Intracytoplasmic sperm injection: a major advance in the management of severe male subfertility

Transcription

1 FERTILITY AND STERILITY Copyright 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Intracytoplasmic sperm injection: a major advance in the management of severe male subfertility Offer Harari, M.D. *t:j: Harold Bourne, B.Sc. * Michele McDonald * Nadine Richings, B.Sc.t Andrew L. Speirs, F.R.C.O.G., C.R.E.I.*t W. Ian H. Johnston, M.G.O., F.R.C.O.G.*t H. W. Gordon Baker, M.D., Ph.D.*tll The Royal Women's Hospital, Carlton; Melbourne IVF, East Melbourne; and University of Melbourne, Parkville, Victoria, Australia Objective: To determine the success of intracytoplasmic sperm injection for severe male infertility. Design: A retrospective survey. Setting: A tertiary infertility service. Patients and Interventions: One hundred fourteen couples had 119 intracytoplasmic sperm injection treatments because of previous failure of standard IVF, poor results with subzonal insemination, sperm concentration < 2 X lo B /ml, other sperm defects, or male genital tract obstruction. Main Outcome Measures: Fertilization, implantation, and pregnancy rates. Results: Of 1,185 oocytes treated by intracytoplasmic sperm injection, normal fertilization and cleavage occurred in 717 of 1,073 that survived (67% normal fertilization rate). Abnormal fertilization occurred in 113 oocytes (11% abnormal fertilization rate) and 112 oocytes did not survive the procedure (survival rate of90%). In 117 couples, 251 embryos were transferred fresh, 409 embryos were cryopreserved, and 224 were transferred after thawing. The implantation rate was 7.4% (fetal heart per embryo transferred). To date 36 clinical pregnancies have been achieved (12% per fresh transfer, 20% per frozen transfer, and 30% overall), 24 are ongoing or delivered (6% per fresh transfer, 14% per frozen transfer, and 20% per intracytoplasmic sperm injection). The fertilization rates were the same (65%) with various sperm defects but higher with genital tract obstructions (75%). Conclusion: Intracytoplasmic sperm injection has improved the prognosis of severe male infertility. Fertil Steril 1995; 64:360-8 Key Words: In vitro fertilization (IVF), intracytoplasmic sperm injection (lcs!), micromanipulation, assisted fertilization, male infertility Standard IVF is used as a treatment for male infertility but fertilization rates are lower than with other causes of infertility (1). Gamete intrafallopian transfer does not appear to improve the pregnancy Received September 22, 1994; revised and accepted February 17,1995. * Reproductive Biology Unit, The Royal Women's Hospital. t Melbourne IVF. :j: Present address: Bnai Zion Medical Center, Department of Obstetrics and Gynecology, Faculty of Medicine, Technion, Haifa, Israel. Reprint requests: Harold Bourne, B.Sc., Reproductive Biology Unit, The Royal Women's Hospital, Carlton, Victoria 3053, Australia (FAX: ). II Department of Obstetrics and Gynaecology, University of Melbourne. 360 Harari et al. Male infertility type and ICSI results rate in subfertile men and the fertilization process cannot be assessed (2). Tubal embryo transfer at different developmental stages: zygote intrafallopian transfer and pronuclear stage transfer for male factor infertility has been claimed to improve pregnancy and implantation rates, but nevertheless average fertilization rates are lower than in nonmale factor patients (3). In vitro fertilization rates may be improved in patients with teratospermia or previous failure of fertilization by inseminating the oocytes with higher numbers of sperm (4, 5). Although this procedure increases the fertilization rates, approximately 40% of patients with moderate to severe oligospermia «5 X 10 6 /ml) or high proportions of sperm with abnormal morphology (>95%) continue to have zero or low

2 fertilization rates «20%) (5). Results with epididymal sperm collection, although reported by one group to be good with standard IVF, have been very poor in our and others' experience (6, 7). Patients with extremely abnormal semen (sperm concentrations <2 X 10 6 /ml, zero sperm motility, or total teratospermia) have such a low chance of success that IVF usually is not offered to them. Finally, increasing numbers of patients are being found who have persistent failure offertilization in vitro despite normal semen by conventional analysis. Assisted fertilization technology had been developed to enhance fertilization in patients with these types of problems and human gamete micromanipulation has been reviewed by Fishel et al. (8). The zona pellucida (ZP) is a barrier to sperm penetration and is an obvious target for micromanipulation techniques. Zona drilling with acid Tyrode's solution followed by