Prediction of pregnancy by intrauterine insemination using CASA estimates and strict criteria in patients with male factor infertility

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1 international journal of andrology, 27:63 68 (2004) Prediction of pregnancy by intrauterine insemination using CASA estimates and strict criteria in patients with male factor infertility HIROAKI SHIBAHARA, HIROMI OBARA, AYUSTAWATI, YUKI HIRANO, TATSUYA SUZUKI, AKIKO OHNO, SATORU TAKAMIZAWA and MITSUAKI SUZUKI Department of Obstetrics and Gynecology, Jichi Medical School, Tochigi, Japan Summary This study was performed to predict pregnancy by intrauterine insemination (IUI) using computer-aided sperm analysis (CASA) estimates and strict criteria in patients with male factor infertility. IUI was performed in 682 cycles on 160 male factor infertile patients. Semen examinations were carried out by CASA and strict criteria before and after sperm preparation using continuous-step density gradient centrifugation. Receiver operating characteristics (ROC) curves were constructed for assessment of the effectiveness of each individual parameter in predicting pregnancy by IUI. A clinically acceptable threshold was calculated when sensitivity plus specificity were maximum. The average cycle of IUI performed was 4.3 ± 2.4. Pregnancy rate per cycle and per patient were 7.2% (49/682) and 28.1% (45/160), respectively. Using ROC curve, it was shown that normal sperm morphology assessed by the strict criteria before sperm separation and five parameters after sperm separation including rapid, progressive motility, average path velocity (VAP), curvilinear velocity (VCL), and straight line velocity (VSL) were able to predict pregnancy by IUI. Correlation between sperm parameters and pregnancy outcome was examined by the logistic regression model. In a multivariate analysis normal morphology before sperm separation 15.5% [odds ratio (OR) ¼ 2.2, p ¼ 0.02], rapid after sperm separation 25.5% [OR ¼ 3.9, p ¼ 0.029], and VCL after sperm separation lm/sec [OR ¼ 3.2, p ¼ 0.002] were the parameters of predictive value for pregnancy outcome. Adjustment of the model for female age, female infertility factors, and the methods of ovulation induction did not change this finding, and the final model still had the same covariates. Pregnancy rates per cycle according to the number (0, 1, 2 and 3) of variables satisfied with the three parameters were 0% (0/110), 1.6% (3/183), 9.7% (21/217) and 15.1% (23/151), respectively. Three semen parameters including normal morphology before sperm separation, rapid and VCL after sperm separation were identified as predictors of pregnancy by IUI. These variables would be helpful when counselling patients before they make the decision to proceed with in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI)-ET. Keywords: computer-aided sperm analysis, intrauterine insemination, male infertility, pregnancy, strict criteria Correspondence: Hiroaki Shibahara, Department of Obstetrics and Gynecology, Jichi Medical School, Yakushiji, Minamikawachimachi, Kawachi-gun, Tochigi , Japan. sibahara@jichi.ac.jp Ó 2004 Blackwell Publishing Ltd.

