Spermac stain analysis of human sperm acrosomes

Transcription

1 FERTILITY AND STERILITY VOL. 72, NO. 1, JULY 1999 Copyright 1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Spermac stain analysis of human sperm acrosomes Philip J. Chan, Ph.D., H.C.L.D.,* Johannah U. Corselli, Ph.D., John D. Jacobson, M.D., William C. Patton, M.D., and Alan King, M.D. Loma Linda University School of Medicine, Loma Linda, California Objective: To study the association between low percentages of intact sperm acrosomes and fertilization failures in conventional IVF procedures. Design: A retrospective study. Setting: Clinical and academic research environment. Patient(s): Patients undergoing treatment of infertility. Intervention(s): Sperm cells were fixed and stained using the Spermac stain. Main Outcome Measure(s): Percentages of intact acrosomes and fertilization. Result(s): There was a significant association between specimens with 40% intact acrosomes and failed conventional IVF procedures. Among the 29 cases with 40% intact acrosomes, 9 cases (31%) resulted in zero penetration of the oocytes. The mean ( SEM) percentage of fertilization was lower in the abnormal acrosome group (43.3% 6.5%) than in the normal acrosome group (64.1% 5.6%). The status of the sperm acrosome was not correlated with the results of fertilization in intracytoplasmic sperm injection procedures. Conclusion(s): Sperm with low percentages of intact acrosomes were associated with failed fertilization. The Spermac stain was useful for assessing acrosomes and identifying possible male factor infertility problems. The results suggested that a minimum percentage of sperm with intact acrosomes are needed for fertilization to occur in vitro. (Fertil Steril 1999;72: by American Society for Reproductive Medicine.) Key Words: IVF-ET, acrosome reaction, spermatozoa, Spermac stain, ICSI Received July 24, 1998; revised and accepted March 12, Reprint requests: Philip J. Chan, Ph.D., Loma Linda University Center for Fertility and In Vitro Fertilization, Anderson Street, Loma Linda, California (FAX: ; E- mail: fertility). * Department of Physiology and Pharmacology. Department of Gynecology and Obstetrics /99/$20.00 PII S (99) The acrosome reaction (1 3) of the ejaculated sperm cell occurs either spontaneously or at the surface of the zona pellucida after binding to the ZP3 receptor protein (4, 5). The mechanism involves fusion of the sperm plasma membrane with the outer acrosomal membrane, calcium influx (4), and vesiculation and exocytosis of the inner acrosomal enzymes (6, 7). The sperm head enters the oocyte by endocytosis, decondenses, and forms the pronucleus, which shortly thereafter participates in syngametic approximation with the oocyte pronucleus (8). Failure to fertilize is thought to occur when the sperm lacks acrosomal enzymes (i.e., the round-headed syndrome [(9)]) or prematurely loses acrosomal enzymes because it undergoes a spontaneous acrosome reaction. The intracytoplasmic sperm injection (ICSI) procedure can bypass acrosomal dysfunction, but because of recent concerns about injecting genetically defective sperm, conventional IVF remains the preferred approach when appropriate. Hence, it is important to determine the status of the sperm acrosome to help in planning the course of treatment with assisted reproductive technology. Methods of assessing the sperm acrosome include the Pisum sativum agglutinin-fluorescein isothiocyanate assay (10), the triple stain procedure (11), the indirect immunofluorescence assay (12), and the chlortetracycline assay (13). However, these methods are laborious to perform and difficult to incorporate into the routine semen analysis. The Spermac stain (14 18), originally developed for domestic animal species, has been shown to be methodologically simple to use and rapid in the assessment of acrosomal status in human sperm. The accuracy of this stain for human sperm analysis has been verified using electron micrographs (15). The objective of this study was to determine whether the status of sperm acrosomes ana- 124

