TECHNIQUES AND INSTRUMENTATION

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1 TECHNIQUES AND INSTRUMENTATION FERTILITY AND STERILITY VOL. 69, NO. 3, MARCH 1998 Copyright 1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Sperm morphology evaluated by computer (IVOS) cannot predict the fertilization rate in vitro after intracytoplasmic sperm injection Nares Sukcharoen, M.D.,* Tippawan Sithipravej, B.Sc.(Biology), Sakchai Promviengchai, B.Sc.(Med. Tech.), Viwat Chinpilas, M.D., and Wisut Boonkasemsanti, M.D.* Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, and Center for Assisted Reproduction and Embryology (CARE), Bangkok, Thailand Received July 30, 1997; revised and accepted October 27, Reprint requests: Nares Sukcharoen, M.D., Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. * Department of Obstetrics and Gynecology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand. Center for Assisted Reproduction and Embryology (CARE), Bangkok 10330, Thailand /98/$19.00 PII S (97) Objective: To evaluate sperm morphology assessment using the IVOS (Hamilton-Thorne Research Version 3 Dimension Program, Beverly, MA) system in prediction of fertilization rate in vitro after intracytoplasmic sperm injection (ICSI). Design: A prospective clinical study. Setting: Diagnostic andrology laboratory and assisted conception service. Patient(s): Thirty-five patients from the ICSI program were evaluated. Semen samples were analyzed using a computerized system for conventional semen parameters, sperm movement characteristics, and sperm morphology. Only patients with three or more metaphase II (MII) oocytes available were studied. Intervention(s): None. Main Outcome Measure(s): Fertilization rates in vitro after ICSI were compared according to the sperm morphology obtained from the IVOS system. Result(s): Linear regression analysis of fertilization rates against the sperm parameters assessed by computer (IVOS), which included conventional semen parameters, sperm movement characteristics, percentage of normal sperm morphology, and percentage of each specific abnormal sperm morphology, did not reveal any significant correlations. The mean ( SEM) fertilization rates in the healthy prognosis group (normal sperm morphology 4%) and poor prognosis group (normal sperm morphology 4%) were % and %, respectively. There was no statistically significant difference in the mean fertilization rate between both groups. Moreover, no statistically significant difference was found in overall fertilization rates in vitro between the two prognosis categories (79.6% versus 78.0%). Conclusion(s): Sperm morphology obtained from the IVOS system is not related to the outcome of ICSI and cannot be used for prediction of fertilization rate in vitro after ICSI. (Fertil Steril 1998;69: by American Society for Reproductive Medicine.) Key Words: Sperm morphology, in vitro fertilization, intracytoplasmic sperm injection, IVOS system Several sperm parameters from conventional semen analysis and sperm function tests have been used to predict the outcome of assisted conceptive technology for male infertility treatment. Among these parameters, the percentage of normal sperm morphology was highly correlated with the in vitro fertilization rate (1). Furthermore, the time delay between testing and IVF did not appear to affect predictive accuracy (2). Since sperm morphology evaluated according to strict criteria was introduced (3), several advantages in the prediction of IVF (4, 5), intrauterine insemination (6), and in vivo reproduction outcomes have been shown (7). However, intraobserver, interobserver, and interlaboratory variations are known to occur in the assessment of sperm morphology (8). Therefore, sperm morphology assessment by computer analysis has been introduced to avoid variation in the interpretation of sperm morphology. The computer gives excellent repeatability of normal and abnormal cells (9, 10). It can identify those patients with a poor prognosis for fertilization in vitro (11). This system 564

2 can be of clinical value both in IVF units and andrology laboratories. Intracytoplasmic sperm injection (ICSI) is a recent and advanced assisted reproductive technology that has been shown to be a highly efficient treatment method for severe male factor infertility (12). The result of ICSI is not related to the sperm parameters (13). A recent study confirms that strict sperm morphology as a routine diagnostic tool is not related to the outcome of ICSI (14). However, information on semen analysis using the computer (IVOS system; Hamilton-Thorne Research Version 3 Dimension Program, Beverly, MA) to predict the fertilization rate after ICSI is lacking, especially with regard to sperm morphology. To address this issue, we conducted a study in which the relation between sperm morphology evaluated by computer (IVOS) and the fertilization rate in vitro after intracytoplasmic sperm injection was examined. MATERIALS AND METHODS Study Population The study population consisted of a cohort of 35 patients undergoing ICSI