De Yi Liu, Ph.D.t Harold Bourne, B.Sc. H. W. Gordon Baker, M.D., Ph.D.

Transcription

1 FERTILITY AND STERILITY Vol. 64, No.1, July 1995 Copyright i) 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. i' I Fertilization and pregnancy with acrosome intact sperm by intracytoplasmic sperm injection in patients with disordered zona pellucida-induced acrosome reaction* De Yi Liu, Ph.D.t Harold Bourne, B.Sc. H. W. Gordon Baker, M.D., Ph.D. Department of Obstetrics and Gynaecology and Reproductive Biology Unit, Royal Women's Hospital, University of Melbourne, Carlton, Victoria, Australia Objective: To determine whether fertilization and pregnancy could be obtained by injection of acrosome-intact sperm into the cytoplasm in patients with persistent failure of fertilization in IVF-ET associated with disordered zona pellucida (ZP)-induced acrosome reaction (AR). Design: Sperm-ZP binding and penetration and ZP-induced AR were compared between patients and fertile donors. Acrosome-intact sperm removed from the ZP were injected into the cytoplasm of the oocytes. Setting: Reproductive Biology Unit, Royal Women's Hospital. Main Outcome Measures: The proportion ofzp penetrated and ZP-induced AR and fertilization and pregnancy after intracytoplasmic sperm injection were analyzed. Results: Most (7/8) of the patients had consistently normal sperm characteristics including concentration, motility and velocity, acrosomes, and morphology. Mean number of sperm bound to the ZP was not significantly different between patients (97 spermlzp) and fertile donor controls (100 spermlzp) However, AR of sperm bound to the ZP was significantly lower in the patients (4%) than in controls (61%). None of the ZP (n = 32) were penetrated by patient sperm whereas all (n = 32) ZP were penetrated by control sperm. Acrosome intact sperm removed from the ZP were used for intracytoplasmic sperm injection. All patients had 50% normal fertilization and embryo development. Three pregnancies (one early aborted, two ongoing) were achieved after the transfer of fresh or frozen embryos. Conclusion: Acrosome-intact human sperm can produce a high fertilization rate and pregnancies after intracytoplasmic sperm injection in patients with disordered ZP-induced AR. The acrosome reaction is unlikely to be important for fertilization with intracytoplasmic sperm injection. Fertil SteriI1995;64: Key Words: Acrosome reaction, intracytoplasmic sperm injection, fertilization, pregnancy It is known that the human acrosome and acrosome reaction (AR) are very important for spermoocyte interactions during the process offertilization (1). Human round-headed acrosomeless sperm do Received September 13, 1994; revised and accepted January 25,1995. * Supported by grant no. 92/03 from the Royal Women's Hospital Research Committee, Carlton, Victoria, and grant no from National Health and Medical Research Council, Canberra, Australian Capital Territory (ACT), Australia. t Reprint requests: De Yi Liu, Ph.D., Department of Obstetrics and Gynaecology, Royal Women's Hospital, University of Melbourne, Carlton, Victoria 3053, Australia (FAX: ). not bind to or penetrate the zona pellucid a (ZP) (2). Although both acrosome-intact and -reacted sperm can be observed on the ZP, it is believed that only acrosome-intact sperm bind to the ZP and that the ZP triggers the AR to facilitate sperm penetration through the ZP (3, 4). In the mouse, two ZP glycoproteins, ZP2 and ZP3, play major roles in sperm-zp interactions. ZP3 is the primary receptor that binds acrosome-intact sperm and induces the AR. ZP2 is the secondary receptor, which binds acrosome-reacted sperm (5,6). Although the human ZP is a very efficient physiological inducer of the AR (7), the mechanism of AR of sperm bound to the ZP is poorly understood. On the other hand, the AR is important 116 Liu et at. ICSI with acrosome intact sperm Fertility and Sterility

