Intracytoplasmic sperm injection (ICSI) for severe semen abnormalities: dissecting the tail of spermatozoa at the tip

Human Reproduction vol 11 no. 12 pp 2640-2644, 1996 Intracytoplasmic sperm injection (ICSI) for severe semen abnormalities: dissecting the tail of spermatozoa at the tip Shee-Uan Chen, Hong-Nerng Ho, Hsin-Fu Chen, Su-Cheng Huang, Tzu-Yao Lee and Yu-Shih Yang 1 Department of Obstetrics and Gynaecology, College of Medicine and the Hospital, National Taiwan University, Taipei, Taiwan 'To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan Recently, several investigators have emphasized that damaging the membrane of spermatozoa by compressing the mid-piece or cutting the mid-portion of the tail prior to injection yields better results than using motile spermatozoa in intracytoplasmic sperm injection (ICSI). Here we report our experience using a modified immobilization technique of dissecting the tail of the spermatozoon at the tip in 78 cycles on 60 patients. In 55 treatment cycles purely using this modified technique, 468 mature oocytes were injected. A total of 35 oocytes (7.5%) were injured. Of the intact oocytes, 282 (65.1%) were normally fertilized and 266 (94-3%) subsequently cleaved. A single pronucleus was observed in 16 (3.7%) oocytes, and three pronuclei were noted in 11 (2.5%) oocytes. Embryo transfers were performed in 54 cycles, and 18 women (32.7%) achieved clinical pregnancies. In 23 cycles, we compared the effects of these three immobilization techniques on the sibling oocytes obtained from the same patient regarding normal fertilization, abnormal fertilization, and embryo cleavage and quality. The results were comparable among them. Seven pregnancies (30.4%) were achieved in this series. Dissecting a sperm tail at the tip is easily and quickly performed and achieves permanent immobilization. Compression of the mid-piece is also easy, but usually takes several actions to achieve immobilization. Cutting the tail at the mid-portion requires more skill. Therefore, dissecting the tail of the spermatozoon at the tip may provide an alternative method to immobilize the spermatozoon permanently prior to ICSI. Key words: immobilization of sperm/intracytoplasmic sperm injection/severe male-factor infertility Introduction In-vitro fertilization (TVF) is widely used in the treatment of infertile couples. Although the average rate of fertilization in most FVF centres is approximately 60-70%, there are cases of low fertilization rates or even fertilization failures, especially in patients with poor semen (Yang et al., 1993). Micromanipulation techniques of partial zona dissection (PZD) (Malter and Cohen, 1989), subzonal sperm insertion (SUZI) (Ng et al, 1988) and intracytoplasmic sperm injection (ICSI) (Palermo et al, 1992) have been introduced to improve fertilization of gametes. Of these, the latter has become prevalent due to its higher success rate (Van Steirteghem et al, 1993). Recently, efforts have been directed towards increasing the efficiency and efficacy of ICSI. One of these involves breaking the tail of the spermatozoon, thus immobilizing it, prior to injection (Antinori et al., 1995; Fishel et al, 1995; Gerris et al, 1995). Breaking the tail is thought to damage the cell membrane and invoke subsequent physiological and biochemical reactions that may promote decondensation of the sperm head and activation of the oocyte (Dozortsev et al, 1994; Sousa and Tesarik, 1994; Tesarik et al, 1994; Parrington et al, 1996). Methods of causing membrane damage using an injection micropipette include compression of the midpiece (Tucker et al., 1995), compression of the end of the mid-piece (Tesarik et al., 1994), cutting the tail below the midpiece region (Fishel et al., 1995) or cutting halfway between the head and the tip of the tail (Gerris et al., 1995). Since the entire spermatozoon is covered with plasma membrane, including the tip of the tail, membrane integrity may be disrupted at any site. Here we present our experience of ICSI using a technique in which the tail is dissected at the tip. It is simple and effective, and may provide an alternative method to damage the membrane and permanently immobilize the spermatozoon. Materials and methods Patients During the period April 1994 to April 1996, 60 men with severe semen abnormalities and their partners, participating in an in-vitro fertilization programme at the National Taiwan University Hospital, were recruited to this study. Severe semen abnormalities were denned as sperm concentration SS5X10 6, motility =s20%, or normal morphology ^4% using Kruger's strict criteria (Yang et al, 1993, 1995). Ovarian stimulation Ovarian stimulation was initiated by the regimen of a combination of a gonadotrophin-releasing hormone analogue (GnRHa; Supremon, buserelin acetate; Hoechst, Frankfurt, Germany) with human menopausal gonadotrophin (HMG; Pergonal; Serono, Rome, Italy) and follicle-stimulating hormone (FSH; Metrodin; Serono). When at least two leading follicles reached a mean diameter of 18 mm or more with corresponding serum oestradiol concentrations, 10000 IU of human chorionic gonadotrophin (HCG; Profasi; Serono) was administered i.m. Thirty-four to 36 h after HCG administration, oocyte retrieval was performed by transvaginal ultrasound-guided aspiration. 2640 O European Society for Human Reproduction and Embryology

