Intracytoplasmic sperm injection for treating infertility associated with sperm autoimmunity

Transcription

1 FERTILITY AND 8TERILITY 1997 American Society for Reproductive Medicine Published by Elsevier Science Inc. Vol. 68, No.1, July 1997 Printed on acid-free paper in U. S. A. Intracytoplasmic sperm injection for treating infertility associated with sperm autoimmunity Gary N. Clarke, M.Sc.*t Harold Bourne, M. Rep. Sci.t H. W. Gordon Baker, M.D., Ph.D. The Royal Women's Hospital, and University of Melbourne, Carlton, Victoria, Australia Objective: To determine whether intracytoplasmic sperm injection (ICS!) can be used to achieve normal fertilization, embryo cleavage, and pregnancies in cases of sperm autoimmunity. Design: A retrospective analysis of ICSI results in sperm antibody-positive and randomly selected antibody-negative groups. Setting: University- and hospital-based reproductive research laboratory and tertiary referral IVF program. Patient(s): Thirty-nine couples selected on the basis of a strongly positive result for sperm antibodies of immunoglobulin (Ig) G and/or IgA immunoglobulin class in the male partner and a control group of 140 antibody-negative couples. Intervention(s): Human menopausal gonadotropin, hcg and Lucrin (Abbott Australasia, Kurnell, NSW, Australia) were given by injection. Oocyte collection was by transvaginal ovarian puncture. Blood was collected for,b-hcg measurement. Main Outcome Measure(s): Normal fertilization, embryo cleavage, establishment of clinical pregnancy, and delivery. Result(s): There were no significant differences in fertilization rates (62% versus 58%) or clinical pregnancy rates (19% versus 12%) between sperm antibody-positive and sperm antibody-negative patient groups. Conclusion: Intracytoplasmic sperm injection is an effective treatment for patients with severe sperm autoimmunity. (Fertil Sterilv 1997;68: by American Society for Reproductive Medicine.) Key Words: Intracytoplasmic sperm injection (ICSn, sperm autoimmunity Previous studies have indicated that high-level sperm autoantibodies are associated with significantly reduced fertilization rates in conventional IVF 0-3). The results of several investigations using human salt-stored zonae pellucidae (ZP) have demonstrated that antibody-coated sperm are much less likely to bind to the zona (4-6). If this is the predominant mechanism by which antibodies block fertilization, then injection of the sperm directly into Received December 16, 1996; revised and accepted March 24, * Andrology Unit, Department of Pathology, The Royal Women's Hospital. t Reprint requests: Gary N. Clarke, M.Sc., Andrology Unit, Department of Pathology, The Royal Women's Hospital, Carlton, Victoria, Australia 3053 (FAX: ). *Reproductive Biology Unit, The Royal Women's Hospital. University of Melbourne. 112 the oocyte cytoplasm should achieve normal fertilization rates for such patients. The first detailed report ofintracytoplasmic sperm injection (lcs!) treatment for sperm autoimmunity was published by Nagy et. al. (7). They reported good fertilization rates and subsequently detected fetal sacs by ultrasonography in 14 patients. They also reported, however, that the embryos were of significantly poorer quality in the sperm antibody-positive patients. This finding prompted us to analyze our results of ICSI treatment for cases of severe sperm autoimmunity, with particular focus on embryo quality, fetal heart-confirmed pregnancy rates, and deliveries. Patient Selection MATERIALS AND METHODS Intracytoplasmic sperm injection treatment commenced at The Royal Women's Hospital on June 23, /97/$17.00 PH (97)

2 1993. The results of treatments up to May 13, 1996, were included in this analysis. Patients were included in the study group if their sperm-antibody test closest to the ovum pick-up cycle indicated that at least 80% of their motile spermatozoa were coated with antibodies of immunoglobulin (Ig) class A or IgG. If cryopreserved sperm were used, then the sperm-antibody test closest to the day of cryopreservation was used to select the patients. In patients in whom the sperm-antibody result varied spontaneously or with corticosteroid treatment, some treatment cycles were included and some were excluded on the basis of the criteria just described. A total of nine men were receiving prednisolone (50 mg/d) at the time when they