2 "1 in humans, as the acrosomal enzymes, mainly acrosin, are involved in sperm penetration of the ZP (1, 4, 8). Inhibition of acrosin activity with a trypsin inhibitor significantly decreases AR of sperm bound to the ZP and completely blocks sperm penetration into the ZP (8, 9). Therefore, it could be predicted that a disordered ZP-induced AR could cause failure of sperm-zp penetration, resulting in failure of fertilization in vitro. We recently have discovered a group of patients with disordered ZP-induced AR (10). These patients usually have normal sperm characteristics, including sperm concentration; motility, velocity, and morphology; a long duration of idiopathic infertility, and persistently low «20%) or zero fertilization with standard IVF -ET. Sperm bind to the ZP but do not undergo the AR normally nor penetrate into the ZP. We report here that normal fertilization and pregnancy can be achieved by injection of acrosome-intact sperm into the cytoplasm in patients with disordered ZP-induced AR. Patients MATERIALS AND METHODS Eight infertile couples had previous IVF treatments with low «20%, two patients) and zero fertilization (six patients) after insemination of the 00- cytes with husband sperm by standard procedures. These couples had an average of two (range one to four) treatment cycles with an average of 26 (range 8 to 57) mature oocytes inseminated. There were large numbers of sperm bound to the ZP of all oocytes that did not fertilize. Previous semen analysis results showed that seven men had consistently normal semen according to World Health Organization criteria (11). One man (patient 2) had variable sperm concentration and motility, possibly associated with intermittent prostatitis. All couples had no antisperm antibodies detected by direct (for husband sperm) and indirect (for husband and wife serum) immunobead tests (11). These couples had an average of 5 years (range 4 to 8 years) of infertility. Female age averaged 33 years (range 26 to 39 years) and male age averaged 37 years (range 31 to 48 years). Couple 1 had a previous natural pregnancy after 4 years of infertility. All the men were in good health. Physical examinations in male partners were normal apart from varicoceles in one man (patient 1). One woman had been treated for mild endometriosis and another one had irregular ovulation. The project was approved by the Royal Women's Hospital Research and Ethics Committees. Semen Samples and Sperm Tests Semen samples were obtained by masturbation after 3 to 5 days abstinence. Fertile donor sperm Vol. 64, No.1, July 1995 were used for control experiments for sperm-zp binding and penetration tests and ZP-induced AR. All sperm tests were performed after liquefaction of the semen within 1 hour. Sperm concentration, viability (eosin Y exclusion), and motility in semen were determined using standard methods (11). Sperm morphology was assessed on stained smears prepared from semen after washing with 0.9% NaCl. The smears were stained with the Shorr method and 200 spermatozoa were assessed under oil immersion with magnification X 1,000 and brightfield illumination. Normal sperm morphology was assessed according to World Health Organization criteria for the silhouette plus internal staining characteristics, with the acrosomal region being seen clearly, regular in shape, and occupying at least half of the sperm head (12). Sperm.ZP Binding and Penetration Tests Oocytes from other patients that failed to fertilize because of severe male infertility were used for sperm-oocyte interaction tests. Each of the patients had more than eight mature oocytes inseminated and none showed any evidence of two pronuclei or cleavage 48 to 60 hours after insemination or had any sperm bound to the ZP. Any remaining corona cells were removed by aspiration in and out of a glass micropipette. Motile sperm selected by swim-up technique from both normal fertile donors (control) and patients were used for sperm-zp binding and penetration tests. Usually 2 X 10 6 motile sperm were incubated with four oocytes in 1 ml of synthetic human tubal fluid (HTF) with 10% human serum. The oocytes used for each test, patient versus control, were from the same failed IVF treatment. After 2 hours ofincubation, the oocytes were transferred to phosphatebuffered saline (PBS), ph 7.4, containing 2 mg/ml bovine serum albumin (BSA) and washed by aspiration in and out of a glass pipette (inside diameter approximately 250 f-tm) to dislodge sperm loosely adheringto the surface ofthe ZP. The number of sperm bound tightly to the ZP was counted with an inverted phase-contrast microscope at a magnification of x250. Sperm numbers> 100 per ZP were recorded and analyzed as 100. All sperm bound to the surface of the ZP were removed by vigorous aspiration in and out of a narrow-gauge micropipette with an inner diameter (approximately 120 f-tm) slightly smaller than the oocyte (8, 13). After this pipetting procedure, sperm with heads in the ZP or previtelline space were counted using an inverted phase-contrast microscope. Liu et ai. ICSI with acrosome intact sperm 117

