Clinical relevance of sperm morphology assessment using strict criteria and relationship with sperm-mucus interaction in vivo and in vitro*

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1 FERTILITY AND STERILITY Vol. 63, No.3, March 1995 Copyright e 1995 American Society for Reproductive Medicine Printed on acid-free paper in U. S. A. Clinical relevance of sperm morphology assessment using strict criteria and relationship with sperm-mucus interaction in vivo and in vitro* Waltraud Eggert-Kruse, M.D.t:l: Jutta Reimann-Andersen, M.D.t Gerhard Rohr, M.D., Ph.D. Sabine Pohl, Ph.D. II Wolfgang Tilgen, M.D.' Benno Runnebaum, M.D.t University of Heidelberg, Heidelberg, Germany Objective: To determine the relationship of the differentiated morphological pattern of semen samples according to strict criteria and sperm-mucus interaction in vivo and in vitro. Patients: One hundred sixty-three randomly chosen couples with long-standing infertility (median duration of infertility 4 years, range 1 to 19 years). Setting: Outpatient clinic of the fertility unit at the Women's University Hospital of the University of Heidelberg, Heidelberg, Germany. Main Outcome Measures: Sperm morphology assessment using strict criteria (Tygerberg or Norfolk classification) parallel to standard methods of sperm analysis: Evaluation of the cervical factor of patients' female partners, including a microbial screening of genital secretions of both partners; Examination of sperm migration ability in vivo under hormonally controlled conditions for the cervical mucus (em) quality and in vitro with the crossed sperm-em penetration test performed with em of patients' partners, as well as with em and spermatozoa of donors; Determination of the selection capacity of em with regard to sperm morphology by means of a biological model; Prospective analysis of the differentiated morphological pattern with respect to couples' subsequent fertility within 6 months. Results: Using stict criteria, amorphous sperm heads were the most frequently found sperm anomaly (severely amorphous forms: median, 28%; range, 4% to 62%). The morphology index offered a median of 45% (range, 7% to 80%). Results correlated significantly with routine sperm analysis, including standard morphology. The morphological pattern differed significantly in samples offering adequate or inadequate ability to penetrate em in the standardized sperm-em penetration test or in the postcoital test, with the percent of severely amorphous heads as the most important parameter. Neck and tail defects did not play an important role. During passage of mucus columns in vitro, the rate of pathological spermatozoal forms was reduced significantly, from a median of 65% to a median of 38%. Better functional capacity of spermatozoa with normal head morphology also was reflected by a significantly higher pregnancy rate under natural conditions of conception. Conclusions: Sperm morphological properties, determined with strict criteria, are important factors for sperm ability to penetrate the mucus barrier at the uterine cervix before reaching the site of fertilization, but sperm morphology is only one among other parameters determining the complex phenomenon of sperm-mucus interaction. Fertil Steril1995;63: Key Words: Sperm-mucus interaction, cervical factor, sperm morphology, in vitro sperm-cervical mucus penetration test, postcoital test (pet), sperm function, sperm fertilizing capacity Received April 27, 1994; revised and accepted September 6, * Presented in part at the 9th Annual Meeting of the European Society of Human Reproduction and Embryology (ESHRE), Thessaloniki, Greece, June 27 to 30, t Department of Gynaecological Endocrinology and Reproductive Medicine, Women's Hospital. :j: Reprint requests: Waltraud Eggert-Kruse, M.D., Department of Gynaecological Endocrinology and Reproductive Medicine, Women's Hospital, University of Heidelberg, VoJ3straJ3e 9, Heidelberg, Germany. Department of Internal Medicine IV, Klinikum Mannheim. II Department of Microbiology and Hygiene. ~ Andrology Division, Department of Dermatology. 612 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction Fertility and Sterility