produced the semen sample used for ICSI. The results of standard IVF treatment cycles for the study group were also analyzed. The control group consisted of all couples with both partners negative for sperm antibodies. These couples were undergoing ICSI treatment because of poor semen quality (count < 1 x 10 6/mL or motility <20% or abnormal sperm 2:95%, or combined problems with count <20 x 10 6/mL, motility <40%, and abnormals >85%). Semen Analysis Semen analyses were performed manually according to World Health Organization (WHO) guidelines (8), except for determination of sperm velocity. The mean straight-line velocity (VSL) was determined using a Hamilton-Thorne Motility Analyzer (HTM-2030; Hamilton-Thorne Research, Beverley, MA) running on version 7.2 software. The analysis was performed at 37 C using 20-J.lm deep Microcell Chambers (Conception Technologies, La Jolla, CA) with the motility analyzer frame rate set at 25/s. Sperm Antibody Detection Sperm antibodies were detected using the direct Immunobead Test (IBT) (2), with slight modifications as described here. Indicator particles (Immunobeads) were obtained from Irvine Scientific (Santa Ana, CA) and consisted of polyacrylamide beads of 5 to 10 us» in diameter with covalently bound rabbit antibodies to human immunoglobulin classes (anti IgG, cat. no ; anti-iga, cat. no ). The lyophilized immunobeads (50 mg) were reconstituted by adding 5 ml of sterile deionized water (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia). After reconstitution, the immunobeads could be stored at 4 C for 4 to 6 weeks. Immediately before use, an aliquot of each reagent was washed once in Tyrode's solution (Commonwealth Serum Laboratories) containing 0.3% bovine serum albumin (Tyrode's BSA) and resuspended to Vol. 68, No.1, July to 10 mg/ml in Tyrode's solution containing 5% BSA (TBSA-5). Sperm were washed (600 x g for 5 minutes at room temperature) twice in TBSA (prewarmed to 30 to 35 C) and resuspended to 10 X 10 6 to 20 x 10 6/mL motile sperm in TBSA-5. A drop of washed immunobead reagent was mixed with an equal drop of washed sperm (7 JLL), covered with a coverslip, and incubated in a moist petri dish for 10 minutes at room temperature. The reaction was observed under phase-contrast optics at a magnification of x400. A motile sperm was scored as positive if two or more beads were bound to its surface. A specimen was classed as positive when 2:50% of motile sperm showed positive binding. This study has focused on patients with 2:80% of motile spermatozoa coated with IgG- or IgA-class antibodies, so only patients with severe sperm autoimmunity were included. Full details of immunobead binding test (IBT) methodology as used in this laboratory have been published elsewhere (9). Ovarian Stimulation and Oocyte Collection In most cases, a short ovarian stimulation protocol (10) was used that consisted ofhmg (Humegon; Organon, Lane Cove, NSW, or Pergonal; Serono, Frenchs Forest, NSW, Australia) and GnRH analogue (Lucrin; Abbott Australasia, Kurnell, NSW, Australia). Oocytes were collected by ultrasound (US)-guided transvaginal ovarian puncture 36 hours after the administration ofhcg (Pregnyl; Organon). Luteal-phase support was given in the form of P pessaries (100 mg/d; The Royal Women's Hospital, Melbourne, Victoria, Australia) or hcg injections (1,000 IU every 3 days) for 12 days after oocyte retrieval. Preparation of Sperm Sperm were isolated from ejaculates produced on the day of oocyte retrieval or from a sample that was cryopreserved before treatment. For the frozen samples, semen was mixed 3:1 with glycerol-egg yolk-citrate medium, sealed in 0.5-mL plastic straws, and cooled at -1.5 C/min from 20 C to -GoC followed by -6 C/min down to -loo C before storing in liquid nitrogen. The cryoprotective medium was based on Clarke et al. (11), but lacked kanamycin. The final mixture contained approximately 30% glycerol, 1.5% glucose, 1.0% sodium citrate, 1.3%glycine, and 17% egg yolk. The diluent (approximately 400 mosmlkg excluding glycerol, ph 7.0) was added to the semen in five aliquots over 10 minutes at room temperature. The final percentage of glycerol in the diluted sample was 7.0% to 7.5%.Prior to use, straws were air thawed and the sperm samples were prepared by swim-up or using standard mini-percoll Clarke et al, Sperm antibodies and ICSI 113