3 Intracytoplasmic Sperm Injection Procedure Figure 1 Acrosome status of sperm removed from the ZP after 2 hours of incubation (Pisum sativum agglutinin-fluorescein isothiocyanate conjugate stain). (A), Fertile donor sperm, six reacted and five intact. (B ), Patient sperm, all sperm with acrosome intact. Assessment of Acrosome Status The acrosome status of sperm was determined with fluorescein-labeled Pisum sativum agglutinin (Sigma Chemical Co., St. Louis, MO) (3). The viability of acrosome-intact and -reacted sperm both in medium and bound to the ZP was not determined because >90% of sperm in the medium had progressive motility and only motile sperm bind to the ZP. Sperm in the swim-up suspension were washed with 0.9% NaCI and centrifuged at 600 X g for 10 minutes and the sperm pellet was resuspended in 20 /-ll of 0.9% NaCI and smeared on a glass slide for acrosome assessment. Sperm bound to surface of the ZP were recovered after aspiration with the narrow-gauge micropipette in 20 /-ll of PBS, ph 7.4, containing 2 mg/ml ofbsa, smeared on a slide and their position marked with a glass pen to help find them after Pisum sativum agglutinin staining (Fig. 1) (8). Our previous study showed that there was no significant difference in ZP-induced AR between groups of oocytes from the same patient. But there is large variability in AR induced by the ZP of oocytes from different patients. Thus, fertile donor sperm must be used to control for this ZP-based variability (10). 118 Liu et al. lcsl with acrosome intact sperm A stimulation protocol using hmg and GnRH analogue was used to induce follicular growth. Oocytes were collected using ultrasound (US)-guided ovarian puncture 36 hours after the administration of hcg. All oocytes had the cumulus and coronal cells removed by brief exposure to 100 IU/mL ofhyaluronidase in HEPES-buffered HTF followed by aspiration through a fine-bore pipette. On the day of oocyte collection, a semen sample was obtained by masturbation after 3 days of abstinence. Motile sperm were prepared by Percoll or swim-up techniques. Motile sperm (1 to 2 X 10 6 ) in 0.5 ml HTF containing 10% wife serum were incubated with only one or two of the patient's own 00- cytes for 2 to 3 hours. Usually immature or morphologically poor oocytes were used. After incubation, the oocyte was washed in fresh medium by aspiration in and out of a glass pipette with a diameter approximately 250 /-lm to dislodge sperm loosely adherent to the ZP. Mter this procedure, only those sperm tightly bound to the ZP remained. These ZPbound sperm then were removed by aspiration in and out of a fine glass pipette with an inner diameter of approximately 120 /-lm, which is slightly smaller than oocyte diameter (Fig. 2). The acrosome status of sperm removed from the ZP was assessed from parallel experiments with oocytes that failed to fertilize in vitro. For injection, a single motile sperm removed from the ZP and immobilized by touching the tail with the injection pipette was aspirated into the pipette and released into the cytoplasm of each oocyte after pushing the pipette through the ZP and puncturing the oolemma. Most of the oocytes used A D ICSI B PiPette c /(lir\'- r( ) Figure 2 Diagram of removal of sperm bound to the ZP for intracytoplasmic sperm injection. (A), Many sperm bound to the ZP; (B) sperm bound to the ZP were removed by aspiration of the oocyte in and out of a fine glass pipette with an inner diameter approximately 120 fj,m, slightly smaller than that of the oocyte; (C), sperm removed from the ZP; (D) sperm used for intracytoplasmic sperm injection. Fertility and Sterility