insemination achieved fertilization but with low rates and also frequent polyspermic fertilization and abnormal cleavage (9). Partial zona dissection improved the fertilization rate in a group of couples with previous failed fertilization in IVF, but again a high rate of polyspermic fertilization occurred (10). An alternative mechanical method of making a hole in the ZP in a mouse model improved fertilization rate after insemination with low sperm concentrations without adversely affecting cleavage or hatching processes (11). In humans this method of zona opening resulted in an improvement in fertilization rates over those obtained with intact control oocytes but there was frequent cleavage arrest and no pregnancies (Bourne H, Hale L, Vassiliadis A, Liu DY, Lopata A, Johnston WIH, et ai., abstract). The next method of micromanipulation to be reported, subzonal sperm insertion (SUZI), should have produced a high fertilization rate with low polyspermic fertilization because the number of sperm inserted under the ZP could be controlled by the operator. However, the polyspermic fertilization rate remained high at approximately 50% of the monospermic fertilization rate (10, 12). A number of groups found SUZI a viable treatment for cases of previous IVF failure and for male infertility (8). The results of these groups may have depended to some extent on the patient selection criteria used by the group. Our experience with very severe male infertility or persistent failure of fertilization in standard IVF was that the monospermic fertilization and normal cleavage rate was only approximately 20% and there were few pregnancies. Although the procedure oftransfer of a single spermatozoon into the cytoplasm of the oocyte had been producing fertilization in animals for some time, trials to apply the same procedure to human oocytes had not been very successful (8). A new era in the treatment of human infertility began with the report of pregnancies after the injection of a single spermatozoon into the ooplasm by Palermo et al. (13). This procedure is called intracytoplasmic sperm injection. Subsequent reports have confirmed the high fertilization rates and indicated that many healthy babies have been born after intracytoplasmic sperm injection (8, 14-16). We report the outcome of our initial experience with intracytoplasmic sperm injection in all patients treated in 1993 categorized by types of male infertility or sperm defect. This also confirms that fertilization results similar to those obtained by standard IVF with normal semen now can be achieved for a variety of types of severe male factor infertility. However, patients with genital tract obstructions do better than those with sperm defects or other causes of failure of fertilization. Patients MATERIALS AND METHODS This study was carried out on 119 treatment cycles performed between July and December Patients were divided into five major groups according to the semen characteristics or previous IVF results. Patients were assigned on the basis of average semen quality from two or more semen analyses in the previous 3 years. Karyotypes were performed in men with average sperm concentrations < 5 X 10 6 /ml (17). Cystic fibrosis gene studies were done in men with absent vasa deferentia and in their partners if abnormal. Patients were grouped into the following categories for analysis: 1. Severe oligospermia (31 patients, 32 treatments), average sperm concentration < 2 X 10 6 /ml. In this group, four patients had previous surgery for genital tract obstruction, three vasoepididymostomies and one vasovasostomy, and the others had severe disorders of spermatogenesis. One of these had mosaic Klinefelter syndrome and has been reported in detail previously (18). 2. Oligoasthenoteratospermia (21 patients, 22 treatments). Average sperm concentration < 20 X 10 6 /ml, with sperm motility < 25% progressive and/or < 40% total and abnormal sperm morphology > 85%. Two of this group had had vasovasostomies and another had semen collected by electroejaculation because of chronic spinal cord injury. 3. Asthenoteratospermia (22 patients, 22 treatments). Average sperm concentration > 20 X 10 6 / ml, with sperm motility < 25% progressive or < 40% total and/or abnormal morphology > 85%. Seven had low sperm motility alone; two associated Harari et al. Male infertility type and lcsl results 361