ICSI with tail-tip damaged spermatozoa (a) Figure 1. Dissecting the tail at the tip (a) The tip of the tail is pressed with the injection micropipette by operating the threedimensional joystick, (b) The micropipette is quickly withdrawn by operating the horizon-directional wheel During this action, the spermatozoon turns from the original perpendicular direction to the horizontal direction. (b) Figure 2. Compressing the mid-piece, (a) The joystick is used to operate the injection micropipette downward to impose pressure on the mid-piece, (b) The pressure is then released by operating the micropipette upward Preparation of sperm Fresh semen was obtained or frozen semen was thawed 2-3 h before ICSI. Motile spermatozoa were collected using a direct swim-up technique or two-layer discontinuous Percoll gradient centrifugation (Chen et al, 1995). The sample was washed, and the pellet was resuspended in human tubal fluid (HTF) medium supplemented with 15% heat-inactivated maternal serum and kept in an incubator at 37 C with an atmosphere of 5% CO2 in air Preparation of oocytes The cumulus cells of oocytes were removed by needle dissection and pipetting in HTF medium containing 80 IU/ml hyaluronidase (type IV-S; Sigma, St Louis, MO, USA). The oocytes were then washed in HTF medium, and transferred back to the culture medium. Metaphase II (Mil) oocytes with the first polar body were selected for microinjection Preparation of micropipettes Micropipettes were made of thin-walled glass tubes (diameter 0 95-1 05 mm, Research Instruments Limited, Cornwall, UK) and Illl (b) Figure 3. Cutting the tail at the mid-portion, (a) The tip of the injection micropipette is sliced across the mid-portion of the tail by operating the joystick. The spermatozoon is pushed forward against the bottom of the dish by a quick and strong force (b) The micropipette is then quickly withdrawn using the same joystick. pulled by a horizontal puller (Sutler Instrument Co., Novato, CA, USA) The tip of the injection micropipette (outer diameter 7 um and inner diameter 5 um) was bevelled at a 35 degree angle and sharpened on both sides by a microgrinder (Research Instruments Limited) The holding micropipette was made with a microforge (Narishige, Tokyo, Japan) with an opening tip of 15-20 um inner diameter and 60-80 um outer diameter. For convenient manipulation on the dish the micropipettes were bent at an angle of 20 degrees These micropipettes were then sterilized by dry heat at 160 C for 2 b. ICSI procedures One drop (5 ul) of 10% poiyvinylpyrrolidone (PVP, molecular weight 360 000, Sigma) in 10 mm Hepes-buffered HTF medium and four drops (5 ul/ drop) of 10 mm Hepes-buffered HTF medium supplemented with 0.5% human serum albumin were placed on the lid of a Petn dish (Falcon 3001; Becton Dickinson and Company, Lincoln Park, NJ, USA) and then covered by pre-equihbrated light-weight mineral oil (embryo tested, Sigma) to prevent evaporation. A 1 ul aliquot of sperm suspension was added to the PVP droplet if the sperm motility was good, or added to one of the HTF droplets if motihty was poor. The oocytes were put into the remaining HTF droplets. Micromanipulation was performed with the aid of two micromanipulators and microsyringes (Narishige) mounted on an inverted microscope (Olympus, Tokyo, Japan) in a modified incubator (Di Shung Instrument Co., Taipei, Taiwan) (Chen et al, 1993). The dissection of the sperm tail was performed in the PVP droplet. Grossly normal spermatozoa were chosen which were perpendicular, or manipulated to be perpendicular, to die injection micropipette. The injection micropipette was pressed against the tip of the tail by operating the three-dimensional joystick (Figure la) and then quickly withdrawn to dissect it by operating the horizon-directional wheel of the hydraulic remote controller. When the tip of the sperm tail was dissected, the spermatozoon turned horizontal to the injection micropipette (Figure lb). The spermatozoon then became permanently immobilized immediately This action led to damage to the membrane and tail-tip structure that it appeared shorter because the flagellum was deformed. The immobilized spermatozoon was then dislodged and aspirated from the bottom of die dish with die injection micropipette The oocyte was firmly held at the 9-o'clock position by suction at the opening of die holding micropipette, and the first polar body was located at the 6- or 12-o'clock position. When die oocyte, 2641