3 gradients. For the latter, samples were diluted with HEPES-buffered human tubal fluid (HTF) medium (Irvine Scientific, Irvine, CA), which was supplemented with 10% patient serum or 4 mg/ml human serum albumin (HSA, Fraction V; Irvine Scientific) (supplemented HEPES-HTF) and centrifuged at 1,800 X g for 5 minutes before separation on a mini Percoll gradient (12). The resultant sperm pellet was washed by one or two centrifugation steps of 1,800 x g for 5 minutes to remove the Percoll. The final pellet was resuspended in a small volume «0.5 ml) of supplemented HEPES-HTF. Preparation of Oocytes Cumuluscells were removedfrom oocytes by brief exposure «1 minute) to 100 IU/mL hyaluronidase (Bovine Type VI; Sigma Chemical Co., St. Louis, MO, or Hyalase; Fisons, Castle Hill, NSW, Australia) in supplemented HEPES-HTF, followed by aspiration through a fine-bore glass pipette. Oocytes that exhibited a polar body after cumulus removal or after a few hours in vitro were injected on the day of collection. Microinjection Procedure Microinjection was based on the methodology described by Palermo et al. (13) but without the use of polyvinylpyrrolidone. Motile sperm were aspirated from the suspension into a fine-injection pipette (outer diameter 6 to 10 /-lm), transferred to a drop containing an oocyte, and immobilized by crushing the tail between the injection pipette and the dish. The freshly immobilized spermatozoon was reaspirated into the injection pipette and released into the cytoplasm of a mature oocyte after pushing the pipette through the ZP and puncturing the oolemma. Prior to expelling the spermatozoon, rupture of the oolemma was confirmed by aspiration of a small volume of the oocyte cytoplasm into the injection pipette. After injection, oocytes were transferred to fresh HTF containing 10% patient serum or 4 mg/ ml HSA (Commonwealth Serum Laboratories) for culture. Survival, Fertilization, and Embryo Cleavage Oocytes were deemedto have survivedicsi ifthey remained intact and failed to show anyobvious signs of lysis or degeneration over the 2 days after injection. Oocytes were assessed for the presence of pronuclei (PN) approximately 15 to 20 hours after injection and were examined for cleavage after a further 24 hours and in some cases, 48 hours. Embryos were classified into five grades on the basis of on their morphology: grade 1, distinct, regu- 114 Clarke et ai. Sperm antibodies and IGSI lar blastomeres with minimal or no fragmentation; grade 2, regular blastomeres and up to 10% fragmentation; grade 3, mostly regular blastomeres and between 10% and 30% fragmentation; grade 4, irregular blastomeres with 30% to 80% fragmentation; and grade 5, indistinct blastomeres with extensive fragmentation (>80%). Embryo Transfer and Cryopreservation Fresh embryos were transferred 2 or 3 days after microinjection, and surplus embryos were frozen in 1,2-propanediol and sucrose (14). Routinely, only two embryos were transferredto minimize the risk of a multiple pregnancy, although up to three embryos were transferred if the embryo quality or post-thaw survival was poor. Usually, the two best embryos were replaced fresh, and the surplus embryos grade 3 and above were frozen. Embryos were transferred after thawing in either a natural or artificial cycle. In natural cycles, embryos were replaced 2 or 3 days (depending on the age of the embryos) after ovulation had occurred as determined using a urine dipstick test for LH (ClearPlan One Step; Fisons). For artificial cycles, endometrial growth was stimulated by E 2 valerate (2 to 6 mg/day). Progesterone pessaries (100 mg/d) were commenced when the endometrial thickness was 2::7 mm, and embryos were transferred 2 or 3 days after the start ofp treatment. In these artificial cycles, luteal-phase support was continued using P pessaries. Pregnancy Assessment A serum,b-hcg measurement was planned for 14 to 18 days after ET. A,B-hCG concentration >250 miu/ml (conversion factor to SI unit, 1.00) was considered positive. Clinical pregnancies were confirmed by the presence of a gestational sac and fetal heart beat on US scan 4 to 6 weeks after ET. Statistical Analysis Differences in the fertilization and pregnancy rates (PRs) were compared using contingency X 2 tests for significance. The distributions of embryo gradings were compared using a X 2 goodness-of-fit test (Statgraphics 6.0, Statistical Graphics Corp., Rockville, MD). RESULTS A total of 39 couples with evidence of strong sperm autoimmunity in the male partner were included in this retrospective study. Prior to commencing ICSI treatment, eight of these couples had conventional IVF treatment with a 2PN fertilization rate of only Fertility and Sterility»

