Factors of importance for the establishment of a successful program of intracytoplasmic sperm injection treatment for male infertility

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1 FERTILITY AND STERILITY Copyright Cl 1995 American Society for Reproductive Medicine Vol. 63, No.4, April'1995 Printed on acid-free paper in U. S. A. Factors of importance for the establishment of a successful program of intracytoplasmic sperm injection treatment for male infertility Peter Svalander, Dr.Med.Sc. * Ann-Sofie Forsberg, B.Sc. Ann-Helene Jakobsson, Ph.D. Matts Wikland, M.D., Ph.D. Fertility Center Scandinavia, Carlanderska Hospital, G6teborg, Sweden Objective: To establish an intracytoplasmic sperm injection treatment program for couples with male infertility and to determine those factors important for success. Design: A retrospective analysis of 171 consecutive cycles of intracytoplasmic sperm injection concerning 145 infertile couples. Setting: Infertility clinic in a private hospital associated with a university hospital. Patients: Couples with infertility in the male partner whose sperm parameters were unacceptable for conventional IVF or in whom fertilization by conventional IVF failed repeatedly. Interventions: One hundred seventy-one transvaginal oocyte retrievals were completed after superovulation with GnRH agonist and gonadotropins. Main Outcome Measures: The parameters evaluated included fertilization, cleavage, implantation, pregnancy, and spontaneous abortion in relation to patient indications and improved procedures. Results: After intracytoplasmic sperm injection, normal fertilization occurred in 45% of the oocytes (n = 1,499). Of 171 treatment cycles, 93% of the couples had fertilization and 86% had ET. Thirty-six pregnancies were achieved. During the period studied, the mean fertilization rate increased from 21.3% during the first 17 weeks to 67.8% during the last 13 weeks, and the pregnancy rate (PR) per started cycle increased from 12.8% to 31.3%. Conclusions: Technical factors critical for achieving high rates of fertilization and pregnancy were the use of standardized intracytoplasmic sperm injection pipettes, the immobilization of sperm before injection, and the aspiration of a minimal amount of ooplasm before reinjection with the sperm. Intracytoplasmic sperm injection appears to be superior to other micromanipulation methods for alleviating male infertility. Fertil Steril 1995;63: Key Words: In vitro fertilization, male factor, infertility, micromanipulation, intracytoplasmic sperm injection Intracytoplasmic sperm injection is a new and promising technique for assisted fertilization. The technique is based on the deposition of a single spermatozoon directly into the ooplasm of a mature Received May 25, 1994; revised and accepted November 1, * Reprint requests: Peter Svalander, Dr.Med.Sc., Fertility Center Scandinavia, Box 5418, G6teborg, Sweden (FAX: ). metaphase II oocyte. After being developed in animals and shown to produce live offspring (1) and after preclinical evaluation in human oocytes (2), intracytoplasmic sperm injection was introduced clinically by Palermo et al. (3, 4) and Van Steirteghem et al. (5, 6). Intracytoplasmic sperm injection bypasses many steps in the fertilization process, such as the spermzona pellucida binding, the acrosome reaction, and the sperm-oolemma fusion. Although the impor- 828 Svalander et al. Establishment of an ICSI program Fertility and Sterility

2 tance of these steps in normal reproduction are rather well known, none of these reactions are required with intracytoplasmic sperm injection for the establishment of pregnancy and the birth of a healthy infant. In our IVF program, micromanipulation with the subzonal insemination (SUZI) technique has been used since 1991 for couples suffering from male infertility. From May 1993 onward, we decided to change our technique from SUZI to intracytoplasmic sperm injection, encouraged by the promising results reported by the Brussels group (3-6). Since then, couples admitted to our unit for male infertility were considered for intracytoplasmic sperm injection treatment. Based on our previous experience of micromanipulation, we believed that the transition from SUZI to intracytoplasmic sperm injection would be easy. However, it turned out that there are certain