4 Table 1 Results of Sperm-ZP Binding and Penetration and Acrosome Reaction of Sperm Bound to the ZP for Patients and Control Fertile Men* Tests Semen samples obtained before intracytoplasmic sperm injection No. of sperm bound per ZP No. of sperm in ZP per ZP ZP penetrated (%) AR on ZP (%) Semen samples obtained on the day of intracytoplasmic sperm injection ARon ZP(%) * Values are means ± SD. t P > *P < P < Patients Fertile men 97 ± ± Ot 0 9± 7* ± 0* 4±4 61 ± 17 3±3 60 ± 15 for intracytoplasmic sperm injection were not exposed to sperm for the ZP selection process. Fertilization was assessed after 18 hours and embryos were transferred to the uterus on 3 days after intracytoplasmic sperm injection or were cryopreserved. The significance of differences between patient and control results for the AR of sperm bound to the ZP and numbers of sperm bound and penetrating into the ZP was examined by Wilcoxon signed rank sum tests. toplasmic sperm injection (Figs. 1 and 3). The percentage of ZP-induced AR were similar in the two tests, which were performed 3 to 6 months apart (Table 1, Fig. 3). There was no significant difference between the mean proportions of acrosome-intact sperm in the insemination medium in patients (80%) and controls (82%). Fertilization and Pregnancy RESULTS Semen Analysis Results of the semen analysis performed before intracytoplasmic sperm injection treatment were as follows: average sperm concentration was 110 X ml (range 30 to 243 X 10 6 /ml), motility 57% (range 33% to 74%), velocity 37.8 f-lm (range 31 to 48 f-lm), and normal morphology in semen 19% (range 10% to 36%). Normal morphology of motile sperm selected by swim-up technique was 37% (range 16% to 55%). Sperm-ZP Binding and Penetration Table 1 shows that the average number of sperm bound to the ZP was not significantly different between patients and controls. However, none of the ZP were penetrated by patient sperm whereas most (98%) of the ZP were penetrated by control sperm for sperm samples obtained before intracytoplasmic sperm injection. The mean percentage AR on the ZP was significantly lower in the patients than in controls both for samples obtained before intracytoplasmic sperm injection and on the day of intracy- Vol. 64, No.1, July 1995 Intracytoplasmic sperm injection results are summarized in Table 2. Two patients had two intracytoplasmic sperm injection cycles with ZP-selected sperm and their results were pooled. An average of 11 (range 4 to 28) oocytes were injected and only 10% ofthe oocytes were damaged. The normal fertilization (two pronuclei at 18 hours) rate averaged 56% (range 33% to 85%). Seven patients had fresh embryos (one to three) transferred, with one (patient 1) ongoing pregnancy established. Patient 3 had an elevated hcg but an empty embryonic sac was found on US at 7 weeks. Patient 8 had all 11 embryos frozen because of hyperstimulation (44 oocytes collected but only 16 oocytes treated with intracytoplasmic sperm injection). Three patients had frozen embryos transferred from which one ongoing pregnancy (patient 8) resulted. Two patients had another intrac: o Q) E o (I) e Fertile men Fertile men Figure 3 Comparison of acrosome reaction of sperm bound to the ZP between patients and fertile men. (A) Sperm samples (n = 8) tested before intracytoplasmic sperm injection; (B) sperm samples (n = 7) tested on the day of intracytoplasmic sperm injection. The bars represent the mean and there is a line for each patient and control sperm sample (P < 0.001). Liu et al. ICSI with acrosome intact sperm 119

5 Table 2 Results ofintracytoplasmic Sperm Injection with ZP-Selected Sperm and Swim-Up Sperm No. of oocytes No. of embryos Patient* Injected Survived Fertilized t Transferred Frozen Cycles with ZP-selected sperm :j: :j: Cycles with swim-up sperm * Patients 1 and 8 have ongoing pregnancies and patient 3 had an empty sac on US. t Normal (two pronuclei at 18 hours) fertilizations only. :j: Results were pooled from two intracytoplasmic sperm injection cycles with ZP-selected sperm. Only frozen embryos were transferred because ofhyperstimulation (44 oocytes collected) in the intracytoplasmic sperm injection cycle. cytoplasmic sperm injection cycle with swim-up sperm. An average of 47% (44% and 50%) normal fertilization rate was obtained but no pregnancy was achieved after ET (Table 2). DISCUSSION The present study shows that high fertilization rates and pregnancy can be achieved by intracytoplasmic sperm injection using acrosome-intact sperm