Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm

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1 FERTILITY AND 8TERILITY Copyright American Society for Reproductive Medicine Published by Elsevier Science Inc. Vol. 68, No. I, July 1997 Printed on acid-free paper in U. S. A Failure of oocyte activation after intracytoplasmic sperm injection using round-headed sperm David E. Battaglia, Ph.D.*t James K. Koehler, Ph.D.t Nancy A. Klein, M.D.* Michael J. Tucker, Ph.D. University of Washington, Seattle, Washington, and Reproductive Biology Associates, Atlanta, Georgia Objective: To examine the outcome of intracytoplasmic sperm injection (ICS!) with roundheaded sperm (globozoospermia). Design: Retrospective analysis. Setting: In vitro fertilization laboratory with extensive ICSI experience. Patient(s): A patient couple with infertility because of globozoospermia seeking ICSI treatment. Main Outcome Measure(s): Fertilization, cleavage, and pregnancy rates. Intervention(s): Intracytoplasmic sperm injection and calcium ionophore. Result(s): This couple experienced only 7% fertilization after ICSI in their first cycle. Treatment of the unfertilized oocytes with calcium ionophore 20 hours after ICSI-induced fertilization and cleavage of 79% of the oocytes. Embryo quality was fair to good. On the second cycle, 8 of the injected oocytes were treated with ionophore immediately after ICSI and the remaining 20 oocytes were untreated. Normal fertilization was achieved in 75% of the treated and 10% of the untreated oocytes. Treatment of these unfertilized oocytes with ionophore 20 hours after ICSI resulted in fertilization in 73%. Pregnancy was not achieved after either ICSI cycle. Ultrastructural analysis indicated multiple structural abnormalities in the sperm. Conclusion(s): These results indicate that the round-headed sperm from this patient were incapable of oocyte activation after ICSI. This may be the reason for the frequent ICSI fertilization failure seen with this condition. Current ICSI procedures may not always overcome the infertility associated with globozoospermia, and further study of the etiology of this condition is needed. (Fertil Steril 1997;68: by American Society for Reproductive Medicine.) Key Words: Globozoospermia, ICSI, fertilization, calcium ionophore, oocyte activation Before the introduction of intracytoplasmic sperm injection (lcs!) into the clinical arena, patients producing only round-headed sperm were considered to be irreversibly infertile, even with lvf (1). This form of teratozoospermia, often called globozoospermia (2), was identified >20 years ago and carries with Received August 2, 1996; revised and accepted March 20, *Department of Obstetrics and Gynecology, Division of Reproduetive Endocrinology, University of Washington. t Reprint requests: David E. Battaglia, Ph.D., Department of Obstetrics and Gynecology, Box , University of Washington, Seattle, Washington (FAX: ; DBATTAG U.WASHINGTON.EDU). t Department of Biological Structure, University of Washington. Reproductive Biology Associates. 118 it a variety of morphological defects (3). The more detailed descriptions of this condition have focused primarily on the absence of acrosomal vesicles and abnormal perinuclear cytoskeleton in the sperm from an affected individual (3-5). Although the absence of the acrosome alone is considered to be the cause of fertilization failure with this condition, it is unclear whether other defects may be present that can influence the functional capacities of these sperm. Because lesl allows us to bypass the inherent problems these sperm have with zona pellucida and oolemma interactions, we are now in a position to examine the ability ofthese sperm to support postfertilization embryonic development. There have been several case reports involving patients with globozoospermia and lcsi (6-9). The /97/$17.00 PH (97)

