Evaluation of polyacrylamide gel as substitute for human cervical mucus in the sperm penetration test*

Transcription

1 FERTILITY AND STERILITY Vol. 60, No.3, September 1993 Copyright 1993 The American Fertility Society Printed on acid-free paper in U. S. A. Evaluation of polyacrylamide gel as substitute for human cervical mucus in the sperm penetration test* Waltraud Eggert-Kruse, M.D.t:j: Beate Schwalbach, M.D.t Gerhard Rohr, M.D., Ph.D. Klaus Klinga, Ph.D.t Wolfgang Tilgen, M.D. II Benno Runnebaum, M.D.t Women's Hospital, University of Heidelberg, Heidelberg, Germany Objective: To compare polyacrylamide gel as synthetic medium with human cervical mucus (CM) for the in vitro sperm-penetration test during infertility investigation. Patients: One hundred sixty-nine randomly chosen couples with a median duration of infertility of 4 (range, 1 to 16) years presenting at the infertility unit of the Women's University Hospital of Heidelberg, Germany. Main Outcome Measures: Evaluation of sperm migration in polyacrylamide gel used in four different concentrations (1.5%, 1.6%, 1.7%, 1.8%) in the capillary tube test in parallel with CM of patients' female partners and CM of fertile donors, obtained under standardized conditions. Correlation of migration test results with outcome of semen analysis including microbial cultures and testing for local antisperm antibodies by means of the mixed antiglobulin reaction, postcoital testing, and the subsequent pregnancy rate after control for female infertility factors in a prospective study. Results: Sperm ability to penetrate the synthetic medium (concerning all concentrations) correlated significantly with the penetration of human CM, although polyacrylamide proved to be a stronger barrier. Sperm velocity and duration of progressive motility were markedly reduced in polyacrylamide. Polyacrylamide results correlated with the outcome of standard sperm analyses but not with sperm antibody testing. No clear differentiation was obtained with regard to subsequent fertility (19% after 6 months), although adequate sperm migration in polyacrylamide 1.8% was significantly more frequent in the fertile group. Conclusions: In analyzing the intrinsic motility, penetration testing with polyacrylamide gel provides important information not obtained by routine sperm analysis. However, particularly with regard to immunological factors and fertility prognosis, human CM should be preferred whenever possible. Fertil Steril 1993;60:540-9 Key Words: Male fertility, sperm-mucus interaction, polyacrylamide gel, sperm function tests Sperm function tests are required during infertility investigation as standard parameters of semen Received December 1, 1992; revised and accepted June 1, * Presented in part at the 8th Annual Meeting of the European Society of Human Reproduction and Embryology (ESHRE), The Hague, The Netherlands, July 5 to 8, t Department of Gynecological Endocrinology and Reproductive Medicine, Women's Hospital. Department of Internal Medicine IV, Klinikum Mannheim. II Division of Andrology, Department of Dermatology. * Reprint requests: Waltraud Eggert-Kruse, M.D., Department of Gynecological Endocrinology and Reproductive Medicine, Women's Hospital, University of Heidelberg, Vo13stral3e 9, 6900 Heidelberg, Germany. analysis are usually insufficient to adequately characterize male fertility potential (1). Sperm migration testing offers valuable information about the functional capacity of spermatozoa (2, 3). The standardized in vitro sperm-cervical mucus (CM) penetration test (SCMPT) has been shown to be of prognostic value for subsequent fertility when CM of patients' female partners was used (4). However, in andrologic practice, fresh CM for the SCMPT is not always available. Furthermore, CM has to be taken under standardized conditions, and the quality of the mucus must be carefully controlled to obtain reproducible results and to evaluate the male factor as the main variable (5). 540 Eggert-Kruse et al. Polyacrylamide as em Substitute Fertility and Sterility

2 Lorton et al. (6) proposed the use of polyacrylamide gel as a substitute for CM in human sperm migration studies to overcome problems associated with the variability of CM. A particular advantage of this medium is that it can be produced in large quantities and equal quality and can be stored without problems. Therefore this synthetic material was used for the in vitro sperm-penetration test (in parallel in 4 different concentrations) and compared with human CM in a large subfertile population. Results were correlated with many other parameters of male fertility: sperm analysis, determination of antisperm antibodies, outcome of microbial cultures of both partners, evaluation of sperm-mucus interaction in vivo by means of the postcoital test (PCT), and, after control for female infertility factors, the subsequent pregnancy rate (PR) in a prospective study. Patients MATERIALS AND METHODS The study population consisted of 169 couples who were randomly chosen from those presenting at the outpatient infertility clinic of the University of Heidelberg. The median duration of infertility was 4 (range, 1 to 16) years. The median age of the male patients was 33.5 (range, 24 to 53) years and of their female partners 29 (range, 21 to 42) years. Primary infertility was found in 68% and secondary infertility in 32%. All patients were asymptomatic in terms of genital tract infection. During infertility investigation before sperm penetration testing, all couples were submitted to a comprehensive evaluation of male and female fertility. A detailed medical