2 The clinical relevance of sperm morphology and its relationship with sperm function and spermmucus interaction are controversial subjects. Kruger and coworkers (1-3) introduced a very strict classification for determination of spermatozoal morphology and showed a significant correlation with the success of IVF. However, under the usual circumstances of conception, the uterine cervix is a main barrier regulating sperm transport to the site of fertilization in the upper female genital tract. Early studies, using conventional criteria for sperm morphology assessment, suggest that cervical mucus (CM) might have a filtering capacity for defective spermatozoal forms (4, 5). Not much is known about the significance of the so-called Tygerberg or Norfolk criteria for sperm penetration ability, although this classification is based partly on the appearance of sperm cells in CM of the upper cervical canal (2). Therefore, the morphological pattern according to these strict criteria, in addition to routine sperm analysis (6), was determined in semen samples of a large number of randomly chosen subfertile couples who presented for infertility investigation and treatment in the general outpatient infertility clinic of the University of Heidelberg. Results were analyzed with regard to sperm ability to penetrate CM in vivo, evaluated by means of the postcoital test (PCT), and sperm-mucus interaction in vitro, using the standardized sperm-cm penetration test with CM of patients' female partners as well as CM of fertile donors (7, 8). In an experimental design, the filtering capacity of CM for the different types of morphological sperm anomalies was determined using a biological model. Cervical mucus is influenced multifactorially, the hormonal factor being of utmost importance (4, 9). Therefore, a standardized hormonal approach was used in this investigation. Furthermore, the differentiated sperm morphology was related to couples' subsequent fertility in this prospective study to determine the specific type of sperm anomaly responsible for impaired functional capacity and fertilizing ability in vivo (10). MATERIALS AND METHODS Patients and Basic Infertility Investigation The study population consisted of 163 randomly chosen couples with a median duration of infertility of 4 years (range, 1 to 19). The median age of the male patients was 32 years (range, 21 to 49), and the age of their female partners 31 years (range, 19 to 45). Primary infertility was found in 73%, and secondary infertility in 27%. All patients were asymptomatic in terms of genital tract infection. Before evaluation of sperm morphology, all couples were submitted to a comprehensive evaluation of male and female fertility. A detailed medical history was obtained, and physical examinations were performed on both partners. Female patients were checked for tubal patency by hysterosalpingography and/or laparoscopy. Follicular growth and hormonal disorders were examined carefully by BBT, ultrasound, multiple determinations of gonadotropins, PRL, E 2, P, T, the adrenal gland hormone pattern including ACTH tests and thyroid function tests and were treated accordingly. Endocrine evaluation was completed by premenstrual endometrial biopsy. Evaluation of Sperm Quality Standard Criteria Semen was obtained in hospital after 5 days of sexual abstinence and examined directly after liquefaction. Sperm volume, ph, count, progressive motility after liquefaction, after 2 hours, and after 4 hours, viability, and number of round cells (undifferentiated, per high-power field [HPF], X400 magnification) were determined. Sperm analysis according to stanoard criteria (6) also included sperm morphology evaluation as recommended by the World Health Organization (WHO). For staining, prepared slides were used (Testsimplets; Boehringer, Mannheim, Germany) (11). Five microliters of semen were applied to the indicator field and processed as recommended by the manufacturer. Spermatozoa were differentiated in percent normal, percent pathological, percent anomalies of the head, the neck, and the tail, respectively (6). Strict Criteria In parallel, sperm morphology was assessed as outlined in detail by Kruger et al. (3). Immediately after liquefaction, semen was applied to carefully cleaned microscopic slides, and a thin layer over the entire surface was obtained. After air drying at room temperature, the prepared slides were stained with the DiffQuick stain set (American Hospital Supply, dell Caribe, Puerto Rico) (12) according to the manufacturer's recommendations, rinsed with distilled water, dried, and read after mounting by Vol. 63, No.3, March 1995 Eggert-Kruse et a1. Relationship of sperm morphology and sperm-mucus interaction 613