4 Table 1 Semen Quality for the Sperm Antibody-Positive Patients Included in the Study (n = 39) Variable Mean Median Minimum Maximum Count (xl0 6/mL) Motility (%) VSL (f.lm/s) Abnormal (%) IBT-IgG (%) IBT-IgA (%) % (311156), resulting in a single fetal heart-confirmed pregnancy, which did not progress to term. Because of this poor fertilization rate, subsequent patients diagnosed with severe sperm autoimmunity were referred immediately for ICSI treatment. Semen quality for the patients included in the study group is presented in Table 1. Most (33/39) patients had normal (~20 X 10 6/mL) sperm counts and many (28/39) had ~25% progressively motile spermatozoa. The majority (21139) had at least 20% morphologically normal spermatozoa. Thus, only six of these couples may have had difficulty with normal IVF treatment because of very poor semen quality. Hence, the primary reason for ICSI treatment in most of the couples was the sperm autoimmunity. The study and control groups were similar in mean female age (33.5 ± 4.3 and, 34.5 ± 4.8, respectively) and median number of oocytes (9 and 8, respectively) recovered at their first cycle. The results of IeSI treatment for the study and control groups are presented in Table 2. There was no difference in normal (2PN) fertilization rates or clinical pregnancy (fetal heart beat confirmed) rates between the groups. There was also no significant difference in the rate of delivered or ongoing (third trimester) pregnancies between the control and sperm autoimmune patients. There was, however, a significantly reduced rate of abnormal (I and 3PN, respectively) fertilization in the sperm antibody-positive groups. A comparison of pregnant (fetal heart beat confirmed) versus nonpregnant cycles within the study group showed no significant differences in fertilization rates (59.4% and 63.5%, respectively), embryo grade (median, 2 for each group), or the result ofthe IBT (mean %coated IgG, 83% and 84%, respectively; mean % coated IgA, 64% and 63%, respectively). Three of 9 men receiving prednisolone achieved pregnancies versus 12 of 30 men without prednisolone (X 2, not significant). With respect to pregnancy loss, there was no difference in the rate of first trimester spontaneous abortions between the three groups. In the control group, there was a clinical termination of pregnancy because of spina bifida, and in each of the sperm autoimmune groups, one pregnancy resulted in peri- natal mortality. In one case, the female partner was 39.4 years of age at the time of conception. In the other case, the female was only 28 years of age but was carrying twins. The parents did not allow a postmortem examination to determine the cause of neonatal death. At the time of this report, the patient was pregnant again. The distributions of embryo grades in the control and sperm antibody-positive groups are presented in Table 3. There was no difference in embryo quality between the patient groups with IgA ~80% and the control (negative) group. The patient group with IgG ~80% and IgA <80%, however, showed a tendency (P = 0.06) toward a difference in overall distribution of embryo quality when compared with the control group. This tendency resulted mainly from a higher proportion of grade 1 embryos, associated with a decrease in the proportion of grade 2 embryos (P = 0.018). DISCUSSION In the past, a number of different approaches have been explored with the hope of developing an effective treatment for infertility associated with sperm autoimmunity (for a review see Marshburn and Kutteh [15]). These have included occlusive therapy, sperm washing, cell affinity chromatography, antibody absorption, enzymatic digestion of spermbound antibodies, T rebound therapy, and various immunosuppressive regimens using glucocorticoids such as prednisolone. Although recent results using chymotrypsin-galactose (16) treatment of sperm before lui look promising, the general consensus from the literature suggests that glucocorticords are the only treatment with demonstrable (but limited) efficacy. Table 2 Results of ICSI for Control (Negative) and Sperm Antibody-Positive Patient Groups Patients Cycles (;;,,1 oocyte) Injected oocytes 2PN oocytes* 1PN + 3PN oocytes* Embryos transferred Transfers Fetal sacs Fetal hearts Delivered Control IgG ;;,,80% (negative) 19A ;;,,80% IgA <80% ,766 1,021 (58) 220 (12) (15) (63) 27 (8)t (18), 6 * Percentage injected in parentheses. t Compared with control, P = (X 2 test). :I: Compared with control, P = (X 2 test). Percentage of cycles in parentheses. II Seven twin fetal hearts., One twin fetal heart (62) 28 (8):1: (24) 8 Vol. 68, No.1, July 1997 Clarke et al, Sperm antibodies and lcsl 115