history was obtained and physical examinations performed in both partners. Female patients were checked for tubal patency by hysterosalpingography and/or laparoscopy. Follicular growth and hormonal disorders were carefully examined by BBT, ultrasound, multiple determinations of gonadotropins, PRL, E 2, P, T, and thyroid function tests and were treated accordingly. Evaluation of Semen Quality In males sperm analyses were performed according to World Health Organization (WHO) criteria (7). Semen was obtained in the hospital after 5 days of sexual abstinence and was examined directly after liquefaction. Sperm volume, ph, number of spermatozoa, progressive motility after liquefac- tion, after 2 hours and after 4 hours, viability (eosine test), morphology (percent normal forms), and the number of round cells (undifferentiated/highpower field [HPF], magnification X400) were analyzed. Additionally, a screening for local antisperm antibodies of the immunoglobulin (Ig)G and/or IgA class was performed simultaneously by means of the mixed antiglobulin reaction (MAR), described in detail elsewhere (8). Briefly, IgG or IgA coated erythrocytes were used as indicator particles to detect surface bound sperm antibodies. Antibody testing was considered positive when >30% of motile spermatozoa were involved in the mixed agglutinates. A microbial screening was performed in all ejacu- 1ates directly after liquefaction. For culture of potentially pathogenic bacteria and species of the physiological flora aliquots were inoculated into a universal transport medium (Port-a-Cul-Universal; Becton Dickinson, Heidelberg, Germany) and identified using standard methods as described previously (Department of Microbiology and Hygiene, University of Heidelberg) (9). Sperm Penetration Testing As synthetic migration medium, polyacrylamide, cross-linked with 0.042% N, N -methylene bis acrylamide was produced as described in detail by Lorton et al. (6) and dialyzed for 24 hours at 4 C against four changes of phosphate-buffered salineglucose solution. Polyacrylamide gel was prepared in four different concentrations (1.5%, 1.6%, 1.7%, 1.8%) that were tested for spinnbarkeit, ferning, and clarity before use. The synthetic medium was slowly aspirated into capillary tubes, and care was taken to prevent air bubbles from disrupting the gel column. Modeling clay was forced into the upper end of the capillary to protrude the gel slightly and thus provide a satisfactory gel-sperm suspension interface. Tubes were then placed onto the migration apparatus (2) with the lower end penetrating into the semen reservoirs, filled with aliquots of freshly obtained semen used directly after liquefaction. The penetration meter was placed in a covered glass dish containing wet cotton to minimize drying of the medium and incubated at 37 C for a period of 6 hours. A light microscope (low-power field [LPF], magnification XI00) was used for evaluation of penetration parameters after 30 minutes, 2 hours, and 6 hours of incubation time. The migration distance Vol. 60, No.3, September 1993 Eggert-Kruse et al. Polyacrylamide as em Substitute 541

3 (penetration depth of the most vanguard spermatozoon) (maximum possible 60 mm), the sperm density (number of spermatozoa [per LPF] reaching the 1-, 2-, 3-, 4-, 5-cm mark in the capillary) (maximum;:::: 300), and the quality of motility (from immotile to highly progressive motility) (motility grades 0 to 3) were recorded at the three time intervals. Because of absolute differences in both the vanguard sperm migration achieved in polyacrylamide and the number of spermatozoa per unit area, and the motility grade, the scoring system used for human CM (4) was not useful for selecting polyacrylamide results. Therefore, for further analyses, samples offering motility of any grade (total motility) in the synthetic medium were combined and the penetration parameters were graded with cutoffs at;::::10 mm for penetration distance and at ;::::50 spermatozoa/lpf for sperm density. Results were summarized and used for selection of groups with good and poor penetration of polyacrylamide (polyacrylamide positive/negative). Samples were regarded as polyacrylamide positive when after 6 hours' incubation time, motile spermatozoa were found at 1 cm in the capillary tube and/or ;::::50 spermatozoa offered a penetration distance of ;::::10 mm. Sperm Penetration Testing With em Results of polyacrylamide testing were correlated with sperm migration in fresh CM of patients' female partners. Cervical mucus was obtained from the endocervix under standardized conditions with regard to site of collection (endocervix), length of sexual abstinence (5 days), time of collection (morning, with a maximum of 2 hours before sperm penetration testing), phase of the menstrual cycle (between days 9 to 14), and after oral treatment with estrogens (80 /lg of ethinyl estradiol [EE 2 ]) for 7 days before mucus collection to control the hormonal influence on the CM properties (4). The cervical index was determined according to Insler et al. (10). The ph was measured with paper strips (ph Indikatorpapier; Merck, Darmstadt, Germany). Sperm migration in CM was evaluated on the sperm penetration meter (2) with regard to penetration distance, sperm density, and quality and duration of motility as described for polyacrylamide. Results in CM were summarized in a cumulative SCMPT score and classified according to previously described criteria as adequate and inadequate (4). With regard to microbial colonization of the cer- 542 Eggert-Kruse et ai. Polyacrylamide as em Substitute vix, a bacterial screening