2 lyzed with the use of the Spermac stain during routine semen analysis would be predictive of fertilization failure in conventional IVF procedures. The information obtained would provide further supporting evidence for the usefulness of this simple staining method as part of the routine semen analysis. MATERIALS AND METHODS Semen Samples In this retrospective study, semen specimens were obtained from the male partners of 63 couples undergoing conventional IVF procedures (n 22), ICSI procedures (n 24), or a combination of both procedures (n 17). The study was approved by the institutional review board at our institution. Signed informed consent forms were obtained from all patients who participated in the study. The sperm were assessed during routine semen analyses (19) performed as part of the pretreatment infertility workup in a retrospective analysis of data design. Cases involving donor sperm or canceled ovulation induction cycles were omitted from the study. Several stains, including the Diff-Quik, the Papanicolaou, and the Spermac stains, were available for use in the morphologic analysis of sperm. In our laboratory (17), the Spermac stain already was being used for morphologic analysis. Hence, nothing different was added to the routine semen analysis procedure except that sperm morphology and acrosomal status were evaluated together for each stained slide. A study of the semen analysis parameters associated with the Spermac stain data has been published previously (17). Spermac Stain Procedure A 100- L aliquot of semen was mixed with an equal volume of culture medium (modified human tubal fluid kept at 23 C) and the diluted semen was smeared on a glass slide. The staining procedure was carried out according to the Spermac kit manufacturer s guidelines (Stain Enterprises, Onderstepoort, South Africa). The sperm smear was allowed to air-dry before being fixed in the formalin solution (Fixative I) provided in the Spermac kit for 5 minutes at room temperature (23 C). Each slide with fixed sperm was washed in tapwater, and stain solution A was pipetted out and used to flood the slide, which was kept in a horizontal position. Stain solution A was kept on the slide for 2 minutes and then rinsed off with water. Stain solution B was used to flood the slide for 1 minute and then was rinsed off with water. Stain solution C was used to flood the slide for 1 minute and then it was rinsed off. The stained slides were air-dried for 10 minutes and then analyzed in oil immersion ( 1,000) and by light microscopy to assess the percentage of sperm with intact acrosomes. Regardless of the shape of the sperm head, sperm were considered to have an intact acrosome when the anterior acrosomal region stained green (14, 15) and the dark green, FIGURE 1 Ejaculated sperm cells after staining with the Spermac acrosomal stain. Sperm with intact acrosomes have a dark green, rubber-band, semicircle cap line with red counterstained postacrosomal regions (left). Acrosome-reacted (center) and acrosome-defective (right) sperm do not have the continuous dark green band of even thickness at the acrosomal cap. thickened, rubber-band border forming a semicircle at the tip of the head remained unbroken or continuous (Fig. 1). The posterior postacrosomal region of each sperm head was red-pink in coloration. Sperm that lacked the red-pink counterstain in the posterior head region were inadequately stained and were not counted. Sperm with nonintact acrosomes (reacted or defective acrosomes) showed peeled acrosomal membranes, spotting, irregular thickness in the green band, or partial green coloration. Another type of sperm with nonintact acrosomes (missing acrosomal enzymes) were stained either white or red at the acrosomal region and had no green color at the head. For each sperm smear, the percentage of sperm with intact acrosomes was calculated by dividing the number of sperm with intact green acrosomes by the total number of sperm analyzed and multiplying by 100. At least 100 sperm cells were assessed for each slide, and the same technician performed all the Spermac stain analyses. The cutoff value of 40%, with higher percentages interpreted as normal percentages of intact sperm acrosomes (Table 1), was based on the lowest percentage that did not result in failed fertilization with conventional IVF. The same Spermac-stained slides also were used to determine the percentage of sperm with strict normal morphology according to the Tygerberg strict criteria developed by Kruger et al. (20) on at least 100 sperm. A sperm was classified as normal when the head was oval, the acrosome occupied 40% 70% of the head, there were no midpiece or tail defects, cytoplasmic droplets were absent or negligible, and the dimensions of the head were appropriate. Assisted Reproductive Technologies The IVF procedures were performed according to established protocols (21). Briefly, female patients and their partners scheduled to undergo IVF procedures at the Center for FERTILITY & STERILITY 125