treatment at the Center for Assisted Reproduction and Embryology, Bangkok, Thailand, between January and June This study was reviewed and approved by the institutional review board. The indications for ICSI were severe male infertility (sperm concentration of /ml or sperm motility of 25% or normal morphology of 14%) and poor fertilization rate ( 20%) in previous IVF cycles. The mean age ( SEM) of the female partners was years (range, years), and the mean age ( SEM) of the male partners was years (range, years). The semen was assessed at the time of an initial consultation with the assisted conception service for sperm concentration, average path velocity (VAP), straight line velocity (VSL), percentage of sperm motility (the percentage of sperm exhibiting a VAP of 10 m/s), percentage rapid (the percentage of cells exhibiting a VAP of 25 m/s), and percentage of progressive motility (the percentage of cells exhibiting a tendency to swim straight [STR], which is VSL/VAP 100, of 75%), using a Hamilton-Thorne motility analyzer (HTMA-IVOS version 10; Hamilton- Thorne Research Inc., Beverly, MA) with an operating frequency of 25 Hz. The HTMA-IVOS setting used during the analysis has already been described (15). Computerized Sperm Morphology Assessment Before making smears, semen was washed with HEPESbuffered Ham s F-10 medium (Biochrom KG, Berlin, Germany) for 10 minutes at 200 g. Supernatant was carefully aspirated. The pellet was resuspended to obtain the final concentration of /ml (16). A sperm suspension (5 10 L) was placed on a clean glass slide. A coverslip was used to pull the drop evenly across the surface of the slide. The slide was then allowed to air dry and was stained with Diff-Quik (Baxter, McGraw Park, IL). Briefly, the slide was fixed by immersion in Diff-Quik fixative for 10 seconds, stained in Diff-Quik solution 1 for 20 seconds, and counterstained with Diff-Quik solution 2 for 20 seconds. The stained slide was rinsed gently in distilled water, blotted, and allowed to dry. A coverslip was applied, and the slides were read on the Hamilton-Thorne Dimensions Morphology Analyzer (11). The images were analyzed by using an interactive mode (an image was captured and processed immediately). The processing of every cell was displayed on the screen. The outline of the cell was superimposed with a color line on the microscope image, and the classification result was shown in a color-coded table, also superimposed on the image. Every object located in the image either was accepted as a valid sperm cell for further evaluation or was rejected if it was not in focus or if it was considered to be cluttered. A cell was analyzed and classified into one of three categories: normal, subnormal, or abnormal (17). The breakdown of the classification was as follows: size (normal, small, or large); shape (normal, slightly amorphous, thin, tapered, elongated, round, midpiece, or severely amorphous); and acrosome (small or large) (10). In addition, numerical values were given for the focal quality of the cell, a shape acceptance factor, and the area of the cell in pixels. For a cell to be classified as normal, all three parameters of size, shape, and acrosome must be considered normal. If both the size and acrosome were normal but the shape was slightly amorphous, the cell was classified into the subnormal category. In any other case, the cell was classified as abnormal. For each semen sample, at least 100 sperm were examined. Occasionally, when an extremely low sperm count was found, the number of sperm examined had to be reduced to 50. The statistic was used to assess agreement of measurement when a categorial measurement was assessed by two repeat readings (18). Excellent results ( 0.85) were obtained in our laboratory. Ovarian Stimulation Briefly, ovarian stimulation was performed with use of two protocols (down-regulation and flare-up) using the GnRH agonist (GnRH-a) buserelin acetate in combination with hmg and FSH. The initial dosage of hmg and FSH was individualized based on day 3 FSH or response to previous stimulations. Changes in gonadotrophin dosage were based on follicular development as reflected by changes in follicular diameter and number assessed by serial transvaginal ultrasonography. Five thousand international units of hcg (Profasi; Serono Laboratories, Randolph, MA) was administered intramuscularly when the leading follicle was 20 mm. Transvaginal follicular aspiration was performed 36 hours later. FERTILITY & STERILITY 565