2 64 H. Shibahara et al. Introduction Although the more sophisticated modern assisted reproductive technologies (ARTs) including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have been developed, artificial intrauterine insemination (IUI) with husbandsõ semen has been playing an important role in the infertility treatment. Indications for IUI have included oligozoospermia, asthenozoospermia, pyospermia, hostile or absent cervical mucus, and immunological infertility. Because of the diversity of its indications, variable degrees of success in IUI depend on the type of male or female problem for which the procedure was used. Our group has been investigating sperm fertilizing ability using several sperm parameters and sperm function tests, including computer-aided sperm analysis (CASA) (Hirano et al., 2001), normal sperm morphology using the strict criteria (Obara et al., 2001), the zona-free hamster egg penetration test (Shibahara et al., 1998), and Sperm Quality Analyzer (SQA) (Shibahara et al., 1997, 1999). Moreover, we have utilized the hemizona assay (HZA) to demonstrate the inhibitory effects of antibodies against sperm (Shibahara et al., 1991, 1993, 1996) or zona-pellucida (Afzalpurkar et al., 1997) on fertilization. However, we have not yet investigated which sperm parameter has the predictive value of the success in IUI treatments. Marshburn et al. (1992) reported that some of the CASA estimates after thawing and washing of cryopreserved sperm could be the predictors of fertility outcome after IUI. A recent review by Van Waart et al. (2001) has shown that the predictive value of normal sperm morphology in IUI has still been controversial. These results may indicate that there is a limitation for predicting fertility outcome after IUI using a single sperm parameter. This study was performed to predict pregnancy after IUI using the CASA estimates and the strict criteria in patients with male factor infertility. Materials and methods Subjects A total of 160 couples with male factor at the hospital in Jichi Medical School were enrolled into this study and their characteristics are summarized in Table 1. Definition of patients with male factor infertility is based on the examinations of ejaculated semen carried out at least twice as assessed by the criteria of World Health Organization (1999). The diagnosis for 160 patients with male factor examined by CASA and strict criteria included 94 teratoasthenozoospermia, 46 asthenozoospermia, nine oligoastheno-teratozoospermia, nine oligozoospermia, and two oligo-asthenozoospermia. IUI was performed in 682 cycles in the study period between May 1995 and December Infertile women aged older than 40, those with sperm immobilizing antibodies in their sera, and those with congenital uterine abnormalities were excluded from this study. Two hundred and thirty-six cycles were treated in the Table 1. Characteristics of 160 patients with male factor treated by intrauterine insemination (IUI) Mean ± SD Range Age Husband (years) 34 ± Wife a (years) 31 ± Infertility period (years) 2.8 ± Infertility Primary (cases) 120 Secondary (cases) 40 No. of IUI performed (times) 4.3 ± PR/cycle (%) 7.2 (49/682) PR/patient (%) 28.1 (45/160) a Women P 40 years uterine anomaly and immunological infertility were excluded; PR, pregnancy rate. spontaneous ovulation cycles, while 446 cycles were treated with the use of ovarian stimulants including bromocriptine or clomiphene citrate or gonadotrophin. The mean age of the male and female subjects were 34 ± 5 years (range 24 52) and 31 ± 4 years (23 39), respectively. The average infertility period was 2.8 ± 2.0 years (range ). One hundred and twenty of 160 couples were diagnosed primary sterility. Semen examinations were carried out using CASA and strict criteria before and after sperm preparation as described below. Clinical pregnancy was diagnosed when the gestational sac was demonstrated under transvaginal ultrasonography. Semen analysis using CASA and strict criteria After liquefaction, semen quality analysis was performed using the CASA system (Hamilton Thorne Research, Beverly, MA, USA) for the 682 IUI treatment cycles. Briefly, a 5-lL aliquot of semen sample was placed on a Makler chamber. At least 200 sperm were counted with CASA to evaluate the sperm concentration, sperm motility, and sperm motion variables including rapid sperm movement (rapid), curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), linearity (LIN ¼ VSL/VCL), and straightness (STR ¼ VSL/VAP). The CASA settings were followed according to the manufacturer s instruction. The sperm morphology was assessed following the method by Kruger et al. (1986, 1988). Briefly, 5 ll semen were used to make the smears using two morphology slides cleaned thoroughly with 70% ethanol before use. The slides were air-dried at room temperature and fixed for 15 sec with Diff-Quik fixative (Diff-Quik, International Reagents Corporation, Kobe, Japan) prior to staining with Diff-Quik solutions. The slides were read and documented on the same day. Spermatozoa were considered as normal when the head had a smooth oval configuration with a well-defined