3 TABLE 1 Relation between sperm specimens with abnormal or normal percentages of intact acrosomes and the outcome of conventional IVF or intracytoplasmic sperm injection. Parameter 40% intact acrosomes group 40% intact acrosomes group Conventional IVF Total no. of cases No. of failed fertilizations/total 9/29 (31) 0/12 (0)* no. of cases (%) Fertilization in vitro (%) * Strict normal morphology (%) * Sperm concentration ( 10 6 / * ml) Total motility (%) Intact acrosomes (%) * ICSI Total no. of cases 34 4 No. of failed fertilizations/total 0/34 (0) 0/4 (0) no. of cases (%) Fertilization by ICSI (%) Strict normal morphology (%) Sperm concentration ( 10 6 / * ml) Total motility (%) * Intact acrosomes (%) * Note: Data are means SEM unless otherwise noted. ICSI intracytoplasmic sperm injection. * P.05. Fertility and In Vitro Fertilization were given routine examinations. For the female patients, ovulation induction was achieved after several days of leuprolide acetate suppression and was initiated using recombinant FSH (an average of four ampules given in split doses daily, 75 IU per ampule) for 9 12 days; 10,000 U of hcg generally was administered on the 10th day if the E 2 level was 500 pg/ml and the largest follicle was 18 mm in diameter. Patients who underwent leuprolide acetate cycles followed a similar induction protocol, except that 0.5 mg of leuprolide acetate was administered beginning on day 21 of the previous cycle and ending on the last day of stimulation (21). The culture medium used for all protocols was human tubal fluid (Irvine Scientific, Santa Ana, CA) supplemented with synthetic serum substitute (Irvine Scientific). At the time of the IVF procedure, the sperm cells were washed according to standard wash procedures and used to inseminate the oocytes at a concentration of 100,000 sperm per milliliter for routine cases. Intracytoplasmic sperm injection was performed in cases of suspected male factor infertility. Day 3 embryos were returned transcervically to the uterine lumen of the originating patients. Each patient was monitored for evidence of clinical pregnancy. The criteria for clinical pregnancy were an elevated serum hcg concentration and detection of a uterine sac with a beating heart on ultrasound examination. Data Analyses Statistical analyses were performed using commercial computerized statistical programs. Student s t-test was used to determine statistically significant differences between means. Categorical data were analyzed using the 2 test statistic. P.05 was considered statistically significant. RESULTS The results showed a significant association between semen specimens with 40% intact acrosomes and failed fertilization with conventional IVF procedures (Table 1). Among the 29 cases in which the sperm of the male partner had 40% intact acrosomes, 9 cases (31%) resulted in no penetration of the oocytes. Two of the 9 cases were in couples with male factor infertility. Altogether, there were only 3 cases of male factor infertility in the 63 patients based on semen parameters (sperm count of /ml, 50% total motility, and 30% normal sperm forms by World Health Organization criteria). The mean percentage of fertilization in the abnormal intact acrosome group was significantly lower than in the group with 40% intact sperm acrosomes. Similarly, there were differences in the percentage of strict normal morphology and the sperm count between the abnormal and normal acrosome groups. There were 16 clinical pregnancies and 2 miscarriages in the abnormal acrosome group and 5 clinical pregnancies and no miscarriages in the normal acrosome group. However, these data were not applicable because some cases involved the transfer of embryos derived from both conventional IVF and ICSI. There were 2 clinical pregnancies from ICSI-derived embryos among the 9 cases of failed conventional IVF. All cases of ICSI yielded fertilized embryos (Table 1). There was no difference in the percentage of fertilization. However, there were differences between the abnormal and normal acrosome groups in the percentage of strict normal morphology, the sperm count, and motility. The abnormal acrosome group had 13 clinical pregnancies and 1 miscarriage, whereas the normal acrosome group had 1 clinical pregnancy and no miscarriages. DISCUSSION The results demonstrated an association between ejaculated sperm with low percentages of intact acrosomes ( 40%) and failed fertilization. This finding suggested that the Spermac acrosome stain procedure may be used to help screen for possible cases of male factor infertility. It is logical that sperm cells with low percentages of intact acrosomes would not fertilize oocytes, and this is supported by the finding of significantly lower fertilization rates in the abnormal acrosome group in the present study. These data are consistent with those in previous reports (10). Other studies (22, 23) have shown that in cases of failed fertiliza- 126 Chan et al. Spermac acrosome stain Vol. 72, No. 1, July 1999