3 Semen Preparation One hour before the scheduled time of oocyte retrieval, a semen sample was collected by masturbation into sterile containers. After liquefaction, the semen sample was mixed with a sterile pipette. Sperm was then prepared by two-layer (40% and 80%) discontinuous Percoll (Pharmacia, Uppsala, Sweden) gradient centrifugation for 20 minutes at 300 g. The pellets were removed carefully, washed twice by centrifugation (5 minutes at 600 g), resuspended at a concentration of /ml in IVF medium (cat. no ; Medicult, Copenhagen, Denmark), and incubated until the time of injection. Oocyte Preparation and Handling The oocyte preparation and handling for complete removal of the corona cells were carried out as described previously (19). Oocytes were assessed for nuclear maturity by examination for the first polar body and the presence or absence of a germinal vesicle. Any cytoplasmic abnormalities were also examined. Intracytoplasmic Sperm Injection Procedure Intracytoplasmic sperm injection was performed as described previously (19). Microinjection was preserved only for the mature oocytes that extruded their first polar bodies. To provide a reliable assessment of the effect of sperm morphology on the fertilization rate in vitro, only cycles that had three or more MII oocytes were included in this study. Assessment of Fertilization About hours after the microinjection, the oocytes were observed under the inverted microscope ( 200 or 400 magnification) for any sign of damage that may have been caused by microinjection and for the presence of pronuclei and polar bodies. Fertilization was considered normal when two clearly distinct pronuclei containing nucleoli were present. Statistical Analysis Two cases were excluded from the analysis because fewer than three MII oocytes were collected. Data are generally presented as means SEM. The relationship between the conventional semen parameters, the percentage of normal sperm morphology, the percentage of each specific abnormal sperm morphology, and IVF rate was analyzed using linear regression analysis. Comparison between two prognosis categories with regard to mean fertilization rates in vitro and overall fertilization rate in vitro were made using an unpaired t-test and a 2 test, respectively. All statistical analyses were performed using the SPSS for Windows version 6.0 (SPSS Inc., Chicago, IL) on an IBM compatible microcomputer. P 0.05 was considered to be statistically significant. RESULTS Sperm Characteristics and Computerized Sperm Morphology Assessment For this cohort of patients, the constituents of the conventional semen profile, expressed as mean SEM, were volume ( ml), sperm concentration ( sperm/ml), percentage of normal sperm morphology ( %), and percentage of sperm motility (44.8% 4.1%). The abnormal sperm were classified as too long (41.1% 3.7%), too short (1.2% 0.2%), too wide (11.8% 1.2%), thin (15.3% 1.4%), tapered (17.1% 1.3%), elongated (8.0% 1.7%), round (12.7% 1.2%), severely amorphous (23.2% 2.1%), having a small acrosome (9.1% 1.7%), and having a large acrosome (0.5% 0.3%). The sperm movement characteristics were VAP ( m/s), percentage rapid (28.9% 3.0%), and percentage progressive motility (17.5% 1.9%). Outcome of ICSI For the 35 cycles studied, the mean number of oocytes retrieved ( SEM) was The mean IVF rate after ICSI ( SEM) was 77.9% 2.8%. In all subsequent analyses, the two cases in which fewer than three oocytes were collected were omitted. Relationship Between Sperm Parameters and Fertilization Rate Simple Linear Regression Analysis Linear regression analysis of the IVF rate after ICSI against the parameters of the sperm characteristics assessed by computer (including conventional semen parameters, sperm movement characteristics, percentage of normal sperm morphology [Fig. 1], percentage of each specific abnormal sperm morphology) for this cohort of patients did not reveal any significant correlations. Moreover, there were no significant correlations between the abnormal fertilization (1 pronuclei and 3 pronuclei) rate and the percentage of each specific abnormal sperm morphology. Comparison of Semen Samples Classified According to the Predictive Categories of the Strict Criteria A second approach for assessment of the relationship between sperm morphology assessed by computer (IVOS) and fertilization rates after ICSI was to compare those semen samples classified according to the two predictive categories of the strict criteria: good prognosis ( 4% morphologically normal sperm) and poor prognosis ( 4% morphologically normal sperm). The semen samples used in 33 ICSI cycles were categorized according to the strict sperm morphologic criteria. It was found that 14 (42.4%) of the ICSI cycles belonged to the good prognosis category and 19 (57.6%) to the poor prognosis category. The IVF rates after ICSI treatment were evaluated and compared with the sperm morphology classification to 566 Sukcharoen et al. Techniques and instrumentation Vol. 69, No. 3, March 1998