5 Table 3 Distribution of Embr yo Quality in Control (Negative ) and Sperm Ant ibody- Positive Patient Groups Embryo quality (%) X 2 (P )* Patient group Grade 1 Grad e 2 Grade 3 Grade 4 Grad e 5 Grades 1-5 Grad es 1-2 Control IgA.,,80% IgG.,,80% IgA < 80% * X 2 goodness-of-fit test. In our clinic, patients are categorized as having significant sperm autoimmunity when at least 80% of motile sperm are coated with antibodies by the direct IBT and theirspermatozoa are unable to penetrate beyond 2 em in 1 hour into normal human periovulatory cervical mucus. Such patients have a severely reduced chance of natural conception and, thereby, warrant consideration as to their medical suita bility for corticosteroid therapy. In a previ ous publi cation (17), we reported th at such treatment significantly reduced sperm immobilization titers in serum and increased both sperm counts and motilities in a group of 14 men, with three pregnancies resulting. Although most patients experienced side effects such as arthralgia, cramps, fluid retention, dyspepsia, and acne, there were no cases of aseptic hip necrosis. Sever al other groups (18, 19) ha ve reported marked reductions in sperm antibody levels in patients tre ated with glucocorticoids, associated with an increased conception rate. Because of the side effects of corticosteroid treatment, however, it can only be given over a limited period to those men who have no significant contraindications. It is, therefore, important that the patient has semen frozen if sperm antibody levels drop and semen quality improves during treatment. We have observed that patients who respond to prednisolone with a significant reduction in the proportion of antibody-coated sperm achieve very good pregnancy rates by IVF us ing cryopreserved sperm. The proportion of patients who can be successfully treated by this approach is limited, however, and so the availability of an effective alternative treatment such as ICSI would be an important advance. The development of ICSI was a major breakthrough in the treatment of severe malefactor infertility (13). The most recent applications ofthis technique have included treatment ofpatients with Schirren-Holstein syndrome (round sperm heads lacking both acrosoma l and postacrosomal den se lamina) (20), patients with defective ZP-induced acrosomal reaction as sociated with otherwise normal semen quality (21), and patients with severe sperm autoimmunity (7,22). 116 Clarke et al, Sperm antibo dies and ICSI The present study has focused on the ICSI treatment of patients with severe sperm autoimmunity, as indicated by a direct IBT result showing at least 80% of motile spermatozoa coated with antibodies of IgG and/or IgA immunoglobulin classes. In a previous study (2), it was observed that patients with ===80% of motile sperm coated with antibodies of both immunoglobulin classes had significantly reduced fertilization rates in conven tional IVF. There have been a number of studies (4, 5) th at have indicated th at sperm antibodies interfere with the spermzona interaction, thus lending support to the belief that ICSI should provide an effective treatment for sperm autoimmunity. For the most part, this conclusion was borne out by the recent report by Nagy et a1. (7). They reported normal fertilization and PRs (fetal sacs) but significan tly poorer embryo quality in sperm antibody-positive patients. A subsequent report by Lah teenmaki et a1. (22) suggested, however, that there may also be a higher rate of pregnancy loss in such patients. Consequently, it was important to re-examine ICSI data in the ligh t of these reports. The results of the present study confirm that fertilization and ongoing PRs are comparable in sperm antibody-positive