3 Prediction of pregnancy by IUI in male factor infertility 65 acrosome involving about 40 70% of the sperm head, as well as an absence of neck, midpiece, or tail defects. No cytoplasmic droplets of more than half the size of the sperm head were allowed to be present. The length of a normal sperm head was 5 6 lm and the diameter lm. At least 200 cells per slide were evaluated ROC curve Normal morphology Procedure of sperm preparation The selective concentration of progressively motile sperm for IUI with husbandsõ semen was carried out using a modified method of continuous-step density gradient centrifugation demonstrated by Kaneko et al. (1987). Briefly, the ejaculated semen was placed on 6.0 ml of 80% Percoll solution, and the density gradient was formed by mixing the semen and Percoll using an L-shaped rod for two or three strokes. After centrifugation at 600 g for 20 min, progressively motile sperm were concentrated in the sediment, whereas the immotile sperm and other types of cells remained in the upper part of the density gradient. The supernatant was removed and the pellet was resuspended in 0.5 ml of Hanks medium. Two 5-lL aliquots were used for CASA and to make the smear for the morphological assessment. The rest of the suspension was used for IUI. Statistical methods A univariate logistic regression model was used to test the above variables for their association with fertility outcome after IUI. Variables that were statistically significant associated with establishing pregnancy were retained for testing in a multivariate logistic regression model (Hosmer & Lemeshow, 1989). Receiver operating characteristics (ROC) curves (Hanley & McNeil, 1982; Zweig & Campbell, 1993) were constructed for assessment of the effectiveness of each individual parameter in predicting pregnancy by IUI. A clinically acceptable threshold was calculated when sensitivity plus specificity was maximum. In the multivariate analyses the results were adjusted for the possible confounding factors. These factors include female age, infertility factors in women, the method of ovulation induction, and bromocriptin administration. The statistical analyses were carried out using the Statistical Package for Social Science (SPSS) for personal computers, version 10.0 for Windows (SPSS Institute Inc., Chicago, IL, USA). A value of p < 0.05 was defined as representing a significant difference. Results Results of IUI treatment As shown in Table 1, the average numbers of cycle treated by IUI were 4.3 ± 2.4 (range 1 12). Forty-five of 160 infertile patients established pregnancy, giving a pregnancy rate per patient of 28.1%. Four patients conceived twice by IUI in the study period. The pregnancy rate per cycle was 7.2% (49/682). Sensitivity Prediction of fertility outcome after IUI ROC curves were constructed for assessment of the effectiveness of each individual parameter in predicting pregnancy by IUI (Fig. 1). A clinically acceptable threshold was calculated when sensitivity plus specificity were maximum. Using ROC curve, it was shown that normal sperm morphology assessed by the strict criteria before sperm preparation and five parameters after sperm preparation including % rapid, progressive motility, VAP, VCL, and VSL were able to predict pregnancy by IUI (Table 2). Correlation between sperm parameters and fertility outcome after IUI was examined using the logistic regression model. In a multivariate analysis, normal morphology before sperm separation 15.5% [odds ratio (OR) ¼ 2.2, p ¼ 0.02], % rapid after sperm separation 25.5% Table 2. Construction of the threshold to predict pregnancy after intrauterine insemination (IUI) using ROC curves Parameter Specificity % rapid (post) % progressive (post) VAP (post) VCL (post) VSL (post) 1.00 Figure 1. Construction of the ROC curves. The ROC curves were constructed for assessment of the effectiveness of each individual parameter in predicting pregnancy by intrauterine pregnancy (IUI). VAP, average path velocity; VCL, curvilinear velocity; VSL, straight line velocity. Threshold a Before preparation Normal morphology (%) 15.5 After preparation Rapid (%) 25.5 Progressive motility (%) 25.5 VAP (lm/sec) VCL (lm/sec) VSL (lm/sec) a A clinically acceptable threshold was calculated when sensitivity plus specificity was maximum.