4 FIGURE 1 The correlation between the percentage of normal sperm morphology and in vitro fertilization rate after ICSI. Dotted line represents the cut-off point (4% morphologically normal sperm) used for classification of the semen samples to the two prognostic categories. TABLE 1 Overall outcome of ICSI cycles (n 33) in relation to prognosis category according to strict sperm morphology criteria. Prognosis category (normal sperm morphology %) Good ( 4%) Poor ( 4%) Number of cycles (oocyte retrievals) Number of oocytes Number of injected oocytes Number of fertilized oocytes (2PN) (%) (79.6%) (78.0%) Note: PN pronuclei. determine whether the strict criteria could aid in predicting the outcome of ICSI. The mean fertilization rates ( SEM) in the good prognosis group and poor prognosis group were 82.4% 4.0% and 75.0% 3.8%, respectively. The mean fertilization rate between both groups was not significantly different (Fig. 2). Moreover, no significant difference was found in overall fertilization rates between the two prognosis categories (79.6% versus 78.0%) (Table 1). DISCUSSION FIGURE 2 In vitro fertilization after ICSI according to the two prognostic categories of the strict criteria: good prognosis and poor prognosis. (Box plot shows the 10 th,25 th,50 th (median), 75 th, and 90 th percentiles). The computer has been shown to serve as an excellent standard for sperm morphology assessment (20). A moreobjective evaluation of morphology based on these criteria may allow all centers specializing in assisted reproduction to analyze sperm using strict criteria in a similar manner and in a more accurate way. In a recent publication of a comparison between computer and manual methods, there was a positive and significant correlation between subjective analysis and computer-based analysis using a Hamilton-Thorne Dimension morphology analyzer for evaluating the sperm morphology. The repeatability of the computerized method was excellent (9, 10). The use of strict criteria for analysis of sperm morphology has been advocated recently for couples undergoing advanced assisted reproductive technologies. The strict criteria indicated that couples with 14% normal forms had decreased fertilization and pregnancy rates when undergoing IVF-ET (3) and that a poor prognosis group ( 4% normal forms) had significantly lower fertilization rates compared with couples with 4% normal forms (5, 17). Sperm morphology assessed by strict criteria can be used as a biomarker of sperm dysfunction. Abnormal morphology is usually associated with abnormal sperm movement, impaired capacity to bind and penetrate zona pellucida, and low incidence of spontaneous and induced acrosome reaction. At a cellular level, abnormal morphology is associated with a higher content of creatine kinase and impaired capacity to undergo appropriate changes in intracellular calcium concentration (21). Furthermore, altered expression of mannosebinding sites, which are putative zona receptors, may be one of the molecular defects causing decreased fertility in morphologically abnormal human sperm (22). Since the introduction of ICSI, patients with severe male factor infertility have been treated successfully. The drawbacks of this new technique, however, are that it is timeconsuming, expensive, and requires specially trained laboratory personnel. Therefore, it would be of great advantage to find a diagnostic method to predict the success rate of ICSI. Apart from overcoming severe male infertility problems, ICSI has also provided another means of assessing the effect of sperm characteristics, especially sperm morphol- FERTILITY & STERILITY 567

5 ogy, on the fertilization rate in vitro after ICSI treatment. With this type of study, we can control many confounding factors. However, this study demonstrates that acceptable rates of fertilization can be achieved with ICSI, even in patients with a poor prognosis ( 4% normal sperm morphology). From linear regression analysis, the sperm characteristics assessed by computer (IVOS) (including conventional semen parameters, sperm movement characteristics, percentage of normal sperm morphology, percentage of each specific abnormal sperm morphology) show no significant correlation with the fertilization rate in vitro after ICSI treatment. It may be because embryologists usually select the best sperm with apparent normal morphology and motility and because the ICSI procedure bypasses many natural processes for fertilization such as hyperactivated motility, zona binding, and penetration, which require many specific sperm characteristics and functions. Therefore, we conclude that evaluating sperm morphology according to strict criteria using IVOS is not related to the fertilization rate in vitro after ICSI. This study confirmed the findings of previous studies that there are no important influences from either the type or the extent of sperm impairment of three basic sperm parameters (total sperm count, sperm motility, and morphology) on the outcome of ICSI (13, 14). This study also showed that the percentage of specific abnormal morphology was not correlated with an abnormal fertilization rate. In previous studies, the failure of the fertilization rate in vitro after ICSI was explained by different factors related to the specific semen characteristics (only immotile or roundheaded spermatozoa), the poor viability of the sperm used for ICSI treatment (13, 23), and the poor quality of oocytes (abnormal morphology or damage after ICSI) (23). The lack of intracytoplasmic sperm oocyte interaction rather than sperm morphologic defects is the most critical role in the fertilization process in intracytoplasmic