Oocytes used in the experimental part of this study came from our IVF program and were those Tesarik and Sousa Intracytoplasmic sperm injection 771

3 Table 1 Clinical Comparison of Two Different Techniques of ICSI Oocytes Technique Patients Injected Damaged 1 pronucleus Gentle Vigorous * Only fresh embryos were transferred. t Both fresh (n = 36) and cryopreserved (n = 29) embryos were transferred. Zygotes Embryos Transfer Clinical 2 pronuclei 3 pronuclei transferred procedures pregnancies ' 15* t 20t 6* * Five pregnancies were obtained after transfer of fresh embryos, and one pregnancy resulted from transfer of cryopreserved embryos. that failed to fertilize by 44 hours after in vitro insemination. The previously described criteria for oocyte allocation to experimental studies (9-11) were applied. Spermatozoa were obtained from healthy volunteers with normal sperm count, concentration, motility, and morphology (13). Before ICSI, oocytes were loaded with the fluorescent Ca 2 + indicator fluo-3 (Molecular Probes, Eugene, OR) as described (9) except for the augmentation of the dye concentration to 10 j.tm. The oocytes then were placed in microdrops of Sperm Preparation Medium previously equilibrated with 5% CO2 in air and covered with embryo-tested light mineral oil (Sigma, St. Quentin Fallavier, France) contained in Falcon 3001 plastic culture dishes (Becton Dickinson, Plymouth, United Kingdom). The dishes were maintained on a heated stage (37 C) of a Nikon (Tokyo, Japan) Diaphot inverted research microscope during all subsequent manipulations. The microscope was equipped with two Narishige (Tokyo, Japan) micromanipulators, MN-151 mechanical joystick manipulator controlling the oocyte holding pipette and MO-202 hydraulic remote control joystick manipulator controlling the microinjection needle, and with two mechanical injectors, IM-5NB connected with the oocyte holding pipette and IM-6 connected with the microinjection needle. The whole micromanipulation unit was incorporated in a Bio-Rad (Richmond, CA) MRC 600 laser scanning confocal microscopy system. Indicator-loaded oocytes were subjected to ICSI with the use of either the gentle aspiration (n = 8) or the vigorous aspiration (n = 12) technique as described in Clinical Part. During the manipulation, confocal images of the fluorescence emitted by the Ca 2 + indicator were recorded from the equatorial plane of the oocytes at 2-second intervals and saved in a Panasonic 1 Gbyte rewritable magneto-optical disk (Panasonic, Osaka, Japan). When the procedure of ICSI was finished, image sampling was continued for an additional 10 minutes. The images were analyzed ex posteriori with the use of a Bio-Rad Time Course Ratiometric Software Measure program. Further details about these techniques were pub- 772 Tesarik and Sousa Intracytoplasmic sperm injection lished earlier (9-11). Success rates obtained with the two ICSI techniques used in the clinical part of this study were compared by X 2 test. Clinical Part RESULTS When success rates oficsi obtained with the gentle aspiration technique and with the vigorous aspiration technique were compared in a first limited series of cases (Table 1), the superiority of the latter protocol was so evident that we felt it unnecessary to compare both techniques on a larger group of patients. In fact, fertilization rates were significantly higher (P < 0.01) with the vigorous aspiration technique (80%) as compared with the gentle aspiration technique (38%), whereas both the ability of twopronucleated zygotes to cleave and that of cleaved embryos to implant was similar for each ofthe techniques (Table 1). Oocyte damage was not a major concern with any of the two techniques, as the percentage of oocytes damaged by the injection procedure did not exceed 4% irrespective of the technique used, and the incidence of abnormal fertilization (one-pronucleated and three-pronucleated zygotes) also was similar in both groups (Table 1). In view of these comparisons, we discontinued the clinical use of the gentle aspiration technique and began to use the vigorous aspiration technique for all patients. Global results of the first 100 treatment cycles after this decision, all performed with the vigorous aspiration technique, are shown in Table 2. From these data it is evident that the improved fertilization and pregnancy rates, obtained with this technique in the first small series of cases (Table 1), can be maintained with larger groups of patients. In fact, both the fertilization rate (87% of total oocytes injected including the damaged ones) and the pregnancy rate (52% of started treatment cycles) were even superior in this second series as compared with the first one, probably because of increased experience with the vigorous aspiration technique. Fertility and Sterility