3 with low level antisperm antibodies insufficient to block sperm mucus penetration. Eight had combined asthenoteratospermia and seven had isolated teratospermia with >95% of sperm with abnormal morphology and previous failure of fertilization. These groups also were divided on the basis of previous IVF results; 4 with severe oligospermia, 15 with oligoasthenoteratospermia, and 16 asthenoteratospermia had average fertilization rates of <20% in one or more previous IVF attempts. 4. Miscellaneous low fertilization rate in vitro (21 patients, 22 treatments). Ten patients with previous failure of fertilization had a specific disorder of the ZP-induced acrosome reaction (19). Eleven patients had poor fertilization of other causes: three with normal semen but low sperm-zona binding in standard IVF of unknown cause, three with variable semen associated with poor fertilization, one with normal semen apart from abnormal DNA determined by acridine orange fluorescence, and four with possible female factors, two women over the age of 42 years and two with low oocyte numbers per treatment. 5. Male genital tract obstructions (19 patients, 21 treatments). Twelve patients had bilateral congenital absence of the vasa deferentia, three had Young syndrome (one of whom had an intratesticular blockage and had a fine-needle tissue aspiration biopsy of the testis [20] to obtain the sperm), two had failed vasoepididymostomies for postinflammatory obstructions in the epididymides, one had a failed vasovasostomy, and one patient with postinflammatory epididymal obstructions who had an artificial spermatocele inserted in Europe; this was nonfunctional but he had developed a large 20- to 25- ml natural spermatocele from which sperm could be obtained by needling percutaneously. Except where already stated these patients had sperm obtained by microsurgical epididymal sperm aspiration and used either fresh or after cryopreservation. The intracytoplasmic sperm injection procedure was introduced with the acquiescence of the Institutional Ethics Committees. Patients were informed about this new assisted fertilization technology by both the medical attendants and specialist infertility counselors and signed a consent form. In view of the report of normal fetal karyotypes with intracytoplasmic sperm injection by Van Steirteghem et al. (Van Steirteghem AC, Joris H, Liu J, Nagy Z, Tournaye H, Wisanto A, et ai., abstract) and after discussion with our medical geneticists, it was decided not to perform prenatal genetic diagnosis because of intracytoplasmic sperm injection but to continue to advise ultrasonographic investigations and chorionic villous sampling according to the usual obstetric indications. 362 Harari et al. Male infertility type and [CS[ results Ovarian Stimulation and Support Ovarian stimulation was achieved by a short desensitization protocol using GnRH analogue (GnRHa; Lucrin; Abbott, Kurnell, New South Wales, Australia), 150 IU/d hmg (Humegon; Organon, Lane Cove, New South Wales, or Pergonal; Serono, French's Forest, New South Wales, Australia) and hcg (Pregnyl; Organon). Luteal phase support was given from the day of oocyte retrieval for 12 days, in the form of 100 mg/d P pessaries (The Royal Women's Hospital, Carlton, Victoria, Australia). Semen Assessment and Preparation For groups 1 to 4, semen was collected by masturbation after 2 to 5 days of abstinence on the day of oocyte retrieval. Sperm concentration and motility were assessed on the raw ejaculate. In most cases semen was diluted 1:1 with carbonate-buffered human tubal fluid (Irvine Scientific, Irvine, CA) containing 10% human serum (HS-HTF) and centrifuged at 1,800 X g for 5 minutes. The resulting pellet was separated on a three-layer mini-percoll gradient followed by removal of the pellet and 95% Percoll layer and dilution with 1 ml HS-HTF. To remove the remaining Percoll, one or two centrifugations of 1,800 X g for 5 minutes were carried out and the pellet was resuspended in 21 mm HEPES-buffered HS-HTF (Irvine Scientific). Some samples were prepared using a standard swim-up method if there were more than approximately 5 X 10 6 progressively motile sperm in the ejaculate. In some patients with extremely low sperm numbers «10 sperm in the whole chamber of a hemocytometer), the sample was spun once at 1,800 X g for 5 minutes and then resuspended in a small volume of HEPES-HS-HTF for use. Oocyte Collection and Preparation Vaginal ultrasound (US)-guided oocyte collection was done 36 hours after hcg injection. The cumulus was removed 1 to 4 hours after retrieval by brief exposure «1 minute) to HEPES-HTF medium containing 100 IU/mL hyaluronidase (Bovine type VI; Sigma Chemical Co., St. Louis, MO) and aspirated several times through a large-bore Pasteur pipette. The remaining corona cells were removed by aspiration through a fine-bore (approximately m diameter) hand-drawn Pasteur pipette in fresh HEPES-HS-HTF medium. Denuded oocytes were washed twice and transferred into fresh HS-HTF. Only oocytes that were scored as mature by the presence of a polar body viewed under an inverted microscope were selected for microinjection.