technical aspects of the method that are crucial for establishing a successful intracytoplasmic sperm injection program. This study describes technical factors necessary for achieving a successful clinical intracytoplasmic sperm injection program. Patients MATERIALS AND METHODS The study period was from May 1993 until February 1994 and included 145 couples treated in 171 consecutive intracytoplasmic sperm injection cycles. The selection of couples for the intracytoplasmic sperm injection method was based on our previ- 0usly established criteria for using the SUZI technique. These criteria were chosen arbitrarily, based on our experience that the chance offertilization was extremely poor in conventional IVF or microdrop IVF. In 84 cycles, intracytoplasmic sperm injection was performed because of a low sperm recovery, i.e., <500,000 motile spermatozoa in total after Percoll preparation and at least one sperm parameter below the World Health Organization (WHO) criteria (7) (group I). In 37 cycles, intracytoplasmic sperm injection was used because of failed fertilization in previous IVF attempts (group II). In 50 cycles, the sperm quality was below the WHO criteria in one, two, or three parameters (group III). In the latter two groups, the recovery of sperm after preparation was >500,000 motile spermatozoa in total. The mean age of the female partners was 34 years (range, 21 to 45 years) and that ofthe male partners was 39 years (range, 26 to 53 years). The median duration of infertility for these couples was 4 years (range, 3 to 15 years). The procedure of assisted fertilization abided in all aspects Swedish legislation and was thoroughly explained to the couples. Before intracytoplasmic sperm injection treatment was started, an agreement was made with the couples that, in the case of pregnancy, a prenatal diagnosis by means of amniocentesis would be done. The study was approved by the Ethics Committee of the University of Goteborg, Sweden. Follicular Stimulation Gonadotropin-releasing hormone analogue (buserelin acetate; Hoechst, Stockholm, Sweden) was given as nasal spray starting in the luteal phase and was continued for 2 weeks until sufficient pituitary down regulation was achieved. Follicular development was then stimulated with highly purified 225 IU/d FSH (Fertinorm/Metrodin HP; Laboratoires Serono S.A., Aubonne, Switzerland) until the mean follicular diameter of the three leading follicles had reached ;:::18 mm. Human chorionic gonadotropin (Profasi; Laboratoires Serono S.A.) was then given and oocyte aspiration was performed 38 hours later. Oocyte Retrieval Oocytes were retrieved under guidance of vaginal ultrasound, as described previously (8). The aspirated follicular fluid was immediately passed to the adjoining laboratory. Oocytes were identified in sterile plastic dishes (Sterilin Ltd, Feltham, United Kingdom), rinsed in Earle's balanced salt solution (EBSS; Medi-Cult a/s, Copenhagen, Denmark) and transferred to EBSS-serum medium (EBSS supplemented with 12 mg/l pyruvate, 100,000 IU /L penicillin G, purchased from Sigma Chemical Co, St Louis, MO, and 10% heat-inactivated human serum screened for human immunodeficiency virus and hepatitis A, B, and C). Oocytes were handled with cleaned and heat-sterilized glass Pasteur pipettes (cat. no ; Maple Leaf Brand, Pro Science Inc., Don Mills, Ontario, Canada). After retrieval, the oocytes were incubated for 2 to 4 hours and thereafter prepared for intracytoplasmic sperm injection. Semen Analysis and Sperm Preparation Men were asked to produce an ejaculate in sterile 50 ml Falcon tubes (cat. no. F-2098; Becton Dick- Vol. 63, No.4, April 1995 Svalander et al. Establishment of an lcsl program 829