3 an aqueous mounting solution (Glycergel; Dako, Hamburg, Germany). Determination of sperm morphology was performed at the high power (X1,000) magnification using oil immersion and a light microscope (Leitz, Wetzlar, Germany). One hundred spermatozoa were counted on each slide and differentiated in strictly normal or pathological forms with the different anomalies of the sperm head, as slightly and severely amorphous types or specific defects of the size or configuration of the sperm head; and in case of a normally shaped head, those with slightly or severely malformed sperm neck or midpiece or tail. Sperm cells with questionable normality that could not be put clearly in the normal or pathological category were excluded (median, 4%). Two slides were prepared from each semen sample, and all readings were done in duplicate. Evaluation of sperm morphology using strict criteria was performed by one consistent observer throughout the whole study (J.R-A.), and sperm analysis using standard criteria by another independent observer. Microbial Screening For microbial screening of ejaculates, 10 JLL of semen were transferred with a disposable sterile plastic loop into Shepard's medium for identification and differentiation of mycoplasmas. For culture of other potentially pathogenic bacteria, aliquots were inoculated into a universal transport medium (Port-a-Cul Universal; Becton Dickinson, Heidelberg, Germany) and identified using standard methods as previously described (13). Both partners ofthe subfertile couples were regarded as a microbial unit. Therefore cervical swabs of patients' female partners were examined simultaneously for mycoplasmas and potentially pathogenic bacteria; additionally, endocervical material was screened for Chlamydia trachomatis and Herpes simplex virus using cell culture technique. Vaginal swabs were examined for Candida albicans and trichomonads and a Gram-stained cervical smear was prepared for determination of leukocytes. Blood was taken from both partners for detection of previous chlamydial infection. Immunoglobulin (Ig) G antibodies (AB) to C. trachomatis were determined using a microimmunofluorescence test, a serum titer ~ 1:256 was considered positive (14). Evaluation of Sperm-Mucus Interaction Postcoital Testing (PCT) For assessment of sperm ability to penetrate CM, couples were submitted to PCT as described in detail previously (8). The condition of the CM was classified according to Insler et al. (15). After the preovulatory CM had been obtained from the endocervix with a special device, 8 to 12 hours after intercourse, the number of motile spermatozoa, using first the low-power field (LPF) and then the HPF magnification (X400) of a phase-contrast microscope, was determined. Postcoital test results were subdivided into four groups as follows: [1] PCT negative (no spermatozoa found in CM-LPF [X100 magnification] but in vaginal secretions); [2] PCT poor «2 motile sperm-hpf); [3] PCT good (2 to 6 motile sperm-hpf); and [4] PCT excellent (~7 motile sperm-hpf). Postcoital testing was performed in the periovulatory period of spontaneous cycles, and in the majority of patients it was repeated (or performed primarily because of late or irregular ovulation, marked luteal insufficiency, or difficulties of timing) after oral administration of estrogens (Es) (80 JLg of ethinylestradiol (EE 2 ) for at least 7 days before PCT, to control the hormonal influence on the mucus quality. Sperm-CM Penetration Test For in vitro testing of sperm penetration, semen specimens and mucus samples were evaluated by means of the standardized in vitro sperm-cm penetration test as described elsewhere (8). Briefly, semen samples were allowed to penetrate capillaries