2 pregnancy rate (PR) with these individuals has been low. These data also reveal that the fertilization rates with round-headed sperm are lower than average for ICSI in general, even in established ICSI laboratories (8). In addition, it appears that cleavage rates are lower than expectedin some cases. Because the etiology ofglobozoospermia is unknown, it is conceivable that this condition may carry a variety of other defects not functionally related to the acrosomal deficiencies. There may be genetic and environmental influences, each of which may create a different set of defects, thereby imparting variable developmental capabilities after ICSI. In this report, we describe the results of two ICSI cycles in a patient with globozoospermia. The sperm were unable to induce the activation of the injected oocytes as revealed by calcium ionophore treatment after ICSI. Electron microscopic analysis was performed on the actual sperm samples used for the ICSI cases to demonstrate the degree of abnormal ultrastructural morphology seen with these sperm. The results from these cycles demonstrate deficiencies in these sperm to sustain specific postfertilization events and show that ICSI alone may not overcome the infertility experienced by every patient presenting with globozoospermia. MATERIALS AND METHODS The patient couple presented with a diagnosis of globozoospermia, with 100% of sperm exhibiting round-headed morphology. The couple underwent two successive ICSI cycles when the female partner was 31 and 32 years of age, respectively. Ovulation induction was accomplished by the luteal phase administration of GnRR analogues followed by gonadotropin administration. Egg retrieval was performed by ultrasound-guided transvaginal follicle aspiration. On the day of oocyte retrieval, oocytes were stripped of surrounding cumulus cells by incubation in REPES-buffered human tubal fluid (RTF; Irvine Scientific, Irvine, CA) containing hyaluronidase (80 IU/mL) for <60 seconds, followed by mechanical disruption of the cumulus matrix by pipetting through narrow-bore pipettes. Only mature oocytes in second meiotic metaphase were selected for ICSI. Preparations for ICSI followed procedures similar to those described by both Tucker et al. (10) and Palermo et al. (11). Briefly, liquefied semen was washed by centrifugation to remove the seminal plasma, and the sperm pellet was resuspended in RTF containing 3% bovine serum albumin (BSA; Sigma Chemical Co., St. Louis, MO). The sperm suspension was layered over a discontinuous Percoll gradient (40% and 80%) and centrifuged at 600 x g for 15 minutes. The resultant sperm pellet was washed three times in RTF containing 3% BSA. The final suspension was maintained at 37 C in a 5% CO 2 incubator until injection. For the ICSI procedure, selected oocytes were placed into REPES-buffered RTF containing 0.5% BSA. Sperm were aliquoted (1:4) into a separate drop of HE PES-buffered HTF containing 10% polyvinylpyrrolidone (ICN Biochemicals, Aurora, OR) for immobilization. Injected sperm were selected from the polyvinylpyrrolidone suspension, compressed at the midpiece by the ICSI needle until they were immotile, and loaded tail-first into the needle for injection. The oocytes for injection were secured by a holding pipette and oriented with the first polar body at the 6 or 12 o'clock position. The injected sperm was moved to the tip of the ICSI needle and the needle was inserted into the oocyte at the 3 o'clock position. After insertion, back pressure was exerted within the needle in an attempt to draw cytoplasm into the needle to ascertain whether the needle was within the cytoplasm. If penetration was not evident, the needle was withdrawn partially and moved tangentially to obtain entry. Only when it was confirmed that the needle had penetrated the oolemma was the sperm expelled into the cytoplasm. After injection, the oocytes were returned to normal culture. Assessment of fertilization was performed 17 hours after injection and was considered to be normal with the presence of two distinct pronuclei (PN) and two polar bodies. An aliquot of sperm was obtained after each ICSI cycle for preparation for transmission electron microscopy (TEM). Sperm from a normal, fertile donor also was obtained for comparison. These sperm were washed once in RTF containing 0.5% BSA and prepared for TEM by the methods of Koehler et al (12). Aliquots of the washed sperm were fixed in 1.25% glutaraldehyde in 0.1 M cacodylate buffer (pr 7.3) at room temperature for 2 to 3 hours. Postfixation in 1% OS04 also in cacodylate buffer was followed by ethanol and propylene oxide dehydration and embedding in Epon 812. Thin sections were prepared by diamond knife on a Sorvall PB-ll microtome (Stovall Inc., Norwalk, CT), stained with uranyl acetate and lead citrate, and examined in a Phillips 420 transmission electron microscope (Phillips Electronics Institute, Mahwah, NJ). Calcium ionophore (A23187; Sigma Chemical Co.) was used after ICSI with some ofthe oocytes because of an apparent failure of fertilization (see Results). The intent was to activate the oocytes artificially to ascertain whether sperm were present within the oocytes but incapable of initiating development. The couple was briefed thoroughly about the known effects of this drug on intracellular Ca 2 + levels on 00 cytes. They also were informed about our lack of Vol. 68, No.1, July 1997 Battaglia et al. Oocyte inactivation with round-headed sperm 119