was performed for a broad spectrum of microorganisms, including mycoplasmas, Chlamydia trachomatis, herpes simplex virus, yeasts, and trichomonads (described in detail elsewhere) (9), and a gram-stained cervical smear was prepared for determination of leucocytes. In addition to SCMPT with CM of patients' wives, CM of fertile donors, obtained under the same conditions and stored at 4 C (with a maximum of 14 days) was used for sperm-mucus interaction testing. Capillary tubes with polyacrylamide gel of four different concentrations and both types of human CM were tested in parallel with aliquots of the same semen samples. All readings were done by a single observer without knowledge of the other variables of sperm quality. Evaluation of Sperm-Mucus Interaction in Vivo Sperm ability to penetrate CM in vivo was evaluated by means of the PCT performed as described previously (5). Briefly, the number of motile spermatozoa in periovulatory CM of good quality was counted 8 to 12 hours after intercourse and after sexual abstinence of 5 days. Postcoital testing was classified as negative (no spermatozoa found in CM, but in vaginal secretions), inadequate «2 motile sperm/lpf), moderate (2 to 6 motile sperm/ HPF), and excellent (>7 motile sperm/hpf). If necessary, because of poor mucus quality on the day ofpct, late or irregular ovulation, or marked luteal insufficiency, PCT was evaluated after standardized treatment with EE2 (5). Statistical Analysis Pregnancy rate was determined 6 months after sperm migration testing in vitro. Data were processed using the Statistical Analysis System (SAS Institute, Heidelberg, Germany). For statistical analysis, Spearman rank correlation, Wilcoxon's rank sum test, X 2 analysis, and Fisher's two-tailed exact tests were used (Institute of Medical Biometry and Statistics, University of Heidelberg). The level of significance was set at P < RESULTS Outcome of Migration Testing With Polyacrylamide Gel Semen samples of a total of 169 patients under infertility investigation were evaluated for their Fertility and Sterility

4 Table 1 Parameters of Sperm Penetration in Capillaries Filled With Polyacrylamide Gel of Different Concentrations Penetration distance (mm)t Penetration density (n/lpf:i:)t Motility grade Polyacrylamide preparation Penetration parameters* 1.5% 1.6% 1.7% 1.8% 10 (0 to 60) 10 (0 to 60) 10 (0 to 60) 5 (0 to 60) 20 (0 to 300) 13 (0 to 300) 10 (0 to 300) 5 (0 to 300) Immotile 85 (50) 95 (56) 113 (67) 142 (84) In situ motility or slow forward progression 26 (15) 26 (15) 28 (17) 16 (10) Highly progressive motility (of >50% of spermatozoa) 58 (35) 48 (28) 28 (16) 11 (6) * Results after an incubation period of 6 hours. t Values are medians with ranges in parentheses. :I: Number of sperm per LPF (magnification Xl00) at the l-cm mark in the capillary tube. Values are numbers of samples with percentage in parentheses. ability to penetrate polyacrylamide gel in comparison with human CM (4 capillary tubes were filled with polyacrylamide of different concentrations and 2 tubes filled with different types of CM). All tests were run in parallel with aliquots of the same semen sample (total 1,014 migration tests). The properties of the synthetic medium were similar to CM with regard to clarity, spinnbarkeit, and ferning, in particular for polyacrylamide gel 1.7%, whereas polyacrylamide gel 1.8% was more viscous and difficult to aspirate. Sperm penetration decreased with the concentration of the synthetic medium (see Table I) and increased with time of incubation. The median penetration distance and the median sperm density were lower in polyacrylamide 1.8% than in polyacrylamide 1.5% to 1.7%. Sperm progressive motility (motility grade 3) after 6 hours' incubation time was found in 35% of samples in polyacrylamide 1.5% to 6% in polyacrylamide 1.8%. A sperm density of ;;:::50 sperm/lpf at the 1 cm mark in the capillary were seen in 36% (polyacrylamide 1.5%) to 21% (polyacrylamide 1.8%) of samples, respectively. With regard to the global migration score, summarizing the different parameters of penetration, results were positive in 60% in the 1.5%, 59% in the 1.6%, 52% in the 1.7%, and 39% in the 1.8% preparation of polyacrylamide. When the four different concentrations of polyacrylamide used to fill the capillaries were compared for sperm ability to penetrate the synthetic medium, significant correlations were found for all comparisons (P < ) (Spearman rank correlation). With regard to penetration distance, the correlation coefficient r ranged from 0.35 to 0.66 and for sperm density from 0.49 to 0.70 (results after 6 hours' incubation). Significant correlations were also found for the penetration parameters in the different polyacrylamide gel preparations at other time intervals. Comparison of Sperm Penetration in Human CM and Polyacrylamide Gel The cervical index according to Insler et al. (10) of the 169 mucus samples of patients' female partners showed a median of 11 (range, 5 to 12); the median ph was 7. Penetration distance in CM of patients' partners and donors' CM showed a median of 60 mm after 30 minutes, progressive motility (grade 3) in partners' CM was found in 84% after 30 minutes and 40% of samples after 6 hours in the sperm migration test. A sperm density of ;;:::50 spermatozoa/lpf (after 6 hours) was found in 54 % of samples in partners' CM and 62% of samples in donors' CM. Both types of human CM correlated significantly with regard to penetrability by spermatozoa (P < ) (Spearman rank correlation), with r = 0.64 for migration distance and 0.84 for sperm density. Therefore the results obtained in CM of patients' female partners were used for further analyses. In polyacrylamide, sperm velocity was markedly reduced compared with human CM, and spermatozoa of only two patients penetrated the synthetic medium (1.5%) more easily. In 14 samples the penetration rate was equal; spermatozoa of the other 153 specimens penetrated human CM faster than polyacrylamide. Although the synthetic medium (depending on concentration) proved to be a stronger barrier, sperm ability to penetrate polyacrylamide gel correlated significantly with the penetration of CM (P < , Spearman rank correlation). Correlation coefficients ranged from 0.43 (P < ) Vol. 60, No.3, September 1993 Eggert-Kruse et ai. Polyacrylamide as em Substitute 543