S.-U.Chen et al holding micropipette, and injection micropipette were all clearly seen at the same focus of the microscope, they were considered to be located in the same plane. The injection micropipette was operated to touch the zona pellucida and to make sure that the equatorial region was in the correct location. The spermatozoon was pushed to the tip of the injection micropipette. The zona pellucida was penetrated by advancing the injection micropipette at the 3-o'clock position, and the oolemma was pressed so that it became uivaginated. The oolemma and ooplasm were aspirated into the injection micropipette to break the oolemma. During this procedure, the oolemma became further invaginated, and the ooplasm slowly flowed into the injection micropipette. When no more invagination of oolemma occurred, or when there was a rush of ooplasmic flow, the oolemma was considered to be broken. The spermatozoa, together with aspirated ooplasm, were then injected into the oocyte, and the injection micropipette was slowly withdrawn. The oocytes were returned to the culture dish and incubated for a further period in HTF medium supplemented with 15% heat-inactivated maternal serum Comparison among three different immobilization techniques In 23 cycles, the sibling oocytes were randomly treated by ICSI using three different immobilization techniques One of these involved compressing the mid-piece, destabilization of the sperm membrane being induced by imposing pressure on the mid-piece with the injection micropipette (Figure 2a, b) (Tucker et al., 1995) The second involved cutting the tail at the mid-portion, the tip of the injection micropipette being sliced across the tail below the mid-piece region to break the tail (Figure 3a, b) (Fishel et al, 1995). The third method involved dissecting the tail at the tip, as described above. After ICSI, the oocytes were cultured individually. The percentages of fertilization and embryo cleavage were calculated in each group, and compared using x 2 or Fisher's exact test The difference was considered statistically significant for P < 0.05. Embryo replacement The ICSI oocytes were observed 16-18 h later, and oocytes with two pronuclei and the second polar body were identified as normally fertilized Those oocytes with a single pronucleus or three pronuclei were considered abnormally fertilized. From the normal zygotes the embryos were examined 24 h later, and the morphological scores were established as grade 1-4: grade 1, equal-sized symmetrical blastomeres; grade 2, uneven blastomeres with <10% fragmentation; grade 3, 10-50% blastomeric fragmentation; and grade 4, >50% blastomeric fragmentation (Steer et al, 1992). The developing embryos at the two-to-four cell stage were transferred into the uterine cavity transvaginally or into the Fallopian tubes laparoscopically. Four embryos at most were transferred to each woman and the supernumerary embryos were frozen for later transfer. Progesterone in oil, 25 mg daily, as well as HCG (Pregnyl; Organon, Holland), 1500 IU on days 2, 5, and 8, were administered i.m after transfer. Clinical pregnancy was defined as the elevation of serum (J-HCG concentrations 14 days after transfer and subsequently the presence of a fetal sac at ultrasound. Results A total of 78 ICSI treatment cycles was performed for 60 couples with severe semen abnormalities, including 15 patients with severe oligozoospermia, 20 with severe asthenozoospermia, six with severe teratozoospermia and 19 with combined defects. Sperm samples were prepared from fresh semen in 73 cycles and from cryopreserved semen in five 2642 Table L The outcome of ICSI for severe semen abnormalities- using the technique of dissecting the tail at the tip Treatment cycles ME oocytes Damaged oocytes (rate) 2 PN (rate) 1 PN (rate) 3 PN (rate) Cleavage (rate) No of pregnancies/treatment cycle No of pregnancies/transfer No of miscarriages/biochemical Ongoing pregnancy rate/transfer Multiple pregnancy rate ICSI 55 468 35 (7.5%) 282 (65 1%) 16 (3.7%) 11 (2.5%) 266 (94 3%) 2CV55 (36 4%) 20/54 (37 0%) 3/2 15/54 (27 8%) 4/15 (26 7%) Tbble IL The fertilization outcome and embryo cleavage of the sibling oocytes aftet ICSI using three different immobilization methods in 23 patients Oocytes injected 69 Damaged oocytes (rate) 4 (5 8%) 2 PN (rate) 43 (66.2%) 1 PN (rate) 1 (1.5%) 3 PN (rate) 2(3 1%) Cleavage (rate) 41 (95 3%) Compress the Cut the tail at Dissect the tail mid-piece the mid-portion at the tip 66 5 (7 6%) 39 (63.9%) 2 (3 3%) 1 (1 6%) 36 (92.3%) Differences statistically non-significant by