The relationship between total motile sperm count and the success of intrauterine insemination

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1 FERTILITY AND STERILITY Copyright e 1994 The American Fertility Society Vol. 62. No.1. July 1994 Printed on acid-free paper in U. S. A. The relationship between total motile sperm count and the success of intrauterine insemination Joel G. Brasch, M.D.* Richard Rawlins, Ph.D. Susan Tarchala, M.S. Ewa Radwanska, M.D., Ph.D. Section of Reproductive Endocrinology and Infertility, Department of Obstetrics Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois Objectives: To determine if the total motile sperm count is predictive of lui success and to apply life-table analysis to a large database of lui procedures. Design: Contingency table and life-table analyses were used to analyze a large group of couples (n = 546) undergoing 1,205 cycles of therapeutic lui with husband's sperm. Setting: Clinical infertility practice at a medical school referral center. Patients: Five hundred forty-six couples with varying etiologies of infertility. Results: There was a trend toward an increasing percentage of conception with increasing total motile sperm count. Significance was reached when the total motile sperm count used for lui exceeded 20 X 10 6 The cumulative pregnancy rate calculated from this data reached 52% after the sixth attempted cycle of lui. Fertil Steril 1994;62:150-4 Key Words: Sperm count, intrauterine insemination, male infertility, pregnancy Therapeutic lui using husband's sperm commonly is performed for male factor infertility as well as empirically to enhance the probability of conception (1-4). The success ofiui has been studied in relation to diagnostic categories and other intergroups with a wide range of varying results. Most reports from fertility centers using therapeutic lui have been composed of relatively small heterogeneous patient populations; observed pregnancy rates have ranged from 7% to 66% (5) but life-table analysis seldom has been applied to the evaluation of fecundity data. One recent report is based upon larger material, representing 208 patients (6). Some investigators have questioned the value of Received August 27, 1993; revised and accepted March 7, * Reprint requests: Joel G. Brasch, M.D., Rush-Presbyterian St. Luke's Medical Center, Department of Obstetrics and Gynecology, 1653 West Congress Parkway, 720 Pavilion, Chicago, Illinois (FAX: ). lui for couples with oligospermic males (7-10). Other reports have described the lowest total motile sperm count at which pregnancy occurred as ranging from 1 to 5 X 10 6 sperm (11-14). Total motile sperm count, as it affects lui outcome, has not been studied systematically. Two studies incidentally mention total motile sperm count (14, 15). DiMarzo et a1. (14) compared the total motile sperm count between pregnant and nonpregnant women, but the difference was not statistically significant (14). Ho et a1. (15) found a significant difference between postwash total motile sperm counts in pregnant and nonpregnant cycles, but their study had only 15 patients. Our study was undertaken to estimate the effect oftotal motile sperm count on the outcome of lui cycles in a large patient population. Study Design MATERIALS AND METHODS Therapeutic lui data obtained between 1989 and 1992 at the Rush-Presbyterian-St. Luke's Medical 150 Brasch et al. Total motile sperm count and lui

2 Center infertility program were analyzed retrospectively. The material represented 546 couples undergoing 1,205 therapeutic lui procedures for male factor infertility or to enhance cycle fecundity during treatment for other fertility problems. Pregnancy rates for five total motile sperm count intervals were calculated and compared statistically. Data ultimately were transformed into a life-table format and a curve representing overall cumulative probability of conception was calculated. Patient Evaluation All couples had pre-lui ovulation testing performed, including serum E 2, LH, and serum P measurements, transvaginal ultrasound (US) evaluation of follicular development, and semen analysis. All women in the study had at least unilateral tubal patency demonstrated by hysterosalpingogram and/or chromopertubation at laparoscopy. The following diagnostic categories were considered: endometriosis' cervical factor, uterine factor, tubal factor, ovulatory dysfunction, male factor, and unexplained infertility. Women with a suspected diagnosis of ovulatory dysfunction were treated with clomiphene citrate or menotropins in standard doses. Both unstimulated and induced ovulation cycles were