filled with fresh CM of patients' female partners, obtained from the endocervix under standardized conditions and after pretreatment with EE 2, Cervical mucus of fertile donors, obtained under the same conditions, was used in parallel on the same penetration meter (5). The clinical characteristics ofthe CM were determined according to Insler (15), and the ph of the CM was measured with ph indicator paper (Merck, Darmstadt, Germany). Any medication with a potentially negative effect on the rheologic properties of the CM was stopped the cycle before. The penetrability of patients' CM samples was confirmed by cross-match testing with spermatozoa of fertile donors. To evaluate sperm functional capacity with the sperm-cm penetration test, semen was used directly after liquefaction. After 0.5, 2, and 6 hours incubation in a moist atmosphere at 37 C, the penetration distance, sperm density, and quality of motility were determined. Results were summarized in a sperm-cm penetration test score and classified based on previ- 0usly described criteria as adequate or inadequate (7). Standard sperm analysis, determination of sperm morphology using strict criteria, microbial 614 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction Fertility and Sterility

4 screening, and assessment of sperm penetration ability by means of the sperm-cm penetration test were performed in parallel with aliquots of the same ejaculates. Assessment of the Filtering Capacity of CM In Vitro U sing a biological model, semen samples of 30 patients were assessed for sperm morphology before and after passage of a mucus column. Only ejaculates with excellent progressive motility (>60%) and CM of high quality (cervical index 12 according to Insler [15]), obtained after pretreatment with Es and as described for the sperm-cm penetration test, were used. Reservoirs of a modified penetration meter were filled with 200,ILL of semen taken directly after liquefaction. In microcapillaries (40,ILL; Brand, Wertheim, Germany) was first aspirated serum, then CM of patients' female partners, so that a column of CM in the lower 30 mm and of serum in the upper 20 mm resulted. Special care was taken to avoid air bubbles with potential interference with the continuity of both media. After incubation of 2 hours at 37 C in a moist Petri chamber, those spermatozoa that had migrated through the CM and had entered the distal part of the capillary filled with serum were assessed, after the contents of the distal 20 mm ofthe capillary had blown out on a precleaned microscopic slide. Slides were prepared and stained as described above, and sperm morphological properties using strict criteria (1-3) were determined. The morphology pattern after passage of the CM column was compared with that in the sperm reservoir before migration into CM. A significant correlation of sperm ability to penetrate both media (CM and serum) had been confirmed in a group of 135 ejaculates with correlation coefficients (r) of for penetration distance and for sperm density (P < ) (Spearman rank correlation). Pregnancy Rate (PR) and Statistical Methods Pregnancy rate was determined 6 months after assessment of sperm morphology. During this period, couples were not submitted to any assisted procreation techniques, such as IVF, GIFT, or lui. Data were processed using the Statistical Analysis System. Wilcoxon's rank sum, Kruskal-Wallis, x 2, Fisher's two-tailed exact, Friedman-Wilcoxon tests, and Spearman rank correlation were used. The level of significance was set at P < Table 1 Results of Sperm Morphology Evaluation Using Strict Criteria* 25% 75% Parameter percentile Median percentile Range to 58 Slightly amorphous to 24 Severely amorphous to 62 Specific defects Small o to 8 Large o to 11 Round o to 18 Tapering o to 45 Duplicate o to 9 Cytoplasmic droplet o to 20 (total) to 97 Slight defects Thickening o to 21 Debris 0 0 o to 6 Severe defects Bending o to 11 Midpiece >30% o to 10 (total) o to 27 Two tails o to 11 Coiled o to 2 (total) o to 12 Morphological index to 80 Total pathological forms to 98 * Number of semen samples was 163. RESULTS Sperm Morphology Assessment Using Strict Criteria Sperm morphology evaluation using strict criteria