3 data regarding any adverse effects that ionophore treatment may have on the oocyte and embryo and that there has been no reported use of this drug in this manner. They also signed a written consent for this situation. When treated with this drug, selected oocytes were incubated in HTF containing 10% synthetic serum substitute (Irvine Scientific) and 10 IlM calcium ionophore for 10 minutes and subsequently were washed four times in culture medium. Treated oocytes were observed for developmental progress at selected intervals after treatment. As with untreated oocytes, fertilization was considered to be normal with the presence of two distinct PN and two polar bodies with each oocyte. The IeSI procedures were accomplished on a Nikon-Narishige micromanipulation setup using a Nikon Diaphot microscope (Nikon Inc., Melville, NY) with Hoffman modulation contrast optics. Light micrographs were obtained on 35-mm Kodak Tri-X film (Eastman Kodak, Rochester, NY) using either this microscope or a Zeiss Universal with differential interference contrast optics. RESULTS Semen parameters for the male partner included sperm counts of 48 x 10 6/mL, 50% motility, and 140 x 10 6/mL, 70% motility, for the two respective IeSI cycles. As expected, light microscopy revealed that the sperm samples from both cycles clearly exhibited the characteristics of globozoospermia, including distinct round-shaped heads in nearly 100% of their sperm. Electron microscopy also revealed that the sperm had the signature characteristics of globozoospermia, including round, compact heads with no evidence of acrosomal vesicles (Fig. la). There was variability in the extent of nuclear condensation, with sperm often exhibiting vacuolar regions within the nucleus. The sperm also exhibited mid piece abnormalities, with multiple profiles of the axoneme being seen in cross section, suggesting coiling, and irregular mitochondrial distributions. This morphology was in contrast to sperm from a normal donor (Fig. IB) showing a fully condensed, elongated nucleus capped by a distinct acrosomal vesicle and a dense mitochondria-ensheathed midpiece. The results ofthe two IeSI cycles are summarized in Table 1. In the first cycle, only one of 15 intact oocytes were fertilized as assessed 18 hours after injection. We treated the unfertilized oocytes with calcium ionophore (10 IlM) for 10 minutes to induce activation and ascertain the developmental capabilities of these oocytes. Within 6 hours after treatment, all of these oocytes had extruded second polar bodies. Eleven of these formed two PN 18 hours after treatment and nine ofthese went on to cleave, developing into two- to four-cell embryos at 38 hours. Several embryos were of good quality. After signing a written consent, the couple elected to have five embryos transferred, including the one developed from spontaneous fertilization. No pregnancy resulted after transfer. In the secondiesi cycle (Table 1), 28 oocytes were injected. With informed consent, the couple agreed to have 8 of the injected oocytes treated with calcium ionophore immediately after injection; the remaining 20 injected oocytes were untreated. Seventeen of the untreated oocytes remained intact after injection, but only 2 exhibited signs of normal fertilization. In contrast, all 8 of the injected, ionophoretreated oocytes remained intact and 6 ofthese exhibited normal fertilization. All of the embryos forming two PN (treated and untreated) cleaved normally and often produced embryos of good quality. Five embryos were transferred ranging from fair to good quality including the two that developed after spontaneous fertilization. This ET resulted in a shortterm chemical pregnancy. DISCUSSION The results of these two IeSI cycles illustrate the distinct inability of these sperm to support oocyte activation after successful sperm injection. In the first lesi cycle, we were surprised to see no evidence of fertilization. Egg quality was excellent and all injections proceeded normally. The addition of calcium ionophore 20 hours after IeSI clearly revealed that most of the oocytes had been injected with sperm successfully and that development could proceed by artificial induction of intracellular calcium ion re- Figure 1 (A), Transmission electron micrograph of sperm from the ICSI procedure showing uniformly small, round nuclei (Nu), greater than normal cytoplasmic residue, lack of acrosomes, and vacuolar regions within the nuclei. Segments of the coiled flagellae (FL) also are visible. Magnification xs,650. (B), Transmission electron micrograph of a sperm from a normal, fertile donor. Note the acrosome (Ac), acorn-shaped nucleus (Nul, and normal midpiece with compact mitochondria. Magnification x22,ooo. 120 Battaglia et ai. Oocyte inactivation with round-headed sperm Fertility and Sterility"