included in the study. Semen Preparation The couples were requested to abstain from intercourse for 3 days before lui. Semen samples were produced by masturbation into sterile containers. After liquefaction, the samples were analyzed according to World Health Organization guidelines (16). Four methods of sperm preparation were used: continuous Percoll separation, discontinuous PercolI gradient, separation by glass-wool filtration, and a sperm wash. Continuous Percoll Separation The neat ejaculate was layered carefully over 1.0 ml of 90% Percoll (Sigma Chemical Company, St. Louis, MO) stock solution in a 15-mL conical tube and centrifuged at 200 X g for 20 minutes. After centrifugation, the Percoll was removed and the resultant pellet was resuspended gently in 1.0 ml of insemination medium Ham's F-10 (GIBCO, Staten Island, NY), supplemented with 3% human serum albumin, penicillin, and streptomycin. This solution was centrifuged at 200 X g for 10 minutes. The final pellet was resuspended in 0.4 ml insemina- tion medium. This method was used for semen samples with a high number of abnormal forms but where the count and motility were essentially normal. Discontinuous Percoll Gradient Separation A reduced-volume Percoll density gradient column was prepared using 90%,70%, and 50% PercolI solution plus insemination medium. The neat ejaculate was layered carefully on top of Percoll gradient and then centrifuged at 200 X g for 20 minutes. After centrifugation, the bottom Percolliayer and the sperm pellet were removed and placed into a clean test tube. The pellet was transferred into a clean test tube, mixed 1:1 (vol:vol) with insemination medium, and then centrifuged at 200 X g for 10 minutes. The supernatant was removed and the pellet was resuspended to 0.4 ml insemination medium. This technique was used in couples with a more severe male factor: sperm count < 20 X 10 6 / ml and motility < 40%. Glass Wool Filtration A glass wool filter was prepared by gently but firmly packing 30 mg of glass wool fiber (Manville Corporation, Denver, CO) into the barrel of a 3-mL syringe to a depth of 3 mm. The filter was placed into a 15-mL conical centrifuge tube and rinsed twice with insemination medium. Seminal plasma was removed from the neat ejaculate by adding an equal volume of insemination medium to the semen sample and centrifuging at 200 X g for 10 minutes. The pellet was resuspended carefully in 0.4 ml insemination medium. The washed sperm was placed over the glass wool column and allowed to flow by gravity into a clean test tube. This fast method was used for semen samples from normospermic males. Sperm Wash Seminal plasma was removed from the semen sample by mixing an equal volume of semen and insemination medium; the specimen was then centrifuged at 200 X g for 10 minutes. The supernatant was removed and the pellet was resuspended in 0.4 ml insemination medium. This traditional method also was used in normospermic cases. After processing, all samples were reanalyzed and total motile sperm count was recorded. Insemination Procedures A single lui procedure was performed during the periovulatory period as judged by serum LH, E 2, Vol. 62, No.1, July 1994 Brasch et al. Total motile sperm count and lui 151

3 Table 1 Characteristics of Pregnant and Nonpregnant Patients Undergoing lui Age* (y) Infertility* (y) Endometriosis (%) Cervical factor (%) Uterine factor (%) Tubal factor (%) Ovulation dysfunction (%) Male factor (%) Unexplained (%) * Values are means ± SD. t P < Pregnant 33.3 ± ± t Not pregnant 33.2 ± ± sonographic follicular measurements, urine LH kits, or a combination of these studies. Human chorionic gonadotropin typically was used in ovulation induction cycles; luis were timed approximately 24 to 36 hours after injection of 5,000 to 10,000 mlu 1M hcg. The O.4-mL sperm suspension was drawn into an insemination catheter (Cook Ob/Gyn, Indianapolis, IN). Using a vaginal speculum, the catheter was passed through the uterine cervix with or without the use of a tenaculum, until high fundal placement was assured. The sample was then deposited. The patient remained supine for approximately 20 minutes. Women were requested to return at midluteal phase for serum E2 and P determinations as well as ovarian US examinations. Luteal support typically was prescribed as injections of P in oil (50 mg/d), P vaginal suppositories (100 mg twice per day), or midluteal injections of hcg (2,500 to 5,000 miu). Serum,6-hCG determination was performed approximately 10 days after the midluteal visit if menses had not ensued. Statistical Analysis Using a relational database, multiple parameters were