was performed in 163 semen samples. All readings were done in duplicate. The different types of pathological forms as well as the morphological index based on the classification of Kruger et al. (1-3) are shown in Table 1. Sperm pathology was dominated by malformations of the sperm head, in particular severely amorphous forms. The distribution of all types of pathological forms was taken for further statistical comparisons; additionally, previously described thresholds, for example, 14%, 7%, or 20% normal spermatozoa were used (1, 3, 10). The so-called "p-pattern" (3) with <4% of spermatozoa was found in only 4.9% (8 of 163); therefore, this group was too small for statistical analysis. When the morphological pattern on the two slides that had been prepared from each semen sample was compared, a significant correlation was found (Spearman rank correlation). The r for the percent normal forms was 0.922, for anomalities of the head r = 0.873, and for abnormalities of the neck r = 0.568, P < for all r. Because of this strong correlation, the results obtained on the first Vol. 63, No.3, March 1995 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction 615

5 slide evaluated of each semen sample were taken for further analyses. Relationship of Sperm Morphology with Medical History and Clinical Findings All couples were submitted to a comprehensive evaluation of female and male fertility. Some sort of minor tubal pathology was found in 31 % of female patients, and a severe tubal factor in 11 %. Uterine pathology was found in 9%, mostly minor anomalies (6%), considerable findings such as uterus duplex or uterus myomatosus in 3%. Because of the differentiated endocrine examination, discrete hormonal disorders were found in the majority of patients. Thirteen percent of women offered irregular ovulation, 38% a luteal insufficiency, and a severe hormonal factor was diagnosed in 17% of patients with oligoamenorrhea. Female factors were not related significantly to sperm morphology of their partners. In males, no significant relationship of sperm morphological properties (analyzed with regard to medians, ranges, and different thresholds) was found with regard to mumps (63%), previous infection in the genital area (10%), genital surgery (most frequently circumcision in 11 %, herniotomy in 6%, and varicocelectomy in 7%), or genital injury (1 %). No marked differences, including the detailed morphology pattern, were found for the subgroup ofpatients with varicocelectomy. Tapering forms, considered to be characteristic in the case of varicocele, were found in nearly the same frequency (median 8% versus 9% in the other patients). Furthermore, there was no significant relationship of any type of sperm morphological pathology with patients' age, nationality, social status, smoking and drinking habits, stress factors, duration of infertility, specific andrologic medication (12%), or general medication (9%). Andrologic examination offered slight abnormalities in 49 patients, mostly clinical findings related to varicocele, epidydimal induration (3%), enlargement of the prostatic gland (2%), or hydrocele (2%). In this group of patients, <14% normal spermatozoa were found more frequently (41% [20 of 49]) than in the group of men with normal findings on examination «14% normal forms in 26% [30 of 114]) (not significant, X 2 analysis). A significant relationship was found between sperm morphology and the testicular volume (on the day of semen analysis determined in 126 patients). A volume of <15 ml on the right side was found in 26% of males with reduced sperm morphology (10 of 38) com- Table 2 Correlation of Sperm Morphology Assessment Using Strict and Standard Criteria in 163 Semen Samples Morphological parameter Total pathological forms Strict criteria* 20 (1 to 58) 62 (24 to 97) 6 (0 to 27) 1 (0 to 12) 73 (36 to 98) Standard criteriat 63 (49 to 87) 16 (5 to 23) 11 (4 to 19) 11 (4 to 19) 37 (13 to 51) r:j: P II Strict Tygerberg or Norfolk criteria (1-3). Values are medians with ranges in parentheses. t Standard criteria of the WHO (6). :j: Correlation coefficient r. Spearman rank correlation. II, not significant. pared with 8% (7 of 88) of men with >14% normal