3 inson, Meylan, France), to maintain the sample at body temperature, and to deliver it to the laboratory no later than 30 minutes after ejaculation. Semen samples were examined by microscopy using a Makler counting chamber (Sefi-Medical Instruments Ltd., Haifa, Israel) and were evaluated according to the WHO criteria (7). The strict criteria of Kruger et al. (9) were used to classify the sperm morphology. Spermatozoa were selected by Percoll density gradient centrifugation (10-12) in conical centrifugation tubes (cat. no ; Nunc a/s, Roskilde, Denmark). The gradients consisted of 1- ml layers of 90% and 40% Percoll (Pharmacia AB, Stockholm, Sweden) prepared in EBSS-HSA medium (EBSS supplemented with 10 mg/ml human serum albumin [HSA; Pharmacia AB, Stockholm, Sweden], 12 mg/lpyruvate, and 100,000 IU/Lpenicillin G). The gradients were centrifuged at 300 X g for 20 minutes in a swing-out rotor. The entire 90% layer was transferred to a new tube and resuspended in 6 ml EBSS-HSA medium. After another centrifugation at 150 X g for 10 minutes, the supernatant was discarded and 1 ml EBSS-HSA medium was added. In cases with very low sperm counts after preparation «0.1 X 10 6 ), the samples were concentrated to a volume of approximately 10 JLL in an Eppendorf centrifuge (model no. 5415C; Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany) at 720 X g for 2 to 3 minutes. After preparation, the sperm sample was evaluated using a Burker hemocytometer and stored on the bench top at room temperature until used for intracytoplasmic sperm injection. Oocyte Preparation Two to four hours after oocyte retrieval, the cumulus cells and the corona radiata were removed by a brief exposure to 80 U /ml hyaluronidase (cat. no. H-4272; Sigma Chemical Co.) diluted in EBSS HSA medium with 15 mm HEPES (cat. no. H-6147; Sigma Chemical Co.). The exposure to hyaluronidase was minimal and the oocytes were denuded completely by repeatedly drawing them through a fine-bore Pasteur pipette, produced by pulling over an open flame, followed by washing three times in EBSS-HSA medium with HEPES. The denudation procedure was done in a four-well culture dish (cat. no ; Nunc a/s) and the denuded oocytes were incubated finally (37 C, 5% CO 2 in air) in EBSS-HSA medium without HEPES until intracytoplasmic sperm injection was performed. All the oocytes were examined by micros- copy to determine their stage of maturation. Only morphologically normal-appearing mature oocytes with a visible first polar body (indicating the metaphase II stage) were micro injected. Equipment for Micromanipulation Intracytoplasmic sperm injection was performed using a Nikon (Nikon Ltd., Tokyo, Japan) Diaphot TMD microscope equipped with Nomarski optics, a 37 C heating stage (Swemed Lab International AB, Viistra Frolunda, Sweden), Narishige MM188 electric coarse movement controls, M0188 3D oil hydraulic micromanipulators, an IM188 injector for the holding pipette, and an IM6 injector for the injection pipette (Narishige Ltd., Tokyo, Japan). Microtools for Intracytoplasmic Sperm Injection The microtools used for intracytoplasmic sperm injection (holding and injection pipettes) were developed in collaboration with Swemed Lab International AB. The purpose was to design high-quality microtools that could be manufactured large scale in a clean-room environment. The glass capillary tubing (cat. no. 1B100-6; World Precision Instruments Inc., New Haven, CT) used in manufacture of the microtools was prerinsed in 18.2 Mrl water (MilliQ-RO-UF system; Millipore AB, V. Frolunda, Sweden), soaked in 2% hydrochloric acid overnight, rinsed in 18.2 Mrl water, soaked in 70% ethanol overnight, rinsed in 18.2 Mrl water, and finally dried in hot air. For manufacture of intracytoplasmic sperm injection pipettes, the pretreated glass tubing was pulled in a Swemed pipette puller (Swemed Lab International AB), ground on a wetted high-speed grinder, and extensively rinsed in 18.2 Mrl water. These pipettes had no distal spike. Every pipette was checked by electronic imaging under X600 microscope magnification. Pipettes having an inner diameter of to mm and an outer diameter of to mm were bent to an angle of 30 at the distal end to facilitate working in a culture dish (Fig. 1). Pipettes that fulfilled these criteria were sterilized by f3-radiation and used for intracytoplasmic sperm injection, after batch-testing was performed for sterility, endotoxins, and DNA. Each pipette carried an evaluation form that was completed at the time of use by the intracytoplasmic sperm injection operator. Data regarding pipette diameter, edge sharpness, bevel, parallelism, and bend angle were compiled retrospectively, and the pipette specifications were adjusted, when necessary, to increase the perfor- 830 Svalander et al. Establishment of an ICSI program Fertility and Sterility