4 Microinjection Technique Intracytoplasmic sperm injection was performed i~ a plastic dish (Falcon 1006; Becton Dickinson, Lmcoln Park, NJ) containing a small drop (5 to 20 j.tl) of sperm suspension in HEPES-HS-HTF surrounded by small drops (5 to 10 j.tl) of HEPES-HS HTF under light mineral oil (M-8410; Sigma Chemical Co.) or light paraffin oil (BDH, Poole, United Kingdom). The oils were tested and shown not to be embryotoxic. Injections were carried out using an inverted microscope (Diaphot-TMD; Nikon, Tokyo, Japan) with Nomarski DIC optics and a warm stage (LEC Instruments, Melbourne, Victoria, Australia) connected to on-stage micromanipulators and microinjectors (ML-188, IM-6; Narishige, Tokyo, Ja ~an).!he injection.pipettes were produced by pullmg thm-walled capillary glass tubes with outer and inner diameters of 1 mm and 0.78 mm, respectively (SDR Chemical Technology, Middle Cove, New South Wales, Australia), using a programmable horizontal micropipette puller (PC-84; Sutter Instrument Co., San Rafael, CA), sharpening with a pipette grinder (EG-4; Narishige), and bending the tip to a 45 angle with a microforge (MF-9; Narishige). The outer diameter of the injection pipette was 6 to 10 j.tm. Holding pipettes were drawn by hand from the same glass tubes to an inner diameter of approximately 10 to 20 j.tm, approximately one tenth the size of the oocyte, to avoid aspirating a large surface area of the oocyte into the pipette. A single motile sperm was aspirated into the injection pipette and transferred from the sperm suspension drop to the HEPES-HS-HTF drop containing the oocyte. The sperm then was immobilized by squeezing its tail against the bottom of the dish with the tip of the injection pipette. The immotile sperm t~en was aspirated back into the injection pipette, either head or tail first, but maintained close to the point of the pipette. The oocyte was held with the holding pipette using gentle negative pressure so that the polar body was at either the 12 or 6 o'clock position. The sperm was injected into the oocyte cytoplasm head or tail first after puncturing both the ZP and the oolemma as indicated by the ability to aspirate cytoplasm into the injection pipette. The injection pipette was withdrawn carefully, the oocyte ~as released from the holding pipette, and the in Jected oocyte was placed into fresh HS-HTF culture medium. Fertilization Assessment Injected oocytes were incubated in HS-HTF medium. Fertilization was assessed 15 to 18 hours after the injection procedure and the presence of pronuclei (PN) was recorded. The zygotes or pre-embryos were viewed again after 40 and 60 hours to record cleavage status. Embryo Transfer, Cryopreservation, and Pregnancy Confirmation Transcervical uterine transfer of embryos was carried out using an end-hole flexible catheter (K-Soft; Cook, Eight Mile Plains, Queensland, Australia). Fresh embryos were transferred 3 days after microinjection. Routinely, two embryos were transferred to decrease the risk of multiple pregnancy. However, iffresh embryo quality was poor, up to three embryos were transferred. This approach is the same as that for all patients undergoing IVF treatment in our program. Allocation of embryos for freezing or fresh transfer was based on morphology. Embryos chosen for freezing were those exhibiting clear distinct cells without any obvious signs of degeneration. The presence of anucleate fragments was tolerated up to approximat~ly one third of the volume of the embryo; although m most cases fragmentation was <20%. If embryo quality varied or only a small number of embryos was suitable for cryopreservation, then embryos of poorer or mixed quality were transferred fresh to maximize embryo use. Em~ryo cryopreservation involved the 1,2-propanedlOl-sucrose slow freeze-rapid thaw protocol described by Lassalle et a1. (21). Briefly, embryos for freezing were transferred at room temperature to phosphate-buffered saline (GIBCO BRL, Life Technologies Inc., Grand Island, NY) supplemented with 20% HS (PBS-HS) for handling and final assessment. The embryos were dehydrated in 1.5 M 1,2- propanediol for 10 minutes, transferred to 1.5 M 1,2- propanediol-0.1 M sucrose in PBSIHS and loaded. ' mto 0.25-mL plastic straws. The straws were cooled at -2 C/min from 15 to 18 down to -7 C using a programmable freezer (Kryo 10; Planar Products Ltd., Sunbury-on-Thames, United Kingdom), held at -7 C for 10 minutes, and seeded manually. Cooling was recommenced at -0.3 C/min down to -30 C and then -50 C/min down to -150 C before placin~ in liquid nitrogen. For thawing, the straws were held at room temperature in air for 30 seconds before being placed in a 30 C water bath for 30 to 40 seconds. The embryos were rehydrated in two 5-minute exposures to reducing 1,2-propanediol concentration (1.0 and 0.5 M 1,2-propanediol-0.2 M sucrose in PBS HS) followed by 10 minutes in 0.2 M sucrose in PBS HS before transfer to PBS-HS at room temperature for 10 minutes, 37 C for 10 minutes, and finally HTF culture medium at 37 C. Embryos were transferred after thawing in either a natural or artificial cycle. Patients having a natural cycle had the embryo transfer 3 days after ovula- Harari et ai. Male infertility type and ICSI results 363