spermatozoa (P < 0.01), a testicular volume of <15 ml on the left side in 38% (15 of 38) when <14% and in 15% (13 of 88) when >14% normal forms were determined in semen samples (P < 0.01, X2 analysis). Sperm Morphology and Other Parameters of Sperm Analysis Standard sperm analysis offered medians for ejaculate volume of 3.6 ml (range, 0.5 to 9.8), sperm count of 40 X 10 6 jml (range, 1 to 198), progressive motility directly after liquefaction of semen of 40% (range, 0% to 80%), viability 70% (range, 20% to 90%), ph 7.2 (range, 6.7 to 8), fructose concentration 1,820 (range, 172 to 3,650) JLgjmL, and number of round cells (per HPF) 5 (range, 1 to 30). Table 2 compares the outcome of sperm morphology assessment using strict and standard criteria and shows the significant relationship between both methods. Significant correlations were found also when other parameters of routine semen analysis and strictly determined sperm morphological properties, in particular head anomalies, were compared, as can be seen in Table 3. Table 3 also demonstrates that standard parameters of semen analysis were interrelated significantly, with strongest correlations found between progressive motility and viability, count, and morphology and viability (P = ) (Spearman rank correlation). In further analyses, the differentiatedmorphological pattern was analyzed after selection of samples with cutoffs at 40 X 10 6 jml for sperm count and 40% for progressive motility. Again, sperm morphology was reduced significantly in males offering a sperm density of <40 X 10 6 jml (n = 66) with a median percent total pathological forms of 80% 616 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction Fertility and Sterility

6 Table 3 Correlation of Different Parameters of Sperm Analysis Using Strict and Standard Criteria Standard criteriat Fructose Morphological Volume Count Motility Morphology Viability concentration parameters:j: (ml) ph (XI0 6 /ml) (%) (% normalt) (%) (ltg/ml) Strict criteria* (%) () () (P ) (P ) (P ) (P ) () (%) () () (P ) (P ) (P ) (P ) () (%) () () (P < 0.01) () () () () (%) () () () (P< 0.05) () () () Pathological forms (%) (total) () () (P ) (P ) (PO.0001) (PO.0001) () Standard criteriat Volume (ml) (P < ) () ph () Sperm count (X10 6 /ml) Motility (%) Morphology (% normalt) Viability (%) () () () (P ) () () () () (P ) (P ) (P ) (P ) (P ) (P ) (P < 0.05) (P ) () () * Strict criteria (1-3). t Standard criteria of the WHO (6). :j: Spearman rank correlation, with rand P shown., not significant. (range, 51% to 98%) compared with 68% (range, 36% to 94%) in men with higher sperm count (n = 97) (P < ), with a significantly higher rate of head anomalies (P < ), particularly severely amorphous forms (median 35% [range, 9% to 62%] versus 25% [range, 4% to 58], P < ), and a reduced morphological index (median 37% [range, 7% to 66%] versus 50% [range, 20% to 80%], respectively [P < ). Strictly determined sperm morphology was impaired also significantly in patients with asthenozoospermia (progressive motility < 40%) (n = 37) with a median percent pathological forms of 82% (range, 53% to 98%) compared with 70% (range, 36% to 97%) in the other men (P < ), significantly more head defects (P < ), especially severely amorphous forms (found in 39% [range, 17% to 62%] versus 26% [range, 4% to 58%], P < ), but also more tapering forms (median 10% [range, 0% to 45%] versus 6% [range, 0% to 45%], P < 0.03) and a reduced morphological index (34 % [range, 7% to 63%] versus 48% [range, 13% to 80%], respectively [P < ) (Wilcoxon tests). Significant morphological differences were found also for sperm motility after 2 hours and 4 hours but not with regard to ejaculate volume, ph, and fructose concentration. Relationship of Microbial Findings and Sperm Morphology All couples included in this study were without symptoms of infection of the lower genital tract. However, a broad microbial screening offered microbial colonization of genital secretions in the majority of patients. The most frequently found bacteria in ejaculates were Staphylococcus epidermidis (44%), enterococci (37%), Streptococcus pyogenes (group A) (10%), and ~-hemolytic streptococci in 7%. As can be seen in Table 4, no significant relationship was found with respect to mycoplasmas, potential pathogenic aerobic or anaerobic bacteria, or species of the physiological flora, and the differentiated morphological pattern. Morphological findings also were not related to elevated titers of IgG AB to C. trachomatis in serum (~1:256 in 14% of males and 22% of females) as marker for previous infection with these microorganisms. When in further steps, semen samples selected at different Vol. 63, No.3, March 1995 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction 617