4 Table 2 Results of 100 Consecutive Treatment Cycles Using the Vigorous Aspiration Technique of ICSI* Cycles Total oocytes injected Oocytes damaged by injection Two-pronucleated zygotes One-pronucleated zygotes Three-pronucleated zygotes Total cleaved embryos Embryos transferred to date (fresh and frozen) Clinical pregnancies 100 (100.0) 644 (100.0) 24 (3.7)t 558 (86.6}t 14 (2.2}t 12 (1.9}t 498 (77.3}t (52.0)* * Values in parentheses are percentages. t These percentages were calculated from the total number of injected oocytes (second line of this table). * This percentage was calculated from the total number of started cycles (first line of this table). Experimental Part When Ca 2 + fluxes produced by each of the two techniques oficsi were compared, the gentle aspiration technique was found to be accompanied by a single major increase in [Ca 2 +]i, occurring at the time of the penetration of the needle tip into the oocyte, and one minor increase at the time of sperm expulsion, exactly the same phenomena as described in a previous study (9). Thus, most of further data concern the vigorous aspiration technique of injection. With this modified technique, the procedure of ICSI produced three major increases and one minor increase in [Ca 2 +]i (Fig. 1). The first increase coincided with the penetration of the oocyte plasma membrane by the needle tip and was analogical to that observed with the gentle aspiration technique. However, the second and the third increases were unique to this modified technique. The second increase was produced during the aspiration of oocyte cytoplasm into the needle, and the third occurred while the needle was being retracted from the oocyte. A minor [Ca 2 +]i increase accompanied the expulsion of the aspirated cytoplasm together with the spermatozoon back to the oocyte (Fig. 1). Spatial analysis of these Ca 2 + fluxes, based on an examination of sequential confocal fluorescence images, was performed to explain how the vigorous aspiration of oocyte cytoplasm into the microinjection needle could increase [Ca 2 +]i in the injected oocytes. A key to understanding the mechanism of this particular phenomenon was provided by following the changes in oocyte deformation that occurred during the aspiration. Just before the aspiration, the oocyte plasma membrane was invaginated slightly at the puncture site, and the position of the needle within the oocyte was seen clearly as a zone oflower [Ca 2 +]i as compared with the adjacent cytoplasm (Fig. 2A). As the aspiration began, the invagination of the oocyte plasma membrane became noticeably deeper and, at the end of the aspiration, it extended for more than three quarter oocyte diameter and approached the tip of the microinjection needle positioned near the opposite pole of the oocyte. Concomitantly with this morphological change, a strong focal augmentation of [Ca 2 +]i appeared around the needle tip (Fig. 2B). As the aspiration continued, most of this Ca 2 + -rich cytoplasm entered the microinjection needle (Fig. 2C) to be reinjected subsequently into the oocyte together with the spermatozoon (Fig. 2D). The retraction of the microinjection needle from the oocyte produced a focal increase in [Ca 2 +]i around the needle tip, quite similar to that provoked by the cytoplasmic aspiration (data not shown). However, this [Ca 2 +]i increase had a greater tendency to spread from this focus throughout the oocyte cytoplasm; a generalized Ca 2 + wave developed in 6 of 12 oocytes injected with the use of the vigorous aspiration technique. It has to be noted that no significant [Ca 2 +]i increase was observed at the time of needle retraction in any of the eight oocytes in which ICSI was performed with the use of the gentle aspiration technique, thus confirming our previous observations (9). At the end of the ICSI procedure, the invagination of the oocyte plasma membrane created by the microinjection needle persisted for several minutes, but it always disappeared by 10 minutes after the intervention. When ICSI was performed with the gentle aspiration technique, no focal or regional increase in [Ca 2 +]i was observed during this period. On the other hand, the use of the vigorous aspiration technique resulted in the persistence of a focus of increased [Ca 2 +]i at the blunt end of the puncture canal in all g ~ o ::J u::: <'? g u::: 100 Ol------~ ~ o 2 3 Time (min) Figure 1 Changes in [Ca 2 +]i occurring in a human oocyte during the ICSI involving the vigorous cytoplasmic aspiration step as revealed by measuring changes in fluo-3 fluorescence intensity (expressed as percentage of maximal pixel intensity value). The points of the record corresponding to the penetration of the microinjection needle tip through the oocyte plasma membrane (puncture), to the beginning of cytoplasmic aspiration, to the beginning of expulsion from the needle (injection), and to the beginning of the needle retraction are shown. This is a typical response representative of those obtained in 11 other oocytes. Tesarik and Sousa Intracytoplasmic sperm injection 773