5 Table 1 Survival and Fertilization Results of 1,185 Oocytes Submitted Intracyloplasmic Sperm Injection* Oocytes Remained intact 2PN Normal cleavage, pronuclei not seen IPN >2PN Not fertilized Fertilized late * Values in parentheses are percentages. Number 1,073 (90.5) 691 (64.4) 26 (2.4) 56 (5.2) 57 (5.3) 225 (21.0) 18 (1.7) tion was estimated to have occurred using a urine dipstick test for LH (Clearplan One Step; Fisons, Castle Hill, New South Wales, Australia). In those patients having an artificial cycle, endometrial growth was induced with 2 mg/d E2 valerate and 100 mg/d of 50 mg P pessaries introduced when the endometrial thickness was 2: 7 mm as determined by US. Embryos were transferred 3 days after starting P treatment. In artificial cycles, luteal phase support was continued with P pessaries. In all cases, a,b-hcg pregnancy test was scheduled 14 to 18 days after embryo transfer with a serum level> 10 lull considered positive. Pregnancy was confirmed by the demonstration of one or more gestation sacs on sonographic examination 4 weeks after transfer. Statistical Methods Results were tabulated and the significance of differences in frequencies between groups examined by X 2 or conditional binomial exact tests for low expected numbers. Differences between means were tested by analysis of variance and least significant differences multiple comparisons. Fertilization Results RESULTS A total of 1,504 oocytes was collected from the 119 oocyte retrievals (mean 12.6 oocytes per treatment cycle). In the first four patients having intracytoplasmic sperm injection, half their oocytes (20 in total) were injected subzonally as a comparison, whereas, in a further seven patients, 41 oocytes were inseminated using normal lvf. During chemical dissection and mechanical removal of the corona cells, cytes were lysed (4.6% damage rate). After maturation assessment, 1,156 oocytes that had extruded their first polar body were selected for intracytoplasmic sperm injection. Another 181 oocytes (12.0%) were immature, either in the germinal vesicle or metaphase I stages of maturation. Of these, 29 that had reached metaphase II by the next day also were injected, making a total of 1,185 oocytes subjected to intracytoplasmic sperm injection (Table 1). The remaining eggs were either degenerate or used for approved research. Of the injected oocytes, 1,073 (91%) remained intact 15 to 18 hours after microinjection and 691 were 2PN in the first fertilization check (Table 1). In another 26 oocytes (2.4%), PN were not seen but extrusion of the second polar body and cleavage occurred at the right times; these oocytes also were considered to be fertilized normally. Of the remaining oocytes, 56 (5.2%) had one PN, 57 (5.3%) had three or more PN, 225 (21%) presented no sign of activation, and 18 (1.7%) oocytes had a delayed start of the fertilization process. Of the 29 oocytes injected after maturing overnight, 25 survived (86%) and 10 fertilized normally (40%). The total normal fertilization rate was 67% per oocyte surviving injection and 61% per oocyte injected (Table 2). In total, 662 embryos developed from the normally fertilized oocytes, giving a cleavage rate of 92% and a mean of 5.5 embryos per Table 2 Fertilization Results With Intracytoplasmic Sperm Injection Intracytoplasmic sperm injection Oocytes Group treatments injected Normal Embryos Oocytes Normal fertilization per oocyte surviving fertilization Embryos rate* injectedt % % Severe oligospermia Oligoasthenoteratospermia Asthenoteratospermia Miscellaneous low fertilization rates in vitro Male genital tract obstruction Total 119 1,185 * Oocytes fertilized normally per oocyte surviving injection. t Normal embryos per oocyte injected. 364 Harari et al. Male infertility type and ICSI results :j: 65:j: 1, :j: The results for the male genital tract obstruction group are significantly different from those of the other groups, P < 0.01.