and -negative patient groups; however, this study found no evidence of an overall decrease in embryo quality after ICSI treatment for patients with sperm autoimmunity. Instead, the main change was a marginal one, involving a decrease in the proportion of grade 2 embryos and an incre ase in grade 1 embryos. There was also a tendency to an increase in grade 5 embryos, but this was not statistically significant because of th e small number of embryos involved. We can offer no definite explanation for the discrepancy with the findings of Nagy et al. (7). Their previous finding of significantly poorer embryo quality in sperm-antibody patients treated by conventional NF indicates, however, tha t this effect is not specifically related to the resr procedure (23). In contrast to th e report by Lahteenmaki et a1. (22), th e results of the present study showed no evidence of an increased incidence offirst trimester pregnancy loss in sperm antibody-posi- Fertility and S terility»

6 tive patients treated by ICSI. The difference in results between the two studies may be related to the relatively small antibody-positive groups involved. The higher rate of abnormal fertilization in the control group may be related to the poor semen quality. Recent work has indicated that the incidence of chromosome structural aberrations was about fourfold higher in sperm with amorphous, round, or elongated heads (24). In conclusion, the results of the present investigation indicate that ICSI, as performed in our clinic, is an effective treatment for patients presenting with infertility associated with severe sperm autoimmunity. Thus, ICSI provides an alternative treatment for patients who have contraindications to corticosteroid treatment or are unwilling to undergo treatment. In addition to improving our capacity to treat "spontaneous" immunoinfertility, the wider availability of ICSI would improve conception rates for vasectomy-reversal patients. In the foreseeable future, it may also benefit men who elect to use antispermatozoal contraceptive vaccines but subsequently decide to have more children. Acknowledgments. We thankthe clinical, laboratory, and nursing staff of The Royal Women's Hospital and Melbourne IVF for their contributions to the development and running of the ICSI program. We also thank Kay Kerrison for typing the manuscript. REFERENCES 1. Witkin SS, Viti D, David SS, Stangel J, Rosenwaks Z. Relation between antisperm antibodies and the rate of fertilization of human oocytes in vitro. J Assist Reprod Genet 1992;9: Clarke GN, Lopata A, McBain JC, Baker HWG, Johnston WIH. Effect of sperm antibodies in males on human in vitro fertilization (IVF). Am J Reprod Immunol 1985;8: Lahteenrnaki A. In-vitro fertilization in the presence of antisperm antibodies detected by the mixed antiglobulin reaction (MAR) and the tray agglutination test (TAT). Hum Reprod 1993;8: Bronson RA, Cooper GW, Rosenfeld DL. 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The use of chymotrypsin/galactose to treat spermatozoa bound with antisperm antibodies prior to intra-uterine insemination. Hum Reprod 1994; 9: Baker HWG, Clarke GN, Hudson B, McBain JC, McGowan MP, Pepperell RJ. Treatment of sperm autoimmunity in men. Clin Reprod Fertil 1983;2: Hendry WF, Stredonska J, Hughes L. Steroid treatment of male subfertility caused by antisperm antibodies. Lancet 1979;2: Shulman JF, Shulman S. Methylprednisolone treatment of immunologic infertility in the male. Fertil Steril 1982;38: Bourne H, Liu DY, Clarke GN, Baker HWG. Normal fertilization and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm. Fertil Steril 1995;63: Liu DY, Bourne H, Baker HWG. Fertilization and pregnancy with acrosome intact sperm by intracytoplasmic sperm injection in patients with disordered zona pellucida-induced acrosome reaction. Fertil Steril 1995;64: Lahteenmaki A, Reima I, Hovatta O. Treatment of severe male immunological infertility by intracytoplasmic sperm injection. Hum Reprod 1995;10: Palermo G, Devroey P, Camus M, Khan I, Wisanto A, Van Steirteghem AC. Assisted procreation in the presence of a positive direct mixed antiglobulin reaction test. Fertil Steril 1989; 52: Lee JD, Kamiguchi Y, Yanagimachi R. Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse 00 cytes. Hum Reprod 1996; 11: Vol. 68, No.1, July 1997 Clarke et al, Sperm antibodies and 1eS1 117