5 Figure 2 Confocal fluorescence images showing the spatial characteristics of major Ca 2 + fluxes in a fluo3-loaded oocyte during and after the ICSI procedure involving the vigorous cytoplasmic aspiration step. The first four images correspond to the times just before the cytoplasmic aspiration (A ), at the beginning of the aspiration (B ), at the end of the aspiration (C ), and in a final phase of the injection (D ). The last image shows the same oocyte 10 minutes after the retraction of the microinjection needle from the ooplasm (E ). Relative changes in [Ca 2 +]i (revealed by differences in fluo-3 fluorescence intensity) are represented in pseudocolors according to the scale bar where the lowest values are coded black and the highest red. The spot of persisting Ca2 + discharge visualized in E corresponds to the site at which the sperm h ead was deposited. This record is representative of those obtained with 11 other oocytes. oocytes. Even though this focus weakened progressively with time, it still was present 10 minutes after ICSI when the puncture canal was no more visible and the plasma membrane invagination, produced by oocyte puncture and subsequent cytoplasmic aspiration, had disappeared totally (Fig. 2E). DISCUSSION Even though the impact of vigorous aspiration of oocyte cytoplasm as part of the ICSI technique on success rates of this method was examined prospectively in a relatively small group of patients, the benefits of this technique modification were quite evident. In fact, these benefits were given by significantly increased fertilization rates that were doubled from approximately 40% to > 80%. Because the chance of a fertilized egg to cleave and that of a cleaved embryo to give a pregnancy appeared to be independent of the injection technique used, the increase in the per patient pregnancy rate was fully attributable to the improvement of fertilization. With respect to possible patient-to-patient variations, it would have been preferable to use sibling oocytes in this clinical evaluation if only fertilization and cleavage rates were to be compared. However, this would make the comparison of pregnancy rates more difficult because embryos resulting from each 774 Tesarik and Sousa Intracytoplasmic sperm injection of the two injection techniques would have to be transferred separately. Anyway, the high clinical efficacy of the vigorous aspiration technique was corroborated by continuing high success rates obtained in the first 100 cases treated by ICSI after the definitive abandon of the gentle aspiration technique and acceptance of the vigorous aspiration technique as unique protocol. Why do oocytes fertilize better when the microsurgical deposition of sperm in their cytoplasm is preceded by vigorous cytoplasmic aspiration? In theory, there are several possible mechanisms that may be at the cause. First of all, the injection of a spermatozoon into the oocyte without previous aspiration can be expected to lead to a slight increase in the cytoplasmic volume, which may facilitate later expulsion of the injected spermatozoon back through the puncture canal out of the oocyte. However, the gentle cytoplasmic aspiration, as performed in our laboratory, apparently is sufficient to cope with this problem because our recent ultrastructural study has shown that the main cause of fertilization failure after ICSI without vigorous aspiration is a failure of oocyte activation rather than sperm expulsion (6). In that study, a total of 15 oocytes that failed to fertilize by 44 hours after ICSI were sectioned serially from pole to pole, and nondecondensed or partly decondensed sperm chromatin was identified by Fertility and Sterility

6 electron microscopy in the cytoplasm of each of them (6). Moreover, most ofthe oocytes that failed to fertilize after ICSI were capable of developing two pronuclei after subsequent treatment with a Ca 2 + ionophore, although the percentage of one-pronucleated eggs (9%) was relatively high (7). Together, these data show that the expulsion ofthe injected spermatozoon from the oocyte may occur in some cases but is not a predominant cause of fertilization failure after ICSI, at least in our hands. Thus, the beneficial effect of the oocyte cytoplasmic aspiration on fertilization has to be looked for elsewhere, probably in examining the relationship between the aspiration and the oocyte activation process. We have shown previously that, when performed without vigorous aspiration of oocyte cytoplasm, the