x 2 or Fisher's exact test 70 4 (5.7%) 44 (66.7%) 2 (3.0%) 1 (1 5%) 42 (95.5%) cycles. The mean age of females was 33 years, with a range of 23-42 years. The total number of oocytes recovered was 820, in which 673 (82 1%) MH oocytes were found after removal of cumulus cells. In 55 cycles, 468 ME oocytes were injected purely using the modified immobilization method. The results regarding oocyte damage, normal fertilization, abnormal fertilization, cleavage and pregnancy are summarized in Table I. Grading of embryos revealed 61 (22.9%) of grade 1; 109 (41.0%) of grade 2; 76 (28.6%) of grade 3; and 20 (7.5%) of grade 4. Transfer of embryos was performed in 54 cycles because one cycle had no fertilized oocyte. The mean number of embryos transferred was 3.4 and the supernumerary embryos (83) were frozen for later use. Among the 18 clinical pregnancies, including four sets of twins, six were delivered with normal babies, three ended in spontaneous abortion and nine pregnancies are ongoing. The implantation rate was 12.0% (22/183). In 23 cycles, 205 Mil oocytes were randomly allocated to be injected using three different immobilization techniques on the sibling oocytes within the same patient. The oocyte damage, normal fertilization, abnormal fertilization, and embryo cleavage were not significantly different among them (Table H). The embryo quality in the three different groups was also similar (Table HI). The embryos from the three groups were mixed for transfer into the individual patient. The mean number of embryos transferred was 3.3 and 33 embryos were cryopreserved. Seven clinical pregnancies (30.4%), including one set of twins, were achieved in this series. One was aborted and seven are ongoing. The implantation rate was 10.5% (8/76).

ICSI with tail-tip damaged spermatozoa Table ILL The details of embryo grading in the groups of three different immobilization techniques Grade of embryos Compress the mid-piece (n = 40) 9 (22 0%) 18 (43 9%) 10 (24 4%) 4 (9 7%) Cut the tail at the mid-portion (n = 36) 10 (27 8%) 14 (38 9%) 9 (25 0%) 3 (8 3%) Dissect the tail at the tip (n = 42) 12 (28 6%) 17 (40 5%) 10 (23 8%) 3(7 1%) Differences statistically non-significant by f} or Fisher's exact test Discussion This study demonstrates that dissecting the tail of a spermatozoon at the tip prior to ICSI achieves a high rate of fertilization and embryo cleavage. Fishel et al. (1995) and Gerris et al. (1995) found that ICSI with permanently immobilized sperm resulted in a significantly higher rate of fertilization than ICSI with motile spermatozoa. The rate of normal fertilization in our study is comparable with that presented their report. Our prospective study further demonstrated no significant difference in normal fertilization, abnormal fertilization, or embryo cleavage among three different methods of immobilizing spermatozoa used for ICSI of sibling oocytes. This implies that dissecting the tail at the tip has the same effect as cutting the tail either at the level below mid-piece or halfway along the tail. The entire spermatozoon, including head, neck, mid-piece and tail is covered with plasma membrane. The integrity of the membrane can be disrupted at the mid-piece (Tesarik et al., 1994; Tucker et al, 1995), midportion (Fishel et al, 1995; Gerris et al, 1995) or tip of the tail. The diameter of the tail narrows at the tip It may be more difficult and require more skill to cut the tail at the midportion or above. The spermatozoon may also sometimes stick to the micropipette during this procedure, and it is troublesome to dislodge it Dissecting a tail at the tip may be easier and as effective as cutting it at the mid-piece to cause permanent immobilization and damage to the membrane. In addition, the technique of dissecting the tail-tip minimizes the likelihood of damage to the mid-piece. This contains the centriole, which plays a major role in the cleavage patterns of the early embryo (Asch et al, 1995; Van Blerkom, 1996). This technique clearly has benefits to circumventing the potential hazard from centriole damage which may occur in compressing the mid-piece. During normal fertilization, the spermatozoon undergoes capacitation, calcium influx, acrosomal reaction, exposure of the inner acrosomal membrane, penetration of the zona pellucida, and becomes lmmobdized before it enters the ooplasm It then undergoes fusion with the oolemma, incorporation into the ooplasm in a demembranated state, release of a cytosolic sperm factor, and decondensation of the head (Sathananthan et al., 1986; Yang et al, 1988; Swann, 1990). Therefore, a plasma membrane-intact motile spermatozoon injected into ooplasm may not be optimal for fertilization. Maleszewski (1990) showed in cell extracts in vitro that only sperm cells with a partially damaged membrane