4 Table 1 Fertilization and Cleavage Results of the Two Successive ICSI Cycles No. of oocytes No. of intact No. of fertilized No. of unfertilized No. of cleaved Cycle Treatment injected oocytes oocytes oocytes oocytes 1 rest * 1 2 ICSI t 2 2 ICSI + ionophore 30 min post-icsi * These apparently unfertilized oocytes were treated with ionophore 20 hours after ICSI. All 14 formed second polar bodies within 6 hours after treatment; 11 formed two PN within 18 hours. Nine embryos cleaved to the four-cell stage within 20 hours. t These apparently unfertilized oocytes were treated with ionophore 20 hours after ICSI. Eleven exhibited two PN and second polar bodies 18 hours after ionophore treatment. All of them cleaved to the two-cell to eight-cell stage within 62 hours. lease. Because most of the ionophore-treated oocytes extruded a second polar body and formed two distinct PN, we assumed that one of the PN was paternally derived in each oocyte. The second ICSI cycle presents even more compelling evidence that these sperm lack the ability to activate oocytes after injection. We treated a small cohort of injected oocytes with ionophore immediately after sperm injection to mimic closely the normal timing of oocyte activation. Only 10% of the injected oocytes that were not treated with calcium ionophore spontaneously showed signs of fertilization. In contrast, 75% of the injectedoocytes thatwere treated with calciumionophore immediately after injection proceeded to develop. They extruded the second polar body and formed two PN during a normal time frame. Every oocyte from both groups that exhibited signs of normal fertilization successfully cleaved, several producing embryos of good morphological character. The sperm used in these cycles, as demonstrated by light microscopy and TEM, exhibited the classic characteristic of globozoospermia by their complete lack of acrosomal vesicles. However, there were other ultrastructural defects, including irregular midpiece morphology and apparent nuclear packaging. These characteristics indicate that variable components may be missing or abnormal in the sperm from this patient. The release of free Ca 2 + from intracellular stores in the oocyte is essential for oocyte activation during fertilization. It has been demonstrated that the fertilizing sperm mediates this release, possibly through the introduction of a soluble factorts) after sperm-oocyte fusion (13, 14). Recently, one of these factors has been identified and characterized (15). This factor, oscillin, has been shown to induce transient calcium ion fluctuations after injection into 00 cytes that mimic the calcium fluctuations seen after normal fertilization. These data provide strong evidence that the activating calcium response needed during fertilization is mediated by the introduction of a soluble sperm factor. With this evidence, coupled with these lcsi data, it is tempting to speculate that sperm from this patient are deficient in oscillin expression. The ultrastructural morphology of these sperm do not provide specific evidence for this possibility. However, further study of this patient with antibody probes to oscillin is currently under way to examine this possibility. It must be stressed that we do not recommend the use of Ca 2 + ionophore treatment as a way routinely to circumvent activation failure after lcsi. This drug was used with this couple in an attempt to salvage unexpected and extremely poor outcomes. There have been no reports of the use of calcium ionophore in this manner and there are no data regarding the direct effects that this drug may have on embryo development. Thus, routine use of this approach to enhance fertilization rates with cases exhibiting poor lcsi fertilization should await further research. The outcomes of these cycles indicate that fertilization failure with some patients presenting with globozoospermia may be because of oocyte activation problems. The low worldwide PRs observed with these patients is