4 0,0068-0, Figure 1 Intracytoplasmic sperm injection pipette specifications in millimeters ,5- ::::!::::::::::::::::::::::::::::::::=:--..:, ,0 mance. The holding pipette had an outer diameter of to mm and an inner diameter of to mm. The holding pipettes also were bent to an angle of 30. The standardized bending angle facilitated horizontal positioning and adjustment of the pipettes. Preparation of Microdroplets for Intracytoplasmic Sperm Injection Intracytoplasmic sperm injection was performed in microdroplets under oil using the lids (covers) of plastic culture dishes (cat. no. 123; Sterilin Ltd). The lids were used because oftheir low edges, which facilitated the positioning ofthe pipettes on the microscope. Mineral oil (cat. no. M-841O; Sigma Chemical Co.) was prepared by shaking twice with 18.2 MQ water and once with EBSS and equilibrating overnight at 37 C in 5% CO 2 in air. To capture a single sperm, a viscous solution of 10% polyvinylpyrrolidone (PVP) in EBSS-HSA medium was used. The viscous ready-to-use PVP solution was produced specifically for human intracytoplasmic sperm injection (lcsi-100; Scandinavian IVF Science AB, G6teborg, Sweden). Each batch is tested for endotoxins, DNA, human sperm survival, and sterility. The PVP solution was stored refrigerated. Using an Eppendorf Varipette (cat. no ; Eppendorf-Netheler-Hinz GmbH) equipped with sterile, pyrogen-free, DNA-free, RNAse-free, adenosine triphosphate-free Biopur pipette tips (cat. no ; Eppendorf Netheler-Hinz GmbH), a 5-ILL droplet of EBSS HSA medium containing 15 mm HEPES was placed in the center of a plastic culture dish cover and five equal droplets were placed closely around it. This arrangement of the droplets minimized movement of the dish on the microscope stage during the intracytoplasmic sperm injection procedure. The droplets were immediately covered by preequilibrated mineral oil to avoid the extremely fast evaporation of these small droplets. Two dishes were prepared for each patient. Shortly before the start of intracytoplasmic sperm injection, the center droplet was replaced with a 5-ILL droplet of PVP solution, and a I-ILL droplet of washed sperm solution was placed in the center of the PVP droplet. Within minutes, motile sperm migrated towards the periphery, passing through the interface between the PVP and the sperm solutions. Two to four oocytes were placed, one in each droplet, in the same dish and the intracytoplasmic sperm injection procedure was started. Intracytoplasmic Sperm Injection Procedure Under the microscope at X200 magnification, the intracytoplasmic sperm injection pipette was lowered directly into the periphery of the PVP solution and filled in the distal end with PVP solution. A single sperm of apparently normal morphology and motility was located. The selected sperm was immobilized by touching the tail with the intracytoplasmic sperm injection pipette and was captured by aspiration into the pipette. The pipette containing the immobilized sperm was moved from the PVP droplet into one of the peripheral droplets containing an oocyte. These steps demanded high precision performance of the oil-hydraulic injection system. The oocyte was held by gentle suction on the holding pipette. The first polar body was positioned in the 6 or 12 o'clock position. With the sperm positioned close to the tip and the edge of the pipette and the oolemma in focus, the intracytoplasmic sperm injection pipette was forced gently horizontally from the 3 o'clock position deep into the ooplasm. Gentle suction was applied carefully to break the oolemmal membrane and to aspirate a minimal amount of ooplasm. This procedure ensured that the sperm was always deposited intracytoplasmically and not in an invagination of the oolemma. The immobilized sperm was inserted together with the withdrawn ooplasm and the smallest volume of PVP solution possible. Thereafter, Vol. 63, No.4, April 1995 Svalander et al. Establishment of an ICSI program 831