6 Table 3 Average Fertilization Results With Intracytoplasmic Sperm Injection* Intracytoplasmic sperm injection Oocytes Oocytes Normal Group treatments injectedt survivedt fertilization:j: Embryos:j: Severe oligospermia (1 to 20) 7.9 (1 to 20) 4.8 (0 to 13) 4.4 (0 to 13) Oligoasthenoteratospermia (1 to 26) 10.2 (1 to 20) 6.4 (1 to 15) 6.0 (1 to 15) Asthenoteratospermia (3 to 22) 10.0 (3 to 20) 7.0 (3 to 15) 6.4 (2 to 13) Miscellaneous low fertilization rates in vitro (1 to 23) 6.6 (0 to 20) 4.4 (0 to 11) 4.0 (0 to 11) Male genital tract obstruction (1 to 20) 10.8 (1 to 20) 8.1 (1 to 17) 7.4(1 to 17) Total (1 to 26) 9.0 (0 to 20) 6.0 (0 to 17) 5.5 (0 to 17) * Values are means with ranges in parentheses. t Mean results of miscellaneous low fertilization rates in vitro are significantly different from those of oligoasthenoteratospermia, asthenoteratospermia, and male genital tract obstruction, P < :j: Mean results of miscellaneous low fertilization rates in vitro and severe oligospermia are significantly different from those of asthenoteratospermia and male genital tract obstruction, P < treatment (Table 3). In 98% ofthe cycles (117 of 119), one or more embryos were formed. In the two cases where no embryos formed, only one and three 00- cytes were retrieved. The single oocyte did not survive after the intracytoplasmic sperm injection procedure. In the other couple only two oocytes were mature and microinjected, but neither fertilized. From the 20 eggs injected sub zonally, 4 fertilized normally (20%), 1 oocyte developed 1PN (5.0%), and 6 oocytes fertilized with 3PN (30%). The SUZI fertilization rate was significantly less than the normal fertilization rate for the sibling oocytes treated by intracytoplasmic sperm injection (20% versus 71%, P = 0.002), therefore, all but seven of the remaining patients had their oocytes treated by intracytoplasmic sperm injection alone. In the seven patients who had both standard IVF and intracytoplasmic sperm injection, normal fertilization occurred in 7 of 42 (17%) oocytes inseminated by standard methods compared with 32 of 46 oocytes (70%, P < 0.001) treated with intracytoplasmic sperm injection. Also only two of the seven patients had fertilization with standard IVF whereas all had fertilization with intracytoplasmic sperm injection. The embryos developed from SUZI and standard IVF were not transferred at the same time as intracytoplasmic sperm injection embryos and are not considered further; the remainder of this report deals with results obtained with intracytoplasmic sperm injection embryos exclusively. Embryo Transfer Results Fresh embryo transfers were performed as follows: 44 (39%) were three embryo transfers, 51 (46%) were two embryo transfers, and 17 (15%) were one embryo transfers. Five patients did not have a fresh embryo transfer because ofthe risk of ovarian hyperstimulation (>20 oocytes collected). Two patients did not have any embryos. In total, 251 fresh embryos were transferred in 112 procedures; 2.2 embryos per transfer. In 114 treatments, 409 embryos were frozen (mean 3.6 embryos per cycle) and 224 were transferred subsequently in 118 procedures before July 1994 as follows: 1 (0.8%) four-embryo transfer, 5 (4.2%) three-embryo transfers, 93 (79%) two-embryo transfers, and 19 (16%) one-embryo transfers. Some (23,9.3%) frozen embryos did not survive after thawing. Remaining in storage are 162 embryos; 103 from couples who are pregnant and 59 still to be transferred in couples not yet pregnant (Table 4). Thirteen clinical pregnancies were achieved after 112 transfers of251 fresh embryos (11.6% per transfer, 9.7% per oocyte collection) and 23 after 118 transfers of 224 frozen-thawed embryos (19.5% per transfer). The implantation rate was significantly lower with fresh (4.8%) than with thawed embryo transfers (10%, P = 0.024). There were three continuing twin pregnancies each from transfer of two thawed embryos. Seven pregnancies occurred with three embryo transfers (5 fresh, 2 thawed) and 29 pregnancies occurred with two embryo transfers (9 fresh, 20 thawed). No pregnancy occurred after the transfer of one embryo. Of the 36 pregnancies, 12 were lost: 6 fetal heart-positive first trimester spontaneous abortions, 1 twin pregnancy second trimester abortion, 1 tubal ectopic pregnancy, 3 with single anembryonic sacs, and 1 with 3 anembryonic sacs. Thus the pregnancy rates were as follows: clinical 11.6% and continuing 6.2% per fresh transfer, clinical 19.5% and continuing 14.4% per frozen transfer, and, for combined fresh and frozen embryo transfers, clinical 30.2% and continuing 20.2% per commenced intracytoplasmic sperm injection treatment. Only one couple conceived naturally during the study period. They had idiopathic asthenoteratospermia and previous low fertilization rates with several stan- Harari et al. Male infertility type and ICSI results 365