further evidence that the developmental capabilities of round-headed sperm may be significantly compromised at many levels, not just with oocyte activation or cleavage arrest. Liu et al. (8) reported the outcomes of 11 ICSI cycles in patients with globozoospermia. They encountered fertilization failure in 7 of these cases, indicating the possibility that these sperm also may be incapable of oocyte activation. In addition to the cycles described here, we also have performed two ICSI cycles with a another patient exhibiting globozoospermia (data not shown). Spontaneous fertilization was low in these cycles (46%), but only one fertilized oocyte cleaved (20%). No pregnancy resulted after the transfer of the cleaving embryo. The cleavage of only one embryo was remarkable and suggests that the sperm of this globozoospermia patient may lack normal centrosomes. Although our suspicions require further Vol. 68, No.1, July 1997 Battaglia et al. Oocyte inactivation with round-headed sperm 121

5 proof, there is ample evidence that normal sperm contribute important centrosomal components that are responsible for mediating embryonic cleavage (16-18). These ICSI cases underscore the concept that globozoospermia has multiple etiologies. Thus, the results achieved with lesl should be expected to reflect variable dysfunction in the sperm. AB additional cases are explored, the etiology of this condition may be understood further and appropriate therapies may be developed to handle them. REFERENCES. 1. Schmiady H, Radke E, Kentenich H. Round-headed spermatozoa-contraindication for IVF. Geburtshilfe Frauenheilkd 1992;52: Wolff HH, Schill WB, Moritz P, Round-headed spermatozoa: a rare andrologic finding ("globe-headed spermatozoa," "globozoospermia"). Hautarzt 1976;27: Holst ein AF, Schirren C, Schirren CG. Human spermatids and spermatozoa lacking acrosomes. J Reprod Fertil 1973; 35: Escalier D. Failure of differentiation of the nuclear-perinuclear skeletal complex in the round-head ed human spermatozoa. Int J Dev BioI 1990;34: Baccetti B, Renieri T, Rosati F, Selmi MG, Casanova S. Further observa tions on the morph ogenesis of the round headed human spermatozoa. Andrologia 1997;9: Lundin K, Sjogren A, Nilsson L, Hamberger L. Fertilization and pregnancy after intracytopl asmic microinjection of aerosomeless spermatozoa, Fertil Steril 1994;62: Bourne H, Liu DY, Clarke GN, Baker HW. Normal fertiliz a tion and embryo development by intracytoplasmic sperm injection of round-headed acrosomeless sperm. Fertil Steril 1995;63: Liu J, Nagy Z, Joris H, Tournaye H, Smitz J, Camus M, et a!' Successful fertilization and esta blishment of pregnancies after intractyoplasmic sperm injection in patients with globezoospermia. Hum Reprod 1995;10: Liu J, Nagy Z, Joris H, Tourn aye H, Smitz J, Camus M, et a!. Analysis of 76 total fertilizati on failure cycles out of 2732 intracytoplas mic sperm injection cycles. Hum Reprod 1995; 10: Tucker MJ, Wright G, Morton PC, Mayer MP, Ingargiola PE, Jones AE. Practical evolution and application of direct intracytoplasmic sperm injection for male factor and idiopathic fertilization failure infertilities. Fertil Steril 1995; 63: Pal ermo G, Joris H, Derde J\.IP, Camus NI, Devroey P, Van Steirteghem A. Sperm cha racteristics and outcome of human assisted fertilization by subzonal insemination and intractyoplasmic sperm injection. Fertil Steril 1993;59: Koehler JK, Platz CC Jr, Waddell W, Jones MH, Smith R, Behrens S. Spermophagy in semen in the red wolf, Canis rufus. Mol Reprod Dev 1994;37: Kline D, Kline JT. Repetitive calcium transients and the role of calcium exocytosis and cell cycle activation in the mouse egg. Dev Bioi 1992;149: Swann K, Ozil JP. Dynamics of the calcium signal that tri g gers mammalian egg activati on. Int Rev Cyto11994; 152: Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA. Calcium oscillations in mamm alian eggs triggered by a soluble sperm protein. Nature 1996;379: Scha tt en G. The centrosome and its mode of inh eritance: the reduction of the centrosome duri ng gametogenesis and its restoration during fertilization. Dev BioI 1994;165: Simerly C, Wu GJ, Zoran S, Ord T, Rawlins R, Jon es J, et a!. The paternal inheritance of the centrosome, the cell's microtubule-organizing center, in humans, and the implications for infertility. Nat Med 1995;1: Palermo G, Munn'e S, Cohen J. The human zygote inherits its mitotic poten tial from th e male gamete. Hum Reprod 1994;9: Battaglia et al. Oocyte inactivation with round-headed sperm Fertility and Sterility»