sperm injection (24). Furthermore, poor chromatin packaging and/or damaged DNA may contribute to failure of sperm decondensation after ICSI and result in failure of fertilization. However, the percentage of sperm that had initiated decondensation in unfertilized oocytes was not influenced by morphology (25). In conclusion, sperm morphology obtained from the IVOS system is not related to the outcome of ICSI and cannot be used to predict fertilization rate in vitro after ICSI. This present study also shows that ICSI is a highly efficient treatment method for cases with poor sperm morphology. References 1. Sukcharoen N, Keith J, Irvine DS, Aitken RJ. Predicting the fertilizing potential of human sperm suspensions in vitro: importance of sperm morphology and leukocyte contamination. Fertil Steril 1995;63: Sukcharoen N, Keith J, Irvine DS, Aitken RJ. Prediction of the in vitro fertilizing potential of human spermatozoa using sperm function tests the effect of the delay between testing and IVF. Hum Reprod 1996;11: Kruger TF, Menkveld R, Stander FSH, Lombard CJ, Van der Merwe JP, van Zyl JA, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1986;46: Vawda AI, Gunby J, Younglai EV. Semen parameters as predictors of in-vitro fertilization: the importance of strict criteria morphology. Hum Reprod 1996;11: Grow DR, Oehninger S, Seltman HJ, Toner JP, Swanson RJ, Kruger TF, et al. Sperm morphology as diagnosed by strict criteria: probing the impact of teratozoospermia on fertilization rate and pregnancy outcome in a large in vitro fertilization population. Fertil Steril 1994;62: Burr RW, Siegberg R, Flaherty SP, Wang X-J, Matthews CD. The influence of sperm morphology and the number of motile sperm inseminated on the outcome of intrauterine insemination combined with mild ovarian stimulation. Fertil Steril 1996;65: Eggert-Kruse W, Schwartz H, Rohr G, Demirakca T, Tilgen W, Runnebaum B. Sperm morphology assessment using strict criteria and male fertility under in-vivo conditions of conception. Hum Reprod 1996;11: Neuwinger J, Behre HM, Nieschlag E. External quality control in the andrology laboratory: an experimental multicenter trial. Fertil Steril 1990;54: Kruger TF, du Toit TC, Franken DR, Acosta AA, Oehninger SC, Menkveld R, et al. A new computerized method of reading sperm morphology (strict criteria) is as efficient as technician reading. Fertil Steril 1993;59: Kruger TF, du Toit TC, Franken DR, Menkveld R, Lombard CJ. Sperm morphology: assessing the agreement between the manual method (strict criteria) and the sperm morphology analyzer IVOS. Fertil Steril 1995;63: Kruger TF, Lacquet FA, Sarmiento CA, Menkveld R, Ozgur K, Lombard CJ, et al. A prospective study on the predictive value of normal sperm morphology as evaluated by computer (IVOS). Fertil Steril 1996;66: Palermo G, Joris H, Derde M-P, Camus M, Devroey P, Van Steirteghem AC. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril 1993;59: Nagy ZP, Liu J, Joris H, Verheyen G, Tournaye H, Camus M, et al. The result of intracytoplasmic sperm injection is not related to any of the three basic sperm parameters. Hum Reprod 1995;10: Svalander P, Jakobsson AH, Forsberg AS, Bengtsson AC, Wikland M. The outcome of intracytoplasmic sperm injection is unrelated to strict criteria sperm morphology. Hum Reprod 1996;11: Sukcharoen N, Keith J, Irvine DS, Aitken RJ. Definition of the optimal criteria for identifying hyperactivated human spermatozoa at 25 Hz using in-vitro fertilization as a functional end-point. Hum Reprod 1995;10: Davis RO, Gravance CG. Standardization of specimen preparation, staining, and sampling methods improves automated sperm-head morphometry analysis. Fertil Steril 1993;59: Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril 1988;49: Maxwell AE, Pillner AEG. Deriving coefficients of reliability and agreement for ratings. Br J Math Stat Psychol 1968;21: Van Steirteghem A, Nagy Z, Joris H, Liu J, Staessen C, Smitz J, et al. High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod 1993;8: Hofmann GE, Santilli BA, Kindig S, Scott RT, Johnson CA. Intraobserver, interobserver variation of sperm critical morphology: comparison of examiner and computer-assisted analysis. Fertil Steril 1996;65: Grow D, Oehninger S. Strict criteria for the evaluation of human sperm morphology and its impact on assisted reproduction. Andrologia 1995; 27: Youssef HM, Doncel GF, Bassiouni BA, Acosta AA. Mannose-binding sites on human spermatozoa and sperm morphology. Fertil Steril 1996; 66: Liu J, Nagy Z, Joris H, Tournaye H, Smitz J, Camus M, et al. Analysis of 76 total fertilization failure cycles out of 2,732 intracytoplasmic sperm injection cycles. Hum Reprod 1995;10: Kupker W, al-hasani S, Schulze W, Kuhnel W, Schill T, Felberbaum R, et al. Morphology in intracytoplasmic sperm injection: preliminary results. J Assist Reprod Genet 1995;12: Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, et al. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod 1996;11: Sukcharoen et al. Techniques and instrumentation Vol. 69, No. 3, March 1998