analyzed in couples achieving pregnancy and those not conceiving. x 2 analysis was used for comparison of single parameters between groups. The separate variance t-test was used for comparison of intergroup means. The two-sample z-test was used for comparison of population proportions. The relationship between total motile sperm count and pregnancies was analyzed by contingency table analysis. Life-table analysis was performed and the curve representing cumulative chance of conception was calculated. RESULTS One hundred twenty-one pregnancies occurred in 546 couples undergoing 1,205 cycles of lui. The overall pregnancy rate per cycle was 10.0% (121/ 1,205), and the couple pregnancy rate was 22.2% (121/546). The couples included in this study underwent an average of 2.2 cycles of lui; pregnant patients underwent an average of 2.0 cycles of lui. A comparison of clinical data between pregnant and nonpregnant patients is presented in Table 1. The only intergroup significant difference was in the prevalence of tubal factor in the nonpregnant versus pregnant women (22.6% and 5.9%, P < 0.05, respectively). Methods of semen preparation (PercoIl, glass wool, wash) were distributed evenly between pregnant and nonpregnant groups. Cycle distribution in relation to total motile sperm count is shown in Figure 1. In almost 30% of all cycles, lui was performed with total motile sperm count < 20 X 10 6 sperm. In approximately 30% of cycles, lui was performed with total motile sperm count> 60 X 10 6 sperm. The majority (54%) of luis were performed with total motile sperm count of ~40 X 10 6 The majority of patients (68%) underwent fewer than three cycles of therapeutic lui. Only 1.8% of patients underwent more than five cycles. Total motile sperm count was evaluated between pregnant and nonpregnant patient groups. The mean ± SD total motile sperm count was 47.7 ± X 10 6 (range 0.8 to X 10 6 ) for pregnant patients and 57.7 ± 68.0 X 10 6 (range 0.2 to 558 X 10 6 ) for nonpregnant patients. This difference was not statistically significant. The threshold total motile sperm count for conception appeared to be approximately 3 X 10 6 A single conception occurred with a total motile sperm count of 800,000; six conceptions occurred with total motile sperm counts between 3 and 5 X 10 6 Analysis of total motile sperm count in relation to cycle pregnancy rate is demonstrated in Figure 2. Figure 1 N.M!ER OF CYa.ES TOTAL MOTLE SPERM CaJNT (Xl08) Distribution of lui cycles by total motile count. 152 Brasch et al. Total motile sperm count and lui

4 There was a trend toward an increasing percentage of conceptions with increasing total motile sperm count. Significance was reached (P < 0.05) when the total motile sperm count used for lui exceeded 20 X 10 6 The data were transformed onto a life-table. Those known not to be pregnant or those lost to follow-up during the study period were censored. The cumulative pregnancy rate calculated from this data reached 52% after the sixth attempted cycle (Fig. 3). DISCUSSION Intrauterine insemination performed for varying indications, with varying techniques and with a wide range of reported pregnancy results. Francavilla et al. (4) studied 86 couples and used canonical discriminate analysis with stepwise variable selection; they determined that total motile sperm count was not predictive of pregnancy rate if normal sperm morphology was >50%. Our study provides evidence that within a sufficiently large group of couples, even with heterogeneous infertility factors, total motile sperm count is largely predictive of lui success. In particular, a postwash total motile sperm count> 20 X 10 6 is associated with significantly improved conception rate over total motile sperm count < 20 X 10 6 (Fig. 2). From the cycle distribution data (Fig. 1), it is evident that almost 30% of lui cycles demonstrated total motile sperm count < 20 X 10 6 Because the diagnosis of male factor (semen analysis with <20 X 10 6 sperm/ml) was prevalent equally in pregnant and nonpregnant patient groups (Table 1), the variable "male factor" should not be considered a confounding variable. Furthermore, as also reported by others (5, 15, 17), most patients in this study (68%) did not undergo more than two cycles of lui. PERCENTAGE OF PREGNANCIES PERCENT PREGNANT ~ m o lui CYCLES Figure 3 Cumulative pregnancy rate after lui calculated by life-table analysis. Timing of lui usually has been reported between 24 and 36 hours after hcg injection (2, 5, 15), consistent with the luis performed in this study. Cycle pregnancy rate (10%) and cumulative pregnancy rate (52% after six cycles) are similar to