4 66 H. Shibahara et al. Table 3. Odds ratio (OR) adjusted for possible confounding factors a in the multivariate analyses B p OR 95% Cl of OR Before preparation Normal morphology (P15.5%) After preparation % Rapid (P25.5%) VCL (P lm/sec) < a The model was adjusted for the following factors: female age, infertile factors in women, method of ovulation induction and bromocriptine administration; B, estimate; Cl, confidence interval. Percentage ** n = 217 n = 151 n = 183 n = [OR ¼ 3.9, p ¼ 0.029], and VCL after sperm separation lm/sec [OR ¼ 3.2, p ¼ 0.002] were the parameters of predictive value for pregnancy outcome (Table 3). Adjustment of the model for female age, infertility factors in women, methods of ovulation induction and bromocriptine administration did not change this finding, and the final model still had the same covariates. Pregnancy rates per cycle according to the number (0, 1, 2 and 3) of variables satisfied with the three parameters were 0% (0/110), 1.6% (3/183), 9.7% (21/217) and 15.1% (23/151), respectively (Fig. 2). There were significant differences of the pregnancy rate per cycle between 0 and 2 variables exceeding the threshold (p < 0.001), and between 0 and 3 variables exceeding the threshold (p < 0.001). There were also significant differences of the pregnancy rate per cycle between 1 and 2 variables exceeding the threshold (p < 0.001), and between 1 and 3 variables exceeding the threshold (p < 0.001). * **** Figure 2. Association between pregnancy rate and number of variables exceeding the threshold in the three independent predictive parameters. Pregnancy rates per cycle according to the number (0, 1, 2 and 3) of variables satisfied with the three parameters were 0% (0/110), 1.6% (3/183), 9.7% (21/217) and 15.1% (23/151), respectively, *, **, ***, ****p < *** Discussion For couples with male factor infertility, the least invasive procedure in ARTs is IUI. IUI should generally be used as a first-line treatment in male factor infertility after treatment of specific factors affecting male fertility but prior to proceeding with other more invasive ARTs. In such infertile patients, the success of IUI is dependent on semen quality. A critical review for IUI by Allen et al. (1985) demonstrated that in the results of 18 series from 1957 to 1984, a total of 714 patients were inseminated, with an overall conception rate per patient of 28% (range 0 62%). In the article, only traditional semen parameters including sperm concentration, sperm motility, sperm morphology, and zona-free hamaster egg penetration rate were analysed to predict pregnancies after IUI in infertile couples with male factor. Later, CASA systems that can identify and track human sperm have been developed to revolutionize the research of the movement of human sperm (Aitken et al., 1985; Jeulin et al., 1986; Katz et al., 1986). The CASA has the advantages of providing objective semen analysis data on sperm kinetics. Our group has been using CASA to predict sperm fertilizing ability in IVF (Hirano et al., 2001), comparing its functions with other devices such as SQA (Suzuki et al., 2002), and analyse the association with multinucleate formation in IVF (Shibahara et al., 2003). There is a study that utilized CASA to investigate the relationship between sperm characteristics from fresh and frozen donor semen with fertility outcome after IUI (Marshburn et al., 1992). They concluded that some of the sperm motion characteristics using CASA after thawing and washing of cryopreserved sperm are the predictors of fertility outcome after IUI. As for the association between sperm morphology and IUI, Van Waart et al. (2001) recently summarized the contents of 18 papers demonstrating the predictive value of normal sperm morphology in IUI. Of the nine publications that used the strict criteria for sperm morphology (Kruger et al., 1986, 1988), six stated a positive predictive value for sperm morphology (Irianni et al., 1993; Toner et al., 1995; Lindheim et al., 1996; Ombelet et al., 1996, 1997; Montanaro Gauci et al., 2001). However, other reports could not show the correlation between normal sperm morphology and the pregnancy outcome after IUI (Matorras et al., 1995; Karabinus & Gelety, 1997; Shulman et al., 1998). Thus, there seems to be a limitation for predicting fertility outcome after IUI using a single sperm parameter. In the present study, a retrospective, univariate, multivariate and ROC analysis of a large series of 682 consecutive IUI in a single centre were carried out. As shown in Table 2, it was shown that normal sperm morphology assessed by the strict criteria before sperm separation and five parameters after sperm separation including rapid, progressive motility, VAP, VCL, and VSL were able to predict pregnancy by IUI. In them, it was shown after an adjustment for possible confounding factors that normal morphology before sperm

5 Prediction of pregnancy by IUI in male factor infertility 67 separation 15.5% [OR ¼ 2.2, p ¼ 0.02], rapid after sperm separation 25.5% [OR ¼ 3.9, p ¼ 0.029], and VCL after sperm separation lm/sec [OR ¼ 3.2, p ¼ 0.002] were the parameters of predictive value for pregnancy outcome (Table 3). We have previously shown that VCL and rapid before and after sperm swim-up provide reliable estimation of the fertilization ability of human sperm (Hirano et al., 2001). It was suggested in the report that the total distance travelled by rapid sperm movement might be important in human sperm fertilizing abilities. The present study also supports the importance of these sperm motion variables for sperm movement to reach the site where sperm meet ovulated oocyte. As shown in Fig. 2, pregnancy rates per cycle according to the number (0, 1, 2 and 3) of variables satisfied with the three parameters were 0% (0/110), 1.6% (3/183), 9.7% (21/217) and 15.1% (23/151), respectively. 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