7 Table 4 Relationship of Sperm Morphology Assessment Using Strict Criteria and Outcome of Microbial Screening in Ejaculates Morphological parameter Pathological forms (total) Pathological forms (total) Pathological forms (total) Microbial findings Mycoplasma hominis and/or Ureaplasma urealyticum Negative Positive (n ~ 143) (n ~ 17) 20 (1 to 58) 24 (2 to 54) 63 (24 to 97) 61 (35 to 89) 6 (0 to 27) 7 (0 to 20) 1 (0 to 12) 0(0 to 3) 74 (36 to 98) 70 (37 to 97) Aerobic cultures Sterile or Potentially physiological pathogenic flora bacteria:j: (n ~ 92) (n ~ 70) 23 (1 to 58) 20 (2 to 56) 62 (31 to 97) 64 (24 to 95) 6 (0 to 23) 7 (0 to 27) 1 (0 to 8) 0(0 to 12) 72 (42 to 96) 74 (36 to 96) Anaerobic cultures Sterile or Potentially physiological pathogenic flora bacteria:j: (n ~ 149) (n ~ 13) 21 (1 to 58) 16 (2 to 43) 62 (24 to 97) 66 (47 to 89) 7 (0 to 27) 4 (0 to 23) 1 (0 to 12) 1 (0 to 3) 72 (36 to 98) 77 (56 to 92) Wilcoxon rank sum test. t, not significant. :j: Additional commensal bacteria in this group not considered. Probability' t thresholds for the percent normal forms, as well as the morphological index, were analyzed for the prevalence of different species, for example, Mycoplasma hominis, Ureaplasma urealyticum, Escherichia coli, Proteus, group A or B streptococci, enterococci, and so on, again no significant relationship was found. Microbial findings in patients' female partners offered a lower prevalence of mycoplasmas (5%), and potential pathogenic aerobes (21 %), mostly enterococci (9%); additionally, commensal aerobes were found in 13%, lactobacilli in 47%, and C. albicans in 10%, confirming previous reports (13). All specimens were negative for Neisseria gonorrhoeae, trichomonads, as well as for C. trachomatis in endocervical material (McCoy cell culture), ;::0:10 leukocytes in Gram-stained cervical smears were seen in 11 %. All microbial results did not offer a relationship with the sperm morphology pattern of their partners. Relationship of Sperm Morphological Properties and Sperm-Mucus Interaction Sperm-Mucus Interaction In Vivo Postcoital testing was performed in 121 couples after hormonal standardization of the mucus quality. The E treatment resulted in a considerable improvement of the cervical factor; for example, a cervical index of ;::0:11 according to Insler (15) was found in 29% and a poor mucus quality (index 0 to 7) in 17% of women when PCT was performed on the day of presumed ovulation in spontaneous cycles (n = 97), whereas a very good Insler-index (;::0:11) was seen in 64% of women after EE2 medication. The significant relationship of PCT using the standardized hormonal approach and sperm morphology assessment using strict criteria is shown in Table 5. In particular, anomalies of the sperm head were significantly more frequent in the case of impaired sperm-mucus interaction in vivo. This could be confirmed when PCT results were combined in those with inadequate (negative and poor, total 50%) and adequate (good and excellent) sperm-mucus interaction. Malformations of the sperm head were associated significantly with inadequate PCT results (P < 0.001), in particular severely amorphous forms (P < 0.05) (Wilcoxon tests). No significant association was found for PCT and anomalies of the sperm neck or tail. Sperm-Mucus Interaction In Vitro Sperm functional capacity in vitro was evaluated by means ofthe in vitro sperm-cm penetration test with CM of patients' female partners, obtained under standardized conditions (n = 150). The majority of CM samples offered a very good Insler-index (median 11), the median ph of the CM was 7.0. Sperm-CM penetration test was evaluated after 30 minutes, 