5 1 the intracytoplasmic sperm injection pipette was gently withdrawn horizontally, and the oocyte was released from the holding pipette. This process was repeated for each oocyte. The injected oocytes were rinsed in EBSS-HSA medium and were placed in an equilibrated culture dish (cat. no. 3037; Becton Dickinson) with EBSS-serum medium. The incidence of oocyte damage was slightly <10% per injected oocyte. When damage occurred, a darkening and shrinkage of the oocyte was observed the next day. Embryology After intracytoplasmic sperm injection, the 00- cytes were incubated in EBSS-serum medium according to conventional IVF procedures (37 C, 5% CO 2 in air). They were observed 16 to 18 hours later by microscopy to assess pronuclei formation. Normal fertilization was defined as the observation of two distinct pronuclei. Twenty-four hours later, cleavage of the fertilized oocytes was assessed. A few hours later, on the 2nd day of culture, the morphologically best embryos were transferred to the uterine cavity of the patient and supernumerary embryos were cryopreserved using a 1,2-propanediol freezing protocol (13). Embryo Transfer and Luteal Supplementation Embryos were transferred to the uterine cavity using a T.D.T. set (Prodimed, Neuilly-en-Thelle, France). Luteal phase support was provided by 1M injections of 25 mg micronized P (Apoteksbolaget AB, Umea, Sweden) two times per day for 2 weeks or as 300 mg vaginal capsules three times per day (Uterogestane; Lab. Besins-Iscovesco, Paris, France) for 2 weeks. Pregnancy was checked with a conventional urinary hcg test. Clinical pregnancy was verified by the observation of a gestational sac by vaginal ultrasound at 6 to 7 weeks of gestation. The predicted take home baby rate was calculated on deliveries and ongoing pregnancies beyond 12 weeks of gestation having a normal ultrasound scan and normal fetal heart activity. Statistical Methods The means, medians, and standard deviations were calculated. For the comparison of fertilization rates between the two periods, the Mann-Whitney U-test was used (14). The pregnancy and spontaneous abortion rates were compared between the two periods using x 2 analysis. Significance was defined as P < RESULTS Clinical Results of Intracytoplasmic Sperm Injection The clinical results of the first 171 consecutive intracytoplasmic sperm injection cycles are shown in Table 1. Of 1,499 injected oocytes, 682 (45%) were normally fertilized (having two visible pronuclei 16 to 18 hours after intracytoplasmic sperm injection). However, the fertilization rate varied considerably during the study period (Fig. 2). During the first 17 weeks, it was 21.3% ± 10.8% (mean ± SD), and the corresponding figure for the next 13 weeks was 67.8% ± 6.7% (P < 0.05). Overall, fertilization was achieved in 93 % of the started cycles and 86% had ET. The pregnancy rate (PR) per oocyte retrieval was 12.8% during the first 17 weeks and 31.3% during the subsequent 13 weeks (P < 0.05). There were 36 clinical pregnancies, representing a PR of 24.5% per ET and 21.0% per started cycle. The spontaneous abortion rate during the first 17 weeks was 40.0%, whereas the corresponding figure for the next 13 weeks was 21.2% (P < 0.05). The predicted take home baby rate for the Table 1 Clinical Results of Intracytoplasmic Sperm Injection No. of couples Started cycles Oocyte retrieval cycles No. of retrieved oocytes Injected oocytes No. of damaged oocytes Fertilized oocytes (two pronuclei) Oocytes with three pronuclei Fertilization rate per injected oocyte (%) Cycles with fertilization Fertilization rate per started cycle (%) Cleavage rate per fertilized oocyte (%) Embryos to ET Embryos to cryopreservation Cycles with cryopreserved embryos No.ofET Cycles with ET (%) Embryos per ET (mean) Clinical pregnancies No. of gestational sacs Implantation rate (%) Pregnancy rate per ET (%) Pregnancy rate per started cycle (%) Pregnancy rate per oocyte retrieval (%) Ongoing pregnancies Predicted take home baby rate per ET (%) Predicted take home baby rate per started cycle (%) ,724 1, Svalander et al. Establishment of an ICSI program Fertility and Sterility

6 ... \J "Cl \J 50 :e-... =,S:: e'ii N :.