5 concerning polyacrylamide 1.6% to 0.24 (P < 0.001) (polyacrylamide 1.8%) with regard to penetration distance and were higher for sperm density with r values ranging from 0.61 (1.6%) to 0.39 (1.8%) (P < ). Sperm density correlated significantly not only at the 1-cm mark in the capillary tubes filled with polyacrylamide and human CM but also with respect to the visual field showing the maximum density in both media (with r values ranging from 0.60 to 0.41) after 6 hours' incubation time (P < ). The percentage of samples with motility (of any grade) was considerably lower in the synthetic medium than in CM. Although 60% of semen samples showed motility 6 hours after penetration of human CM (89% after an incubation period of 30 minutes), less than half ofthese also remained motile in polyacrylamide: 45% in the 1.5%, 36% in the 1.6%, 27% in the 1.7%, and only 19% in the 1.8% preparation. The relationship for sperm motility in the synthetic medium and CM was significant between polyacrylamide 1.6% and 1.7% and human CM after 6 hours (P < 0.05, x 2 analysis) and with regard to polyacrylamide 1.5% after 2 hours (P < 0.01). In a further step, statistical comparison was made after selection of samples with adequate (71 %) and inadequate (29%) outcome of SCMPT performed with human CM, based on the cumulative penetration score (4) after 6 hours. Although the median penetration distance and sperm density in polyacrylamide were higher in samples exhibiting an adequate outcome of SCMPT with hormonally standardized mucus of patients' wives, differences of sperm migration in polyacrylamide (concerning all concentrations) in both groups did not reach statistical significance (Wilcoxon test). Correlation of Polyacrylamide Migration Testing With Sperm Analysis Standard sperm analysis offered a wide range of findings. Sperm count ranged from 1 to 212 X 10 6 / ml, with a median of 45 X 10 6 /ml; progressive motility directly after liquefaction ranged from 1 % to 80% (median 45%), from 1% to 80% (median 40%) after 2 hours, and from 0% to 70% (median 40%) after 4 hours. The percentage of normal forms according to WHO criteria (7) varied from 10% to 85% (median 61%) and the viability from 10% to 80% (median 65%). Results correlated significantly with sperm ability to penetrate the synthetic medium as well as CM, with r values decreasing with higher concentration of polyacrylamide (P < , Spearman rank correlation) (see Table 2). No significant 544 Eggert-Kruse et al. Polyacrylamide as em Substitute Table 2 Correlation of Standard Parameters of Sperm Analysis and Sperm Ability to Penetrate Polyacrylamide Gel* Polyacrylamide preparation Semen parameters 1.5% 1.6% 1.7% Correlation with regard to penetration distancet:j: Sperm count (X10 6 /ml) Progressive motility (%) Morphology (% normal formslll Viability (%) Correlation with regard to sperm densityt:j: Sperm count (X10 6 /ml) Progressive motility (%) Morphology (% normal forms III Viability (%) * Results after 6 hours' incubation time. t Spearman rank correlation, correlation coefficient r shown. :j: P < for all r values, except for two. P < II Morphology according to WHO criteria (7). 1.8% correlations were found with regard to sperm volume, ph, fructose concentration, and number of round cells. After selection of patients in those with reduced sperm counts «40 X 10 6 /ml) and/or asthenozoospermia (progressive motility of <40%) and those with normal findings, significant differences were found again for sperm ability to penetrate the synthetic medium (all concentrations) (P < 0.001, Wilcoxon test). Significant differences were also found between both groups with regard to motility in the synthetic medium (P < 0.001, x 2 analysis). However, as the median penetration distance as well as the sperm density was 0 in samples with asthenozoospermia or oligozoospermia, the use of polyacrylamide testing in these patients was limited. Significant correlations were also found between standard sperm parameters and migration testing in human CM with lower r values, e.g., 0.37 with regard to sperm count. However, in samples with reduced sperm count and in asthenozoospermic patients median penetration distance (51 and 55 mm, respectively) as well as sperm density were much higher than in polyacrylamide. Furthermore, there was no significant relationship of sperm motility in human CM in the migration test and sperm count and motility determined in the seminal fluid analysis, as found in the synthetic medium. Relationship of Polyacrylamide Testing With Local Antisperm Antibodies For evaluation of local antisperm antibodies, the MAR was performed with Ig-coated erythrocytes allowing differentiation of antisperm antibodies of Fertility and Sterility