ICSI procedure by itself does not activate the oocyte (9). From another~eriesof experiments (Tesarik J, unpublished data), it was concluded that the vigorous aspiration technique of ICSI does not activate either. However, changes in the intracellular concentration of free Ca 2 + ions, the key regulatory element of oocyte activation (14-16), accompanying ICSI were influenced profoundly by the technique modification. So, in the absence of vigorous aspiration, there is only one major increase in [Ca 2 +]i during the ICSI procedure; this increase coincides with the penetration of the microinjection needle into the oocyte and is a sequela of an influx of Ca 2 + ions from the oocyte bathing medium rather that from the medium present inside the needle (9). When the vigorous cytoplasmic aspiration step is added, further major [Ca 2 +]i increases accompany the aspiration and the retraction of the needle from the oocyte. How to explain these additional [Ca 2 +]i increases? The most probable explanation is a purely hydrodynamic one. The vigorous aspiration of the oocyte cytoplasm produces a negative pressure within the oocyte (also evidenced by concomitant deformation of the puncture canal) leading to aspiration of the external Ca 2 + -rich medium through the most leaky zone that is actually that of the apposition of the oocyte plasma membrane to the outer surface of the inserted microinjection needle. Because of the deep invagination of the oocyte plasma membrane along the puncture canal, the resulting [Ca 2 +]i increase is first detectable near the needle tip and most of the influxed Ca 2 + is aspirated immediately into the needle. Mter subsequent reinjection into the oocyte, this Ca 2 + is resorbed by the oocyte's Ca 2 + stores, but the retraction of the microinjection needle makes the contact between the needle and the oocyte plasma membrane leaky again and entails a new Ca 2 + influx. With the gentle aspiration technique, the needle retraction fails to produce a similar influx, possi- bly because ofthe persisting slight positive pressure in the interior ofthe oocyte, caused by a larger excess of the injected fluid volume as compared with the volume of aspirated cytoplasm. As a corollary, the inclusion of vigorous aspiration of oocyte cytoplasm in the ICSI technique makes the oocyte undergo a substantially higher Ca 2 + load as compared with the gentle aspiration technique. This increased Ca 2 + load may support the action of a sperm cytosolic factor supposed to trigger oocyte activation after ICSI (14, 15). Interestingly, when human oocytes are treated by a Ca 2 + ionophore subsequent to ICSI, the time required for the injected spermatozoon to trigger [Ca 2 +]i oscillations typical of the normal oocyte activation process is reduced significantly (17). Based on these data, it has been suggested that the primary [Ca 2 +]i increase produced by the ionophoreactivates a latent process in. the oocyte, rendering it more responsive to the sperm factor (17). This increased responsiveness may be related to an increased loading of the oocyte Ca 2 + stores because the propensity of Ca 2 + stores to discharge the stored Ca 2 + is known to increase proportionally with their Ca 2 + load (18-20). It also is possible that the ionophore-induced [Ca 2 +]i increase supports the action of the sperm factor by activating phospholipase C, leading to increased production of inositol 1,4,5-trisphosphate with subsequent effects of this agonist on inositol trisphosphate-sensitive Ca 2 + stores (17). Though originally suggested to explain the action ofionophore, both of these hypothetical mechanisms also can be implicated in the beneficial effect of oocyte cytoplasmic aspiration before sperm deposition because both the ionophore and the aspiration produce similar increases in [Ca 2 +]i. Although the hypothesis that vigorous aspiration of oocyte cytoplasm improves fertilization rates after ICSI by increasing the Ca 2 + load of oocytes at the time of injection is substantiated by previous experimental data, the beneficial effect of the aspiration may be m.ore complex and may be related at least in part to the resulting dislocation of the oocyte cytoplasm. The aspiration obviously makes the contact between the injected sperm head and the oocyte cytoplasm more intimate. Moreover, the aspirated cytoplasm becomes highly enriched in Ca 2 +. Mter reinjection, this modified cytoplasm persists as a focus of increased [Ca 2 +]i in which the sperm head is deposited. Last, but not least, it appears that most of the aspirated cytoplasm comes from a region close to the oocyte cortex where oocyte activation normally is triggered and where the responsiveness of local Ca 2 + stores is different from the rest of the oocyte cytoplasm (10). By virtue ofthe aspiration, it is thus possible to place the injected spermatozoon in the Tesarik and Sousa Intracytoplasmic sperm injection 775