= ',c Figure 2 Fertilization rate (%) per microinjected oocyte with the intracytoplasmic 10 sperm injection technique. The arrow at week 17 indi- cates where technical modifications caused an increase in the outcome of intracytoplas- mic sperm injection Week whole period and per started intracytoplasmic sperm injection cycle is 15.2%. For comparison, during the same time period, 156 couples were treated in 185 cycles by conventional IVF because of tubal infertility and without a sperm factor. This group had a fertilization rate of 74.4% and a PR of 36.6% per ET and 32.4% per started cycle. The predicted take home baby rate per started cycle in this group is 24.3%. For intracytoplasmic sperm injection, the average implantation rate was 10.5%; this rate varied in the three groups, being 8.2% in the low sperm recovery group (group I), 11.6% in the failed fertilization group (group II), and 13.4% in the group with abnormal sperm (group III) (Table 2). As a comparison, the implantation rate was 16.7% for conventional IVF during the same time period. In 68 of 171 intracytoplasmic sperm injection cycles (39.8%), embryos were also cryopreserved. Thus far, 13 thaw-et cycles of cryopreserved intracytoplasmic sperm injection embryos have resulted in four ongoing pregnancies. Results of Intracytoplasmic Sperm Injection in Relation to Semen Characteristics The outcome of intracytoplasmic sperm injection related to the number of sperm defects present is shown in Table 3. Although the number of patients in each group is small, there was a tendency for the chance of obtaining an ongoing pregnancy to be reduced in men with several sperm defects according to WHO and Kruger criteria (7, 9). However, this conclusion requires confirmation in a larger series of intracytoplasmic sperm injection cycles. The results of intracytoplasmic sperm injection treatment in relation to sperm count and the presence of mot ility is presented in Table 4. One clinical pregnancy was obtained in the most severe group «100,000 spermatozoa lacking motility); unfortunately, it did not proceed to term. Influence of Technical Improvements on the Results of Intracytoplasmic Sperm Injection In Figure 2, the fertilization rate per week during the 9-month period is shown. The fertilization rate was improved significantly from week 17 onward (Fig. 2, arrow). This increase coincided with certain technical modifications of the intracytoplasmic sperm injection procedure: [1] the use of standardized intracytoplasmic sperm injection pipettes (the variables considered are outlined in Table 5), [2] the immobilization of sperm before injection, and [3] the aspiration of a minimal amount of ooplasm before reinjection with the sperm. DISCUSSION The high fertilization and PRs obtained with intracytoplasmic sperm injection are remarkable, especially considering that 55.5% (95/171) of the started cycles had very poor sperm quality, i.e., two or three sperm parameters below the normal WHO Vol. 63, No.4, April 1995 Svalander et al. Establishment of an ICSI program 833

7 1 Table 2 Results of Intracytoplasmic Sperm Injection in Relation to Clinical Indication Group I <0.5 X 1Q6/mL after preparation Group II Previous failed fertilization Group III Abnormal sperm quality but >0.5 X 1Q6/mL after preparation ICSI cycles 84 Sperm recovery (XI06) Mean 0.19 Median 0.20 Range 0.01 to 0.5 Abnormal morphology (%) Mean 95 Median 94 Range 85 to 99 Cycles with fertilization 77 No. retrieved oocytes 940 Injected oocytes 792 Fertilized oocytes (two pronuclei) 337 Oocytes with three pronuclei 1 Fertilization rate (%) 42.5 Cleavage rate (%) 98 Embryo transfers 72 Embryos to ET 158 Embryos to ET (mean) 2.2 Clinical pregnancies 15 No. of gestational sags 13 Implantation rate (%) 8.2 Ongoing pregnancies 11 Pregnancy rate per started cycle (%) 17.9 Pregnancy rate per oocyte retrieval (%) 17.9 Pregnancy rate per ET (%) to to to to criteria (see Table 3). Although the average fertilization and PRs initially were not as high as reported by the Brussels group (3-6), it is reassuring so see that these figures increased considerably during the course of the study period, reaching the reported levels during the last 13 weeks. During the establishment of intracytoplasmic sperm injection, the fertilization and PRs increased as the spontaneous abortion rate decreased, without changing the selection criteria of the patients. This was taken as a sign of accumulation of the technical skill considered crucial for the outcome of the procedure. Thirteen thaw-et cycles of cryopreserved intracytoplasmic sperm injection embryos resulted in four Table 3 Results of Intracytoplasmic Sperm Injection in Relation to Semen Characteristics (WHO and Kruger Criteria)* Semen characteristics Normal sperm (failed fertilization) Single sperm defect Oligozoospermia Asthenozoospermia Teratozoospermia Total Double sperm defects Oligoasthenozoospermia Asthenoteratozoospermia o ligoteratozoospermia Total Triple sperm defects Oligoasthenoteratozoospermia Total * See references 7 and 9. Cycles Clinical Ongoing Embryo transfers pregnanciest pregnanciest 21 5 (20.0) 5 (20.0) 13 4 (22.2) 3 (16.7) 24 7 (23.3) 6 (20.0) (21.6) 9 (17.6) (22.4) 9 (13.4) 4 2 (50.0) 1 (25.0) (23.6) 10 (13.9) 21 3 (13.0) 2 (8.7) (21.1) 26 (15.2) t Values in parentheses are percent per started cycle. 834 Svalander et al. Establishment of an ICSI program Fertility and Sterility