6 Table 3 No Influence of Local Antisperm Antibodies on Sperm Ability to Penetrate Polyacrylamide Gel Outcome of Outcome of antisperm migration testing* antibody testing Probabilityt IgG antibodies:!: Negative Positive 1I Positive results of polyacrylamide testing* 1.5% 41 (57) 4 (36) NSlI 1.6% 45 (62) 7 (64) NS 1.7% 40 (56) 7 (64) NS 1.8% 18 (25) 3 (27) NS No. of samples 72 (87) 11 (13) IgA antibodies:!: Negative Positive II Positive results of polyacrylamide testing* 1.5% 43 (54) 2 (50) NS 1.6% 50 (63) 2 (50) NS 1.7% 45 (57) 2 (50) NS 1.8% 21 (27) 0(0) NS No. of samples 79 (95) 4 (5) * Determined with the cumulative penetration score in the synthetic medium after 6 hours. t Fisher's two-tailed exact test. :!: Evaluated with the MAR test. Values are numbers of patients per group with percentages in parentheses. II MAR %;:;,: 30%. 11 NS, not significant. the IgG and/or IgA class. In 83 of169 samples MAR testing was performed simultaneously with sperm migration testing (49%). These were taken for further analysis: 13% of patients were positive for MAR IgG and 5% for IgA class antisperm antibodies. Statistical analysis did not reveal significant differences for the percentage of polyacrylamide positive results in samples with or without local antisperm antibodies as shown in Table 3. However, none of the samples with local IgA antibodies was polyacrylamide 1.8% positive. When instead of the summarized outcome of the polyacrylamide test using the cumulative score, the different migration parameters were analyzed, no significant difference was found with regard to penetration distance and sperm density in polyacrylamide (Wilcoxon test), as well as for sperm motility in the synthetic medium (all concentrations) at the different time intervals (Fisher's 2-tailed exact test) when MAR positive and negative ejaculates were compared. Relationship of Microbial Colonization and Sperm Migration Testing Microbial colonization of semen samples did not interfere with the outcome of polyacrylamide penetration testing with regard to potentially pathogenic aerobic (41 %) or anaerobic bacteria as well as species of the physiological flora. Semen cultures offered commensal bacteria in the majority of semen samples; most frequently isolated was Staphylococcus epididermidis in 25%. Only 8% of semen specimens were sterile in the different microbial cultures. Positive bacterial cultures concerning different species as well as groups of microorganisms were found in equal frequency in samples with positive or negative polyacrylamide test outcome. With regard to sperm-mucus interaction testing using CM of patients' female partners, the microbial colonization of the cervix did not interfere with the quality of the CM, determined with the cervical index, and the results of the SCMPT. Most frequently isolated were lactobacilli in the majority of women, potentially pathogenic aerobes were found in 31%, mycoplasmas in 11%, yeasts in 10%, and herpes simplex virus in 5%. Chlamydia trachomatis and Trichomonas vaginalis were found in none of the women. Correlation of Polyacrylamide Gel Migration With Outcome of PCT When the relationship of sperm migration in the synthetic medium and sperm-mucus interaction in vivo evaluated by means of the PCT (n = 160) was analyzed, significantly more samples of patients with moderate or excellent results of PCT were polyacrylamide positive (see Table 4). However, a considerable percentage of samples of patients with negative or inadequate outcome of PCT also offered positive results of the polyacrylamide migration test, e.g., 43 % of semen samples with negative PCT results (polyacrylamide 1.5%), and 26% in polyacrylamide 1.8%, respectively. Relationship With Subsequent Fertility Pregnancy rate 6 months after sperm migration testing was 19% (32/169). No clear differentiation with respect to subsequent fertility was obtained when the synthetic material was used as penetration medium for the capillary tube test. Positive polyacrylamide results were found in nearly the same percentage in the later fertile and not fertile group of patients (Table 5). Vol. 60, No.3, September 1993 Eggert-Kruse et al. Polyacrylamide as em Substitute 545

7 Table 4 Relationship of Sperm-CM Interaction Testing in Vivo and Sperm Migration in Polyacrylamide Gel Outcome of PCT Outcome of migration testing Negativet Inadequatet Moderatet Excellentt Probability+ Positive* polyacrylamide results in 1.5% 15 (43) 1.6% 14 (40) 1.7% 12 (34) 1.8% 9 (26) No. of patients 35 (22) * Determined with the cumulative penetration score in the synthetic medium after 6 hours. t Values are numbers of patients per group with percentages in parentheses. 23 (52) 34 (67) 23 (77) < (52) 33 (65) 23 (77) < (41) 28 (55) 24 (80) < (27) 24 (47) 18 (60) < (27) 51 (32) 30 (19) + X 2 analysis, compared with polyacrylamide gel negative groups. In the total population, positive results in polyacrylamide of the highest concentration (1.8%) used in this study, were more frequent in samples of patients who later achieved a pregnancy (56% versus 37%). This difference was statistically significant (P < 0.02, x 2 analysis). However, it has to be taken into consideration that 34% of semen speci- Table 5 Outcome of Polyacrylamide Migration Testing and Couples' Subsequent Fertility* Outcome of Not migration testing fertile*t Fertile*t Probability:j: All couples Positive polyacrylamide results (after 2 hours) in 1.5% 1.6% 1.7% 1.8% Positive polyacrylamide results (after 6 hours) in 1.5% 1.6% 1.7% 1.8% No. of patients Couples without female infertility factors Positive polyacrylamide results (after 2 hours) in 1.5% 1.6% 1.7% 1.8% Positive polyacrylamide results (after 6 hours) in 1.5% 1.6% 1.7% 1.8% No. of patients 48 (35) 54 (39) 56 (41) 34 (25) 84 (61) 78 (57) 71 (52) 47 (34) 137 (81) 23 (32) 30 (42) 27 (38) 17 (24) 47 (66) 41 (58) 39 (55) 26 (37) 71 (75) 14 (44) NS:j: 14 (44) NS 14 (44) NS 15 (47) < (53) NS 21 (66) NS 16 (50) NS 18 (56) < (19) 11 (46) NS 10 (42) NS 9 (38) NS 11 (46) < (54) NS 14 (58) NS 11 (46) NS 14 (58) NS 24 (25) * Pregnancy rate determined 6 months after migration testing. t Values are numbers of patients per group with percentages in parentheses. :j: NS, not significant; X 2 analysis. Determined with the cumulative penetration score in the synthetic medium. mens in the group without subsequent fertility also showed adequate penetration of this synthetic medium. On the other hand, 44% of couples (14/32) with negative outcome of polyacrylamide migration testing later achieved a pregnancy. No additional information was obtained when the different parameters of penetration (migration distance, sperm density, and motility grade) were analyzed separately (concerning all concentrations of polyacrylamide and the different time intervals of reading). After exclusion of those patients with female factors of infertility, again no significant relationship was found between PR and outcome of polyacrylamide migration testing with respect to polyacrylamide 1.5%, 1.6%, and 1.7%. In couples with patency of both fallopian tubes, normal uterine configuration and regular ovulations without any hormonal imbalances of the female partner (n = 95), subsequent fertility was significantly higher when the results of migration testing with polyacrylamide 1.8% (after 2 hours) were positive (P < 0.05, x 2 analysis). With respect to polyacrylamide (1.8%) outcome after 6 hours, PR was 35% in case ofpositive migration testing versus 18% when polyacrylamide results were negative (not significant). DISCUSSION Cervical mucus has been demonstrated to have potential as a medium to test the quality of human semen in terms of sperm function (2, 3, 11) and a number of independent studies have indicated that the fertilizing capacity of human spermatozoa is correlated with CM penetration (4, 12, 13). Evaluation of sperm function in vitro with partners' CM proved to be superior to in vivo testing by means of a standardized PCT with regard to fertility prognosis (5). The in vitro system allows cross-testing with 546 Eggert-Kruse et al. Polyacrylamide as CM Substitute Fertility and Sterility

8 material of fertile donors and, furthermore, can be used with various penetration media as mucus surrogate. Artificial media for sperm migration testing are required because there are various problems in working with human CM. The main problem for most andrologists might be the availability of high quality human CM because they do usually only see the male partner of the infertile couple, and the variability of the CM during the menstrual cycle with regard to amount, viscoelastic properties and composition (14, 15). Influencing factors such as microbial colonization (9), antisperm antibodies (16, 17), and ph of the cervical secretions (18) also have to be regarded. Further disadvantages are the limitation of amount of mucus per collection, the variability because of storage, and the impossibility to store human CM over longer time periods without loss of quality. Therefore, attempts have been made to identify an alternative standardized source of mucus in developing an artificial CM from polyacrylamide gel with reagents commonly used for gel electrophoresis (6, 19). Polyacrylamide gel is relatively easy to formulate, can be obtained in large quantities, is uniform, and can be stored for months in an ordinary refrigerator at 4 C without changes in sperm penetrating characteristics (6, 20). The reproducibility of results for testing of sperm migration and the uniformity of this mucus surrogate has been shown in previous studies (6, 20). Polyacrylamide gel has physical characteristics similar to human CM, shows spinnbarkeit, clarity, and ferning comparable with CM, and can be easily aspirated into capillary tubes. As could be confirmed in the present study, the artificial medium is receptive to human sperm. The gel causes most spermatozoa to migrate along parallel lines in a unidirectional manner (6), and comparable with periovulatory human CM the synthetic material consists of >98% of water and has distinct rheologic properties depending on the acrylamide concentration. In the present study the synthetic medium was prepared in four different concentrations (polyacrylamide 1.5%, 1.6%, 1.7%, 1.8%) that were tested in parallel on the same semen sample. It could be demonstrated that sperm migration depended on the acrylamide concentration determining the viscosity of the material. Although, in general, the synthetic medium proved to be a much stronger barrier to sperm penetration than CM, significant correlations for all polyacrylamide preparations were found for the migration distance, when compared with human CM (P < 0.001). The penetration distance in polyacrylamide revealed in the present study was slightly lower than that reported by Lorton et al. (6) who used bull spermatozoa. Significant correlations were also found for the sperm density (P < ) with r values ranging from 0.35 to 0.61, depending on time of incubation and polyacrylamide concentration, although the number of spermatozoa capable of penetrating the synthetic medium was markedly reduced compared with human CM. Consistent with the reports ofurry et al. (21) who compared bovine CM and polyacrylamide as migration medium, an earlier decrease of sperm motility in the synthetic medium was observed. Less than half of the samples with motile