7 Table 4 Embryos and Embryo Transfer Results with Intracytoplasmic Sperm Injection Cryopreserved Continuing embryos pregnancy Intracytoplas- remaining Fetal heart Pregnancies rate per mic sperm Embryos (FH)im- intracytoplasinjection transferred Preg- Not plantation FHI Bio- mic sperm Group* treatments (ET) nant pregnant rates ET chemical Clinical Lost injection Severe oligospermia Oligoasthenoteratospermia ABthenoteratospermia Miscellaneous low fertilization rates in vitro Male genital tract obstruction Total % % dard IVF treatments. Six embryos resulted from injection of nine oocytes, three were transferred fresh without a pregnancy and three remain frozen. Effect of Cause of Infertility Fertilization results were significantly higher with obstructions than in the other groups combined (Table 2) for both normal fertilization rates (75% versus 65%, P = 0.003) and proportions of embryos developing per oocyte injected (65% versus 54%, P = 0.001). The fetal heart implantation rate, the quotient of number of fetal hearts detected and number of embryos transferred (Table 4), was 10% in the obstruction group and was not significantly different from that of the other groups combined (6.6%, P = 0.23). The loss of clinical pregnancies was lower in the obstruction group (0/9) than with the others (12/27, P = 0.014, binomial exact test). Patients with severe oligospermia and previous low IVF ofmiscellaneous causes had significantly fewer embryos develop than did the other groups (Table 3). Considering the nonobstruction groups separately, there were 57 treatments in patients with previous poor results with standard IVF and 41 in patients without prior IVF. The normal fertilization rates were similar (63% and 67%) but the proportion of transferable embryos developing per oocyte injected was significantly lower (P = 0.026) in patients with previous low IVF (295 of 583, 51%) than in those having their first IVF with intracytoplasmic sperm injection (210 of 362, 58%). Pregnancy loss was not significantly different in the two groups: 6 of 15 (40%) in patients with previous IVF and 6 of 12 (50%) in new patients. Analysis of smaller subgroups did not reveal any other significant differences. DISCUSSION In this report of intracytoplasmic sperm injection in 119 couples with severe male factor infertility or persistent failure of fertilization in vitro, the normal 366 Harari et al. Male infertility type and lcsl results fertilization rate was 67% and there was a low rate of loss of oocytes because of the procedure (9.5%). The fertilization, implantation, and continuing pregnancy rates are comparable with those of our standard IVF results with normal semen (60%, 7%, and 23%) (5). The fertilization rates compare favorably with those of previous reports of intracytoplasmic sperm injection (8, 13-16). The fetal heart implantation rate of 7.4% is lower than achieved by the Brussels group (14). Pregnancy and implantation rates generally seem to be lower in Australia than reported by groups in other countries (22). With our patients having intracytoplasmic sperm injection there was a high probability (98%) of getting an embryo for transfer and most of the couples (85%) had more than one embryo transferred. Continuing pregnancies occurred at a rate of 20% per intracytoplasmic sperm injection procedure; but as yet, not all the frozen oocytes have been transferred. Pregnancies were achieved with the transfer of fresh and frozen thawed embryos with fetal heart implantation rates of 4.8% and 10.0%, respectively. This probably results from the practice of cryopreserving the best embryos and transferring fresh embryos considered unsuitable for freezing to maximize the cumulative chance of a singleton pregnancy per procedure. This confirms that intracytoplasmic sperm injection is a major breakthrough in the management of severe male infertility as previously patients with these types of problems were unable to produce pregnancies naturally or did so at very low rates. The remarkable change in outlook for these patients is exemplified by the following historical perspective of assisted fertilization endeavors in our programs. In the period between February 1988 and June 1990 we used mechanical zona opening for couples with failure of fertilization in standard IVF or with motile sperm number < 2 X 10 6 per ejaculate. A controlled trial involving 93 treatments was conducted with 615 oocytes being zona opened and 145 sibling control oocytes remaining intact. After in-

8 II semination, none of the zona intact and 130 of the zona opened oocytes were fertilized (21%),83 being monospermic (13%). In 35 cycles, embryo transfer was carried out (38%) but no pregnancy occurred. Patients with the same criteria were treated by SUZI between August 1991 and June In 152 treatment cycles, 1,477 oocytes were inseminated subzonally. Fertilization occurred in 94 cycles (62%) but only 229 oocytes were fertilized normally (16%). Clinical pregnancies were achieved on five occasions, i.e., a pregnancy rate of 5.3% per treatment. There were also seven biochemical pregnancies. Of the five clinical pregnancies, three had only SUZI embryos transferred and two had mixed SUZI and conventional IVF embryos transferred. From June 1993 patients were recruited for a controlled trial of SUZI and intracytoplasmic sperm injection using the techniques learned from Palermo et ai. (13). Sibling 00- cytes were allocated randomly to SUZI or intracytoplasmic sperm injection but the significantly better monospermic fertilization rate with intracytoplasmic sperm injection and lack of difference in proportions of oocytes damaged and cleavage rate was so obvious that the trial was abandoned after the first four patients had been treated. The intracytoplasmic sperm injection technique in our