7 , cortical-type cytoplasm, although its actual deposition site may be near the center of the oocyte. In conclusion, the first part of this study has demonstrated the crucial importance of vigorous aspiration of oocyte cytoplasm for success rates of ICSI in a clinical assisted reproduction program. It is clear that the nature of this move is difficult to explain by merely descriptive means because individual oocytes respond differently in terms of resistance and deformation. It seems that adapting the ICSI technique for every individual oocyte is an important element of the technical skill required for high success rates. The second part of this study has pointed out several differences, related to the oocyte Ca 2 + metabolism, between this technique and another one that uses only gentle cytoplasmic aspiration. This experimental part was performed with the aged oocyte model whose possibilities and limitations have been discussed earlier (9-11). Further research will show which of these differences is decisive for the higher fertilization rates obtained with the vigorous aspiration technique. Acknowledgments. We thank all members of the assisted reproduction team of the American Hospital of Paris who were involved in the clinical application of these findings. They were Jacques Testart, Ph.D., Ms. Beatrice Berdrin, Ms. Catherine Carre, Ms. Marie Ouvrard, and Ms. Benedicte Rouby for the laboratory part, and Alain Thebault, M.D., Charles Brami, M.D., Jean-Marc Emmanuelli, M.D., Alain Gauthier, M.D., Gerard Heluin, M.D., Fran~ois Rolet, M.D., Eric Sedbon, M.D., Jean Thorel, M.D., and Charles Tibi, M.D. for the clinical part. REFERENCES 1. Uehara T, Yanagimachi R. Microsurgical injection of sperm a tozoa into hamster eggs with subsequent transformation of sperm nuclei into male pronuclei. BioI Reprod 1976; 15: Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of a single spermatozoon into an oocyte. Lancet 1992;340: Van Steirteghem A, Devroey P, Joris H, Nagy P, Liu J, Camus M, et al. Criteria of patient selection for micromanipulation assisted conception. In: Tesarik J, editor. Male factor in human infertility. Rome: Ares-Serono Symposia, 1994: Silber SJ. The use of epididymal sperm in assisted reproduc- tion. In: Tesarik J, editor. Male factor in human infertility. Rome: Ares-Serono Symposia, 1994: Tesarik J. Subzonal sperm insertion with aged human 00- cytes from failed IVF attempts: fertilization results and some applications. Hum Reprod 1993;8: Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum Reprod 1994; 9: Tesarik J, Sousa M. More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil Steril 1995;63: Taylor CT, Lawrence YM, Kingsland CR, Biljan MM, Cuthbertson KSR. Oscillations in intracellular free calcium induced by spermatozoa in human oocytes at fertilization. Hum Reprod 1993;8: Tesarik J, Sousa M, Testart J. Human oocyte activation after intracytoplasmic sperm injection. Hum Reprod 1994;9: Tesarik J, Sousa M, Mendoza C. Sperm-induced calcium oscillations of human oocytes show distinct features in oocyte center and periphery. Mol Reprod Dev. In press. 11. Tesarik J, Sousa M. Comparison of Ca 2 + responses in human oocytes fertilized by subzonal insemination and by intracytoplasmic sperm injection. Fertil SteriI1994;62: Tesarik J, Mendoza C. Sperm treatment with pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency. Fertil Steril 1993;60: World Health Organization. WHO Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 3rd ed. Cambridge: The Press Syndicate of the University of Cambridge, 1993: Tesarik J. How the spermatozoon awakens the oocyte: lessons from intracytoplasmic sperm injection. Hum Reprod 1994; 9: Swann K, Homa S, Carroll J. An inside job: the results of injecting whole sperm into eggs supports one view of signal transduction at fertilization. Hum Reprod 1994; 9: Taylor CT. Calcium signals and human oocyte activation: implications for assisted conception. Hum Reprod 1994; 9: Tesarik J, Testart J. Treatment of sperm-injected human 00- cytes with Ca 2 + ionophore supports the development of Ca 2 + oscillations. BioI Reprod 1994;51: Missiaen L, De Smedt H, Droogmans G, Casteels R. Ca 2 + release induced by inositol 1,4,5-trisphosphate is a steadystate phenomenon controlled by luminal Ca 2 + in permeabilized cells. Nature 1992;357: Oldershaw KA, Taylor CWo Luminal Ca 2 + increases the affinity ofinositol 1,4,5-trisphosphate for its receptor. Biochem J 1993;292: Camacho P, Lechleiter JD. Increased frequency of calcium waves in Xenopus laevis oocytes that express a calcium-at Pase. Science 1993;260: Tesarik and Sousa Intracytoplasmic sperm injection Fertility and Sterility