spermatozoa in human CM after 6 hours also offered motility in polyacrylamide which further decreased with increasing concentration of the artificial medium «20% with regard to polyacrylamide 1.8%). Therefore, when instead of the single parameters of penetration, the cumulative SCMPT score (4) was used to select samples in those with adequate and inadequate sperm-mucus interaction in vitro (using CM ofpatients' partners obtained under standardized conditions)' no significant relationship was found with polyacrylamide results. Polyacrylamide testing was further limited by the fact that the majority of sampies of the inadequate SCMPT group did not, or at a very low rate «10 mm) penetrate the artificial medium (concerning all concentrations). The present study also revealed highly significant correlations between the outcome of routine semen analysis and penetration testing with polyacrylamide gel. The relationship between sperm parameters in the seminal fluid analysis and the outcome of sperm penetration test in different viscous media is consistent with other reports (4, 11, 20, 22), e.g., Goldstein et al. (20) showed that motility of spermatozoa was a major factor influencing migration distance in polyacrylamide gel with r values of 0.57 to 0.62, which are comparable with those of the present investigation (with regard to polyacrylamide gel 1.5% to 1.7%). These authors (20), by using regression analysis, showed that <40% of the variation in migration distance (in polyacrylamide gel) was explained by the differences in sperm motility. A normal sperm count by no means ensured good penetration ability, and furthermore, migration distance decreased as a result of freezing much more than did sperm motility, maybe because of influences of the freezing process on sperm Vol. 60, No.3, September 1993 Eggert-Kruse et al. Polyacrylamide as em Substitute 547

9 functional characteristics. In the present study, in spite of good semen quality, determined by standard semen analysis (>40 X 10 6 /ml, >40% progressive motility, >60% normal forms) a considerable amount of semen samples offered poor migration in the synthetic medium (polyacrylamide negative): 25% in the 1.5% to 50% in the 1.8% preparation. The fact that the penetration distance as well as the sperm density showed a median of 0 in patients with reduced sperm counts, motility, or impaired morphology is a major drawback for the use of polyacrylamide for penetration testing because evaluation of sperm functional capacity is particularly important in patients with reduced results of ejaculate analysis as a basic examination of male factor infertility. Semen cultures offered positive results with regard to potentially pathogenic bacteria as well as to species of the physiological flora in the majority of patients of the present study. This did not interfere with sperm ability to penetrate polyacrylamide. However, as could be shown in a much larger population (9), microorganisms in genital secretions of the male and/or female member of the infertile partnership also did not markedly influence sperm interaction with em in patients without symptoms of genital tract infection. Postcoital testing is widely used during infertility investigation as a screening method for local compatibility and sperm-mucus interaction (3, 5, 14). In the present study, the rate of semen samples offering positive polyacrylamide results increased significantly in parallel to a better outcome of pet in particular with regard to gel preparations with higher concentration (polyacrylamide 1.7% and 1.8%). This might suggest the use of the artificial medium for in vitro migration testing when em of good quality is not available and when information about sperm-mucus interaction in vivo cannot be obtained. However, the local compatibility of genital secretions is significantly determined by antisperm antibodies. In particular, sperm surface antibodies of the IgA class are important for subsequent fertility (16, 17). Although em of patients' partners and also donors' em is an immunological barrier for antibody-covered spermatozoa (17), no significant selection (with regard to local IgG as well as IgA antibodies) could be obtained when polyacrylamide was used as penetration medium in the present study that clearly limits the clinical significance of polyacrylamide migration testing during infertility investigation. Pregnancy is the only irrefutable proof that a sperm can fertilize. In this prospective study, polyacrylamide testing offered no clear differentiation with regard to subsequent fertility. No significant difference of PR was found between polyacrylamide (1.5% to 1.7%) positive and negative patients. The 1.8% preparation, in being the strongest barrier to sperm penetration, was able to select in some way the functionally most competent sperm cells. Fertility rate was significantly higher in patients with semen samples able to adequately penetrate this viscous medium in the in vitro capillary tube test. However, this has to be interpreted with caution as a considerable amount of those patients with negative outcome of polyacrylamide testing also achieved a pregnancy within a 6 months' observation period. Although the findings suggest that the synthetic medium, depending on concentration, acts in some way as a filter selecting the strongest group of spermatozoa, the conditions to allow penetration and to maintain sperm motility are quite different comparing the artificial medium and human em. The composition of the mucus surrogate obviously differs with regard to macromolecular components, proteins, electrolytes, sources