8 Table 4 Results of Intracytoplasmic Sperm Injection in Relation to Sperm Count and Presence of Motility Initial sperm count <100, ,000 to 500,000 >500,000 Motile Nonmotile Motile Nonmotile Motile Intracytoplasmic sperm injection cycles Sperm recovery (X10 6 ) Mean Median Range Abnormal morphology (%) Mean Median Range Cycles with fertilization Injected oocytes Fertilized oocytes Fertilization rate (%) Cleavage rate (%) Embryo transfers Embryos to ET (mean) Clinical pregnancies Implantation rate (%) Ongoing pregnancies Prgnancy rate (%) Per started cycle PerET to to to to to 25.0 ND* 90 ND to * ND, not determined. ongoing pregnancies, indicating that routine cryopreservation of intracytoplasmic sperm injection embryos is possible. Thus, to date, the first series of 145 infertile couples treated with intracytoplasmic sperm injection have resulted in the birth of two children and 28 ongoing pregnancies, including the first 13 ETs with cryopreserved intracytoplasmic sperm injection embryos. Table 5 Microtool Variables and Their Possible Influence on Intracytoplasmic Sperm Injection Microtool variables Inner diameter Outer diameter Grinding wheel quality Bevel angle Bending angle Siliconization Length of the narrow part Spike Washing protocol Sterilization protocol Quality testing Possible influence on intracytoplasmic sperm injection Sperm capture Volume load Oocyte-oolemma damage Edge sharpness Edge sharpness and penetration Microscope adjustment Horisontal orientation Adhesion Blunting of the edge Longitudinal "stability Penetration Contamination Sterility Reliability and safety It is noteworthy that during the last 13 weeks, the fertilization and PRs approached the results for conventional IVF in couples with tubal factor infertility without sperm factor. This confirms the high efficacy of the intracytoplasmic sperm injection method, as reported previously by the Brussels group. Using the correct technique and high-quality equipment, such as standardized microtools and PVP solution, seem to be important for reaching consistent good results with intracytoplasmic sperm injection. After experiencing limited success during the first 17 weeks, there was a threefold increase in the fertilization rate (Fig. 2) after modifying three technical details of the procedure. First, we adhered strictly to uniform pipette specifications (see Materials and Methods). Second, the sperm were immobilized by gently touching the tail with the intracytoplasmic sperm injection pipette. Otherwise, a motile sperm inside the oocyte will form a vacuole, which is detrimental to the outcome of the procedure. Third, to ensure a correct deposition of the sperm, a minimal amount of the ooplasm was aspirated into the pipette before reinjection with the sperm, thereby confirming penetration of the oolemma. The simplifications of the sperm preparation method used for intracytoplasmic sperm injection Vol. 63, No.4, April 1995 Svalander et al. Establishment of an ICSI program 835

9 compared with SUZI save time that can be used for performing the intracytoplasmic sperm injection procedure. In our laboratory, having a total staff of three embryologists, two to three intracytoplasmic sperm injection treatment cycles are performed each day, while a similar number of conventional IVF cycles also are performed daily. Thus, once established, the intracytoplasmic sperm injection procedure can be integrated into the normal IVF laboratory routine. Encouraged by the high fertilization, ET, and PRs of intracytoplasmic sperm injection reported by the Brussels group (3-6) and their controlled comparison of SUZI and intracytoplasmic sperm injection on sibling oocytes, we decided to abandon the SUZI technique in May In retrospect, it can be argued that, before changing completely to the intracytoplasmic sperm injection method, it would have been wise to conduct a randomized study of the two