2 hours, and 6 hours. The good reproducibility of this in vitro test was confirmed when sperm-cm penetration test with CM of patients' female partners was performed in duplicate (n = 21 couples), with r > 0.9 with regard to the different parameters of penetration (migration distance, sperm density, and motility in CM) and the three time intervals of reading (P < for all r, Spearman rank correlation). When performed as a cross-matching penetrability test with CM and semen of fertile donors, results of sperm-cm penetration test were considerably better when donor spermatozoa were allowed to penetrate the capillaries filled with CM of female 618 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction Fertility and Sterility

8 Table 5 Relationship of Sperm Morphology Assessment Using Strict Criteria and Outcome of PCT* Result of PCT* Negative (n = 41) Poor (n = 20) Good (n = 42) Excellent (n = 18) Probabilityt % of couples Morphological parameters* Pathological forms (total) (2 to 44) 68 (33 to 95) 6 (0 to 23) 1 (0 to 12) 77 (50 to 96) 15 (5 to 48) 76 (41 to 90) 6 (0 to 16) 1 (0 to 4) 81 (49 to 91) (5 to 56) 60 (32 to 89) 8 (0 to 23) 1 (0 to 9) 71 (41 to 92) (9 to 58) 46 (24 to 78) 9 (0 to 27) 0.5 (0 to 6) 55 (36 to 83) <0.001 < :I: < * Performed after standardized treatment with Es (n = 121 couples). Values are medians with ranges in parentheses. patients, confirming previous findings (7). A poor sperm-em penetration test outcome (score < 3) was never found in the crossed sperm-em penetration test with donor spermatozoa that demonstrates the penetrability of patients' em. The relationship of sperm migration ability in vitro and the morphological pattern is shown in Table 6. The percent pathological forms was significantly lower in the group with adequate sperm-em penetration test outcome (cumulative score :2: 6) than in the group with inadequate sperm-mucus interaction (sperm-em penetration test score < 6). The rate of severely amorphous heads offered a median of 27% (range, 4% to 58%) in case of adequate sperm-em penetration test outcome compared with a median of36% (range, 19% to 62%) in case of inadequate sperm penetration ability in vitro (P < 0.005). Small heads were also less frequent in case of adequate sperm -em penetration test (P < 0.05), and no significant differences were found with regard to slightly amorphous heads, big heads, round heads, duplicate or tapering forms, cytoplasmic droplets, nor with regard to minor or severe defects of the sperm neck or midpiece or tail. The influence of sperm morphological properties on their functional capacity could be demonstrated also when donors' em was taken for performance ofthe crossed sperm penetration test (see Table 6). Again, differences were most pronounced for severely amorphous forms that offered a median of 25% (range, 6% to 57%) in the group with adaequate, compared with 35% (range, 4% to 62%) in the group with inadaequte outcome of the crossed sperm-em penetration test (P < ) (Wilcoxon tests). Neck and tail abnormalities were not associated with poor results of sperm penetration testing with em of fertile donors. The relevance of sperm morphological normality for mucus penetration ability was confirmed when sperm-em penet Kruskal-Wallis test. :I:, not significant. tration test outcome was related to the percent normal forms with thresholds at 14% as well as 7% or 20% normal forms (P < 0.001, x 2 analysis). Filtering Capacity of CM In an experimental design, sperm morphology was compared before and after passage through a Table 6 Relationship of Sperm Morphology Assessment Using Strict Criteria and Outcome of the In Vitro Sperm CM Penetration Test Morphological parameter Pathological forms (total) Morphological parameter Pathological forms (total) Outcome of sperm-cm penetration test Sperm-CM penetration test with CM' of patients' female partners Inadequatet Adequatet (n = 23) (n = 127) 12 (3 to 34) 23 (2 to 58) 76 (40 to 95) 60 (24 to 89) 5 (1 to 23) 6 (0 to 27) 1 (0 to 8) 1 (0 to 12) 82 (60 to 96) 71 (36 to 97) Crossed sperm-cm penetration test with CM of fertile donors Inadequatet Adequatet (n = 29) (n = 123) 15 (1 to 56) 28 (2 to 58) 72 (25 to 97) 57 (24 to 89) 5 (0 to 27) 8 