4 tion, the sperm cells do not have appropriate head-directed mannose ligand activity and membrane cholesterol levels, and that they have low percentages of spontaneous acrosome loss. Such cases have been referred to as nonresponders by Benoff et al. (22, 23). In the present study, explanations for the observed low percentages of intact acrosomes in sperm include abnormal spermiogenesis with a disrupted Golgi transformation step, premature discharge of acrosomal contents caused by luminal receptors and autoantibodies, or an artifact resulting from genetic defects (8). Sperm with abnormally low percentages of intact acrosomes also had low percentages of strict normal morphology. As expected, the acrosomal status of the sperm was not associated with the fertilization results at ICSI. When comparing Spermac staining with advanced tests such as the mannose binding assay, several advantages of Spermac staining are apparent: extra preparatory steps are not needed because Spermac staining already is being used for sperm morphology assessment; the method is simple and rapid, yielding results within 20 minutes; a fluorescent microscope and darkroom are not needed; and repeated low-cost analyses can be performed on specimens at different times (14, 17, 18). Further, assessment of the sperm acrosomal status using Spermac staining has been shown to correlate with the results of electron microscopy studies (15, 16). Sperm acrosomes stained by the Spermac stain (Fig. 1) can be categorized as intact (the green acrosome band is continuous), reacted (the band appears as spots or dashes, or it is missing), or defective (the band appears uneven in thickness). Colored photographs are available on the internet website, One concern with the staining procedure is disruption of the acrosome during the air-drying process. The procedure may be improved by diluting the semen in the Spermac fixative solution (formalin) provided, making the smear, and then air-drying. Other concerns, such as distinguishing between dead and viable sperm, have been addressed by combining the Spermac stain with the hypo-osmotic swelling solution (18) or multiplying the percentage of intact acrosomes by the fraction of viable sperm determined with the use of the supravital stain during the routine semen analysis (19). Although the traditional semen analysis parameters of sperm count, motility, and morphology have been criticized severely for their lack of correlation with sperm function and fertilizing capacity, little has been done to improve this problem (19). Adding the evaluation of acrosomal status to the semen analysis provides some improvement with minimal effort. The 31% incidence of failed fertilization in men with low percentages of intact acrosomes suggests that the Spermac stain cannot predict with 100% accuracy and is not meant to be used alone. The collective interpretation of several other sperm tests, such as strict criteria morphology, is necessary. In addition, variations in sperm quality occur with different ejaculates, so repeated testing may be required (19). The consistent identification of male infertility factors in sperm specimens using the Spermac stain as part of routine semen analyses would assist clinicians in deciding between conventional infertility treatments and the need for ICSI. Further, the detection of sperm with low percentages of intact acrosomes would trigger a thorough evaluation for possible round-headed sperm syndrome. In summary, our findings suggest that the minimal extra effort expended for sperm acrosome evaluation may help improve the traditional semen analysis for the detection of male factor infertility. Acknowledgments: The authors thank members of the Loma Linda University Gynecology and Obstetrics Medical Group, Inc., and the Loma Linda University Center for Fertility and In Vitro Fertilization, for their help and support in the preparation of this manuscript. References 1. Barros C. Capacitation of mammalian spermatozoa. In: Coutinho E, Fuchs F, editors. Physiology and genetics of reproduction, part B. New York: Plenum Publishing Corp., 1974: Bedford JM. Sperm capacitation and fertilization in mammals. Biol Reprod 1970;(Suppl 2): Meizel S. The mammalian sperm acrosome reaction: a biochemical approach. In: Johnson MH, editor. Development in mammals. vol. 3. New York: Elsevier-North Holland, 1978: Bleil JD, Wassarman PM. 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