removed from the ZP in patients with disordered ZP-induced AR; a condition that causes failure of sperm-zp penetration and fertilization with standard IVF procedures despite normal-appearing sperm, normal sperm-zp binding, and normal acrosomal proteolytic activity (10). These results show that patients with this condition can be treated by intracytoplasmic sperm injection. Also, human 00- cytes can process sperm heads introduced into the cytoplasm with intact acrosomes. Furthermore, the human acrosome and the AR is important for sperm binding and penetration of the ZP but not important for fertilization with intracytoplasmic sperm injection. To demonstrate this, acrosome-intact sperm removed from the ZP were used for intracytoplasmic sperm injection. Because these patients had disordered ZP-induced AR, on average >96% sperm removed from the ZP had an intact acrosome and there was little chance of picking up an acrosome-reacted sperm for intracytoplasmic sperm injection. The average chance of an acrosome-reacted sperm being injected is 4% each time but the chance of a number of oocytes fertilizing with injection of an acrosomereacted sperm is 4% raised to the power of the number of oocytes fertilized. Thus, it is most unlikely that all the fertilizations observed were from acro- 120 Liu et al. lcsl with acrosome intact sperm some-reacted sperm. However, it could be argued that the procedures of removing the sperm from the ZP and immobilizing them for intracytoplasmic sperm injection could induce the AR. Although these will cause changes in the plasma membrane, it is clear from the results of Pisum sativum agglutinin staining and electron microscopy of sperm from these patients that there is not a major release of acrosomal contents after their removal from the ZP (10). The use of dislodged ZP-bound sperm for intracytoplasmic sperm injection did not appear to alter the fertilization rates from those obtained with sperm selected for intracytoplasmic sperm injection by swim-up in two of the patients. Ng et al. (14) have shown by electron microscopy that the majority of sperm obtained from a variety of subjects and injected into the cytoplasm of the oocytes with a similar technique had intact acrosomes at 2': 1 hour after intracytoplasmic sperm injection. Therefore, the mechanical immobilization of sperm for intracytoplasmic sperm injection usually does not provoke a rapid massive release of the acrosome contents. Thus, intravitelline sperm processing must be able to cope with the bulk of acrosomal enzymes from a single injected sperm. Patients found so far with disordered ZP-induced AR have a long duration of infertility and persistent low or zero fertilization in vitro. The condition either causes severe but not absolute sterility or fluctuates in severity. Nonpaternity also is possible but this was not tested. Although most of them have normal sperm characteristics and sperm-zp binding, their sperm can not penetrate into the ZP associated with failure of the AR to occur on the ZP (10). The frequency of this defect in couples with failure of fertilization in Fertility and Sterility

6 vitro or idiopathic infertility requires further investigation. However, our preliminary study suggests that the majority of patients with normal semen but persistent failure offertilization in vitro may have it (Liu DY, Baker HWG, unpublished data). Because patients with disordered ZP-induced AR have little or no chance offertilization with standard IVF, they should be assigned directly to intracytoplasmic sperm injection if they are identified before commencement of IVF treatment. At present, assessment of sperm-zp penetration and the AR of sperm bound to the ZP are needed for diagnosis. However, human ZP materials for routine assessment of sperm-zp penetration and ZP-induced AR are in limited supply. Recombinant human ZP3 might be useful for diagnosing this condition and for further investigation ofthe pathophysiology (15). Our previous study showed that the majority of patients with disordered ZP-induced AR also had low calcium ionophore A23187-induced AR (10). Others have reported that ionophore A23187-induced AR is related to fertilization rate in vitro (16, 17). Further work is needed to determine if this test would be useful