other published reports (11, 14). Not surprisingly, the only intergroup difference detected in our study was in the existence of tubal factor, which was more prevalent in the nonpregnant than in the pregnant group (22.6% versus 5.9%). In epidemiologic terms, tubal factor is a confounding variable. Association of the total motile sperm count with pregnancy rates might be affected somewhat by this confounding variable. However, with data sufficiently large, variables other than total motile sperm count appear to be obscured statistically. The cumulative pregnancy curve presented in Figure 3 is similar to other recently published estimates (11, 14). DiMarzo et al. (14) calculated 21.0%,32.7%, and 60.7% pregnancy rates after six cycles for unstimulated, clomiphene citrate-, and menotropin-treated patients, respectively. Using the data of DiMarzo et al. (14) and weighing them according to the reported group sizes, a sixth cycle pregnancy rate of 34.7% for a heterogenous patient population could be calculated. Because our patient data were composed of stimulated and unstimu- 1ated cycles, this extrapolated six-cycle pregnancy rate could be comparable to that of our patient pop':' ulation. The main conclusion of this study is that a final total motile count in sperm samples used for lui may be considered predictive of the potential for pregnancy. It appears that studies investigating other parameters with regard to lui success should control for total motile sperm count. lutal MOTILE SPERM COUNT (MILLION) Figure 2 Distribution of pregnancy rates after lui by total motile count. REFERENCES 1. Allen NC, Herbert CM III, Maxson WS, Rogers BJ, Diamond MP, Wentz AC. Intrauterine insemination: a critical review. Fertil SteriI1985;44: Vol. 62, NO.1, July 1994 Brasch et al. Total motile sperm count and lui 153

5 2. Bachus KE, Walmer DK. Superovulation and washed intrauterine insemination. Contemp Ob Gyn 1992; KerinJFP, PeekJ, Warnes GM, Kirby C, Jeffrey R, Matthews CD, Cox LW. Improved conception rate after intrauterine insemination of washed spermatozoa from men with poor quality semen. Lancet 1984;310: Francavilla F, Romano R, Santucci R, Poccia G. Effect of sperm morphology and motile sperm count on outcome of intrauterine insemination in oligozoospermia and/or asthenozoospermia. Fertil Steril 1990;53: Hewitt J, Cohen J, Krishnaswamy V, Fehilly CB, Steptoe PC, Walters DE. Treatment of idiopathic infertility, cervical mucus hostility, and male infertility: artificial insemination with husband's semen or in vitro fertilization? Fertil Steril 1985;44: Stovall DW, Christman GM, Hammond MG, Talbert LM. Abnormal findings on hysterosalpingography: effects on fecundity in a donor insemination program using frozen semen. Obstet Gynecol 1992;80: Dodson WC, Haney AF. Controlled ovarian hyperstimulation and intrauterine insemination for treatment of infertility. Fertil Steril 1991;55: Velde ER, van Kooy RJ, Waterreus JJH. Intrauterine insemination of washed husband's spermatozoa: a controlled study. Fertil Steril 1989;51: Kirby CA, Flaherty SP, Godfrey BM, Warnes GM, Matthews CD. A prospective trial of intrauterine insemination of motile spermatozoa versus timed intercourse. Fertil Steril 1991;56: Ho P-C, Poon IML, Chan SYW, Wang C. Intrauterine in- semination is not useful in oligoasthenospermia. Fertil Steril 1989;51: Berger T, Marrs RP, Moyer DL. Comparison of techniques for selection of motile spermatozoa. Fertil Steril 1985; 43: Cruz RI, Kemmann E, Brandeis VT, Becker KA, Beck, M, Beardsley M, et al. A prospective study of intrauterine insemination of processed sperm from men with oligoasthenospermia in superovulated women. Fertil Steril 1986;46: Confino E, Friberg J, Dudkiewicz AB, Gleicher N. Intrauterine inseminations with washed human spermatozoa. Fertil Steril 1986;46: DiMarzo SJ, Kennedy JF, Young PE, Herbert SA, Rosenberg DC, Villanueva B. Effect of controlled ovarian hyperstimulation on pregnancy rates intrauterine insemination. Am J Obstet GynecoI1992;166: Ho P-C, So W-K, Chan Y-F, Yeung WS-B. Intrauterine insemination after ovarian stimulation as a treatment for subfertility because of subnormal semen: a prospective randomized controlled trial. Fertil Steril 1992;58: World Health Organization. WHO laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, Martinez AR, Bernardus RE, Voorhorst FJ, Vermeiden JPW, Schoemaker J. Intrauterine insemination does and clomiphene citrate does not improve fecundity in couples with infertility due to male or idiopathic factors: a prospective, randomized, controlled study. Fertil Steril 1990; 53: Brasch et al. Total motile sperm count and lui