units is slightly different from that of the Brussels group (13-15). Sperm preparation usually involved washing, separation on a mini-percoll gradient, centrifugation, and resuspension, but on occasion only washing was possible. High speed centrifugation was not always used. These modifications did not appear to affect the results. Sperm were immobilized by placing the injection pipette tip on the sperm tail and crushing it. We did not use polyvinyl pyrrolidone (PVP). Polyvinyl pyrrolidone has been introduced to aid capture of highly motile sperm and to overcome sticking of the sperm head in the lumen of the microinjection pipette, as well as a means of getting a better control on the injection flow during the procedure. Although we found it difficult initially to aspirate highly motile sperm into the injection pipette, this was overcome easily with experience. Not using PVP eliminates exposing sperm and oocytes to a potentially toxic compound. The necessity of the acrosome and acrosome reaction in normal mammalian fertilization is well defined. Also, acrosome-reacted sperm may be important for SUZI, as injecting acrosome-reacted sperm sub zonally increases the fertilization rate in the mouse. However, results are conflicting in the human (8, 23). We decided to avoid special techniques to induce the acrosome reaction before intracytoplasmic sperm injection. It may be that mechanical factors involving sperm aspiration, immobilization, and injection initiate the acrosome reaction in the injected sperm. Similarly, calcium plays a significant role in oocyte activation in mammals, including humans, but its role in intracytoplasmic sperm injection is unclear. The concentration of calcium in the sperm suspension that we used for intracytoplasmic sperm injection was 2.04 mm and we did not alter the calcium concentration during sperm preparation as suggested originally by the Brussels group (Van Steirteghem AC, Joris H, Liu J, Nagy Z, Bocken G, Vankelecom A, et ai., 1st International Course on Assisted Fertilization by intracytoplasmic sperm injection, Brussels, 1993). More recently they have reported that such special preparation of the sperm for intracytoplasmic sperm injection is not necessary (24). Fertilization and embryo development with intracytoplasmic sperm injection are slightly different from those with standard IVF. When the fertilization assessment was carried out 15 to 18 hours after intracytoplasmic sperm injection the PN stage of development was missed in 2.7% of the oocytes. Fertilized oocytes in this group showed regular cleavage similar to their sibling 2PN zygotes and were considered normally fertilized. In two patients in which earlier observations were done, 2PN were seen in two oocytes 6 hours after injection and, in one, the PN were no longer evident by 18 hours. Second polar body extrusion was observed in some oocytes as early as 4 hours after injection. Early stages of development may occur more quickly and be shorter in intracytoplasmic sperm injection-fertilized oocytes than in standard IVF -fertilized oocytes. Further studies are needed to define the extent and significance of early and abnormal fertilization with intracytoplasmic sperm injection. The results of intracytoplasmic sperm injection were related to the cause of infertility in that fertilization rates were higher and pregnancy loss lower with sperm from men with genital tract obstruction than from the other groups. It is possible these results are fortuitous because of the small numbers of subjects studied. For example, it is likely that some spontaneous abortions will occur in the obstruction group as more patients are treated. Furthermore, Tournaye et ai. (25) reported four biochemical pregnancies or miscarriages out of seven pregnancies in 12 couples having intracytoplasmic sperm injection for congenital absence of the vasa. However, real differences in fertilization and continuing pregnancy rates could arise because the gametes and the uterine environment are more likely to be normal in couples with male genital tract obstruction than in those with other causes of infertility. Apart from a significantly higher proportion of embryos developing in new patients, whether the patients had previous poor results with standard IVF or were having Harari et al. Male infertility type and ICSI results 367

9 intracytoplasmic sperm injection with their first IVF treatment, did not influence fertilization or continuing pregnancy rates. These results confirm that intracytoplasmic sperm injection is a valuable treatment for patients with severe male factor infertility of diverse types who have no other proven treatment options except the use of donor sperm. In general, intracytoplasmic sperm injection in these patients can now produce results comparable with those of standard IVF with normal semen. A possible increase in pregnancy loss in patients without male genital tract obstruction requires further study. Another important task for the future is to develop efficient methods for allocating patients to intracytoplasmic sperm injection or standard IVF. Acknowledgments. We thank the medical, laboratory, and nursing staff of the Reproductive Biology Unit and Melbourne IVF for their assistance in the clinical management of the patients. 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