were able to undergo swelling. In ICSI, whether damaging the plasma membrane activates the spermatozoon and invokes similar events to those occurring during natural fusion of gametes, or whether immobilizing the spermatozoon prevents disruption of the ooplasm by an active sperm tail (Gerris et al, 1995) deserves further research. Activation of oocytes is a series of events which may be triggered by fusion with a fertilizing spermatozoon (Tesarik et al, 1994), although the mechanism is still controversial. However, ICSI circumvents the fusion of gametes, and thus leads to successful fertilization and cleavage. The causes of activation of the oocyte are also uncertain in ICSI, but may include penetration of the oocyte and aspiration of the ooplasm by an injection micropipette, injection of spermatozoa, and release of a cytosobc sperm factor which leads to physiological and biochemical changes, such as calcium oscillations and membrane hyperpolarizauon, necessary for the activation of oocytes (H<5ma and Swann, 1994; Tesank et al, 1994). After activation, mammalian oocytes possess the capability to induce sperm decondensation during a narrow time-span only (Komar, 1982). A motile spermatozoon injected into the ooplasm may be incapable of completely activating the oocyte or decondensing the head in the time available (Dozortsev et al, 1994; Tesarik et al, 1994). Damage to the tail of the injected spermatozoon results in depolymerization of the sperm membrane which may permit ooplasm enzymes to easily penetrate the sperm nucleus to initiate decondensation (Sousa and Tesarik, 1994). Oscillin, a calcium oscillation-inducing factor released from the depolymerized spermatozoon, is also able to activate the oocytes (Pamngton et al, 1996). It is speculated that the injection of membrane-damaged spermatozoa may not only increase the chance of normal fertilization but may also decrease the risk of abnormal fertilization. It has been noticed that the incidence of single pronucleus is higher in ICSI than in conventional FVF (Winston et al, 1991; Palermo et al, 1993). A study of chromosomal status has revealed that the single pronucleus in IVF is usually derived from normal fertilization; however, in ICSI it is mainly due to parthenogenetic activation (Sultan et al, 1995). The activation of the oocyte during ICSI without synchronized activation of spermatozoa may result in sole formation of a female pronucleus (Dozortsev et al, 1994). Gerris et al. (1995) observed a significant decrease in the percentage of single pronuclei with tail-broken spermatozoa, compared with tailintact spermatozoa; however, Antinori et al. (1995) did not make the same observation. The rate of formation of a single pronucleus in the injected oocytes with tail-dissected spermatozoa was not high (3.2%) in our series. The effect of damage to the sperm membrane prior to ICSI upon the incidence of single pronuclei deserves further investigation. ICSI is a powerful method to treat severe male-factor infertility. Immobilization of spermatozoa prior to injection is gaining wide acceptance and appears to achieve better results. Dissecting the tail at the tip is an easy and efficient method and may provide an alternative to compressing the mid-piece and cutting the mid-portion of the tail. The effects of breaking the tail of spermatozoa on the incidence of increased normal fertilization and decreased incidence of abnormal fertilization need further investigation. The relationship between damage 2643

S.-U.Chen et al to the sperm membrane and activation of spermatozoa, as well as subsequent activation of oocytes, is currently under study in our laboratory. Acknowledgements This study is supported in part by grants from the Nauonal Science Council (NSC-82-412-B002-343), Taiwan and the National Taiwan University Hospital (NTUH-85175-07). The authors are grateful to Ms Heng-Ru Lin, Ms Jane-Ru Lin and Ms Li-Jung Chang for their technical assistance and Dr Chin-Der Chen for his help in sketching the figures. References Antinori, S., Panel, C, Caffa, B. et al. (1995) The R.APRUL Center experience: from SUZI, through lasers to ICSI using spermatozoa with broken tails. Hum. Reprod., 10, 489-491 Asch, R, Simerly, C, Old, T et al (1995) The stages at which human fertilization arrests, microtubule and chromosome configurations m inseminated oocytes which failed to complete fertilization and development in humans MoL Hum. Reprod, 1, see Hum. Reprod., 10, 1897-1906. 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Assoc, 92, 122-127 Yang, YS., Chen, S U., Ho, H.N. et al (1995) The correlation between sperm morphology using strict criteria in original semen and swim-up inseminate and human in vitro fertilization Arch. AndroL, 34, 105-113. Received on June 5, 1996; accepted on October 5, 1996