for identifying patients with this condition. General tests of acrosome status and the AR induced by stimuli other than the ZP may have high false positive rates if used uncritically. For instance, sperm with defects of morphology commonly also have acrosomal abnormalities. The relationship of disordered ZP-induced AR to other reported acrosomal defects is not clear at present. Preliminary results (Liu DY, Baker HWG, unpublished data) showed no effect of pentoxifylline on disordered ZP-induced AR, suggesting that it is different from the AR insufficiency reported by Tesarik and Mendoza (18). In conclusion, intracytoplasmic sperm injection is useful for treating patients with disordered ZP-induced AR. High fertilization rates and pregnancies are achieved by intracytoplasmic sperm injection with acrosome-intact sperm removed from the ZP. Thus, the human acrosome and the AR probably are not important for fertilization after intracytoplasmic sperm injection. Acknowledgments. We thank Mingli Liu, B.Sc., for technical assistance and all the clinicians and scientists in both Royal Women's Hospital and Melbourne IVF Laboratories for referring the patients and collecting the oocytes. REFERENCES 1. Yanagimachi R. Mammalian fertilisation. In: Knobil E, Neill J, editors. The physiology of reproduction. New York: Raven Press, 1988: von Bernhardi R, de Ioannes AE, Blanco LP, Herrera E, Bustos-Obregon E, Vigil P. Round-headed spermatozoa: a model to study the role of the acrosome in early events of gamete interaction. Andrologia 1990;22: Liu DY, Baker HWG. Inducing the human acrosome reaction with a calcium ionophore A23187 decrease sperm-zona pellucida binding with oocytes that failed to fertilise in vitro. J Reprod FertiI1990;89: Tesarik J. Appropriate time of the acrosome reaction is a major requirement for the fertilizing spermatozoa. Hum Reprod 1989;4: Bleil JD, Wassarman PM. Identification ofa secondary sperm receptor in the mouse egg zonae pellucidae: role in maintenance of binding of acrosome-reacted sperm to eggs. Dev BioI 1988; 128: Wassarman PM. Regulation of mammalian fertilisation by zona pellucida glycoproteins. J Reprod FertiI1990;42(Suppl): Cross NL, Morales P, Overstreet JW, Hanson FW. Induction of acrosome reaction by the human zona pellucida. BioI Reprod 1988; 38: Liu DY, Baker HWG. Inhibition of acrosin activity with a trypsin inhibitor blocks human sperm penetration ofthe zona pellucida. BioI Reprod 1993;48: Llanos M, Vigil P, Salgado AM, Morales P. Inhibition of the acrosome reaction by trypsin inhibitors and prevention of penetration of spermatozoa through the human zona pellucida. J Reprod FertiI1993;97: Liu DY, Baker HWG. Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro. Hum Reprod 1994;9: World Health Organization. WHO laboratory manual for examination of human semen and semen-cervical mucus interaction. New York: Cambridge University Press, Liu DY, Lopata A, Johnston WIH, Baker HWG. Human sperm-zona binding, sperm characteristics and in-vitro fertilisation. Hum Reprod 1989;4: Liu DY, Baker HWG. A new test for the assessment of spermzona pellucida penetration: relationship with results of other sperm tests and fertilization in vitro. Hum Reprod 1994;9: Ng SC, Liow SL, Sathananthan H, Bongso A, Ratnam SS. Review: microinjection of human sperm directly into human oocytes. J Assist Reprod Genet 1993; 10: van Duin M, Polman JEM, de Breet ITM, va Ginneken K, Bunschoten H, Grootenhuis A, et al. Recombinant human zona pellucida protein ZP3 produced in Chinese hamster ovary cell induces the human sperm acrosome reaction and promotes sperm-egg fusion. BioI Reprod 1994;50: Cummins JM, Pember SM, Jequier AM, Yovich JL, Hartmann PE. A test of the human sperm acrosome reaction following ionophore challenge (ARIC). Relationship to fertility and other seminal parameters. J Androl 1991; 12: Yovich JM, Edirisinghe WR, Yovich JL. Use of the acrosome reaction to ionophore challenge test in managing patients in an assisted reproduction program: a prospective, doubleblind, randomized controlled study. Fertil Steril 1994; 61: Tesarik J, Mendoza C. Sperm treatment with pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency. Fertil Steril1993;60: Vol. 64, No.1, July 1995 Liu et al. lcsl with acrosome intact sperm 121