of energy (14, 23), and many other factors, e.g., steroid hormones. In this respect, natural media as a substitute for human em, such as estrous bovine em (13), fresh hen's egg white (24), or hyaluronate (22) would seem to be more favorable. However, these also have considerable disadvantages (4, 17,25). A major limitation is that they differ from human em in the lack of responsiveness to the presence of antisperm antibodies (17). On the other hand, viscous media of natural or synthetic origin that are receptive to sperm offer information about sperm intrinsic motility (3). In - providing a filter of varying specificity, these substitute media, when used in migration tests, can select subgroups of spermatozoa. It is postulated that those sperm able to penetrate these complex substances are endowed with higher energy. This in some way reflects a characteristic related to functional competence necessary to reach the site of fertilization in the upper female genital tract, although when polyacrylamide gel was used as migration medium, it could not be proven that this was also strictly related to the fertilizing capacity that depends on a multiplicity of different factors. For clinical andrologic investigation as well as for research purposes, a material is needed for standardized migration testing with properties similar to those of human em that could be prepared sim- 548 Eggert-Kruse et al. Polyacrylamide as em Substitute Fertility and Sterility

10 ply with uniform stable characteristics. Special regard has to be paid to metabolic properties of the mucus surrogate and sources of energy for sperm migration, e.g., the sugar composition, the protein balance, and the amount of different types of highenergy phosphates. Future efforts are necessary, on the one hand, to improve the technology for storage of human em, and on the other hand, to clearly define its composition to synthesize a comparable medium for sperm migration testing. REFERENCES 1. Polansky FF, Lamb EJ. Do the results of semen analysis predict future fertility? A survival analysis study. Fertil Steril1988;49: Kremer J. A simple sperm penetration test. Int J Fertil 1965;10: Blasco L. Clinical tests of sperm fertilizing ability. Fertil Steril1984;41: Eggert-Kruse W, Leinhos G, Gerhard I, Tilgen W, Runnebaum B. Prognostic value of in vitro sperm penetration into hormonally standardized human cervical mucus. Fertil Steril1989;51: Eggert-Kruse W, Gerhard I, Tilgen W, Runnebaum B. Clinical significance of crossed in vitro sperm-cervical mucus penetration test in infertility investigation. Fertil Steril 1989;52: Lorton SP, Kummerfeld HL, Foote RH. Polyacrylamide as a substitute for cervical mucus in sperm migration tests. Fertil Steril 1981;35: 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, 1987: Jager S, Kremer J, Slochteren-Draaisma T van. A simple method of screening for antisperm antibodies in the human male: detection of spermatozoal surface IgG with the direct mixed antiglobulin reaction carried out on untreated fresh human semen. Int J Fertil1978;23: 'Eggert-Kruse W, Pohl S, Naher H, Tilgen W, Runnebaum B. Microbial colonization and sperm-mucus interactionresults in 1000 infertile couples. Hum Reprod 1992;7: Insler V, Melmed H, Eichenbrenner I, Serr DM, Lunenfeld B. The cervical score. A simple semiquantitative method for monitoring of the menstrual cycle. Int J Gynaecol Obstet 1972;10: Insler V, Bernstein D, Glezerman M, Misgav N. Correlation of seminal fluid analysis with mucus-penetrating ability of spermatozoa. Fertil Steril 1979;32: Ulstein M. Sperm penetration of cervical mucus as a criterion of male infertility. Acta Obstet Gynecol Scand 1972;51: Alexander NJ. Evaluation of male infertility with an in vitro cervical mucus penetration test. Fertil Steril 1981; 36: Moghissi KS. The function of the cervix in fertility. Fertil Steril 1972;23: Wolf DP, Blasco L, Khan MA, Litt M. Human cervical mucus. IV. Viscoelasticity and sperm penetrability during the ovulatory menstrual cycle. Fertil Steril 1977;30: Kremer J, Jager S. The significance of antisperm antibodies for sperm-cervical mucus interaction. Hum Reprod 1992; 7: Eggert-Kruse W, Hofsa13 A, Haury E, Tilgen W, Gerhard I, Runnebaum B. Relationship of local antisperm antibodies and sperm-mucus interaction in vitro and in vivo. Hum Reprod 1991;6: Eggert-Kruse W, Kohler A, Rohr G, Runnebaum B. The ph as an important determinant of sperm-mucus interaction. Fertil Steril 1993;59: Bissett DL. Development of a model of human cervical mucus. Fertil Steril1980;33: Goldstein MC, Wix LS, Foote RH, Feldschuh R, Feldschuh J. Migration of fresh and cryopreserved human spermatozoa in polyacrylamide gel. Fertil Steril1982;37: Urry RL, Middleton RG, Mayo D. A comparison ofthe penetration of human sperm into bovine and artificial cervical mucus. Fertil Steril 1986;45: Mortimer D, Mortimer ST, Shu MA, Swart R. A simplified approach to sperm-cervical mucus interaction testing using a hyaluronate migration test. Hum Reprod 1990;5: Rohr G, Eggert-Kruse W, Pehlke A, Sahrbacher U, Runnebaum B, Kalbitzer HR. Biochemical analysis of cervical mucus by nuclear magnetic resonance spectroscopy. Hum Reprod 1992;7: Eggert-Kruse W, Gerhard I, Tilgen W, Runnebaum B. The use of hen's egg white (HEW) as a substitute for human cervical mucus in assessing human infertility. Int J Androl 1990;13: Marshburn PB, Dodson WC, Haney AF. Bovine cervical mucus penetration by human spermatozoa: lack of association with conception. Arch Androl 1989;22: Vol. 60, No.3, September 1993 Eggert-Kruse et al. Polyacrylamide as em Substitute 549