procedures, intracytoplasmic sperm injection or SUZI, in the couples with male infertility. However, because the Brussels group recently had carried out such a comparison showing an advantage for intracytoplasmic sperm injection, we considered it unethical to allocate couples to SUZI, knowing it to be less effective. During the introduction of intracytoplasmic sperm injection, we soon experienced that several technical difficulties existed when performing the technique. During the first 4 months, the fertilization rate was below expectation, although it was comparable to that obtained previously by our group with the SUZI technique. In attempting to improve the results, we re-evaluated the technical procedures. One key factor in intracytoplasmic sperm injection is the design and quality of the microtools. It is crucial to have uniform microtool quality to obtain consistent results. The quality of the microtools may influence the outcome of intracytoplasmic sperm injection in several ways. For example, a blunt injection pipette can damage the oocyte by compression, whereas a pipette with a diameter too large could cause an overload of injected fluid. The consequences of overloading the oocyte with fluid have been presented recently by Tesarik and co-workers (15), who provided data showing that an excessively high calcium load may induce a deficiency in the regenerating mechanisms of the injected oocyte. To attain the technical skill required for intracytoplasmic sperm injection, our previous experience with other micromanipulation techniques was a great asset. A new assisted fertilization technique, such as in- tracytoplasmic sperm injection, must be applied with caution before large clinical follow-up studies can establish its safety. Because it is likely that many laboratories will begin using intracytoplasmic sperm injection within the near future, it is of utmost importance to reach a uniform safety level for this procedure. One aspect of the safety problem is the possibility that foreign DNA may be introduced accidentally into human oocytes. This could occur because of contamination from the microtools or the PVP solution. Because sperm are able to bind DNA to their cell surfaces and then internalize it (16), it is also important to consider the use of proper sperm preparation methods for intracytoplasmic sperm injection. For example, the spontaneous capacity of sperm to take up and internalize exogenous DNA is exaggerated by membrane damage, and this is known to occur under high centrifugation forces. To reduce such risks, it is important to avoid DNA contamination in everything that comes into contact with the sperm. This excludes, for instance, the use of human serum in any step of the intracytoplasmic sperm injection procedure. However, it may be that the fertilized oocyte can inactivate naturally transmitted and internalized foreign DNA. An even more disturbing mechanism for introduction offoreign DNA or RNA is via virus contamination, particularly those viruses carrying oncogenes. To further reduce the potential risks with intracytoplasmic sperm injection, animal material such as bovine serum albumin should never be used for human intracytoplasmic sperm injection. Instead, it is recommended that only Food and Drug Administration-approved pharmaceutical substances be used in intracytoplasmic sperm injection procedures. Consequently, to guarantee a completely safe procedure, it is necessary to perform intracytoplasmic sperm injection with precautions that well exceed those necessary for conventional IVF routines. These considerations were the driving force behind the decision to enter developmental projects for new standardized microtools and for pharmaceutical quality PVP solution, with the ultimate aim of reducing the contamination risks of the intracytoplasmic sperm injection procedure. In conclusion, we believe that the improvements in fertilization and PRs seen with intracytoplasmic sperm injection in the middle of the study period primarily were due to technical improvements. Our opinion is that the intracytoplasmic sperm injection technique has revolutionized the treatment of male infertility and probably will become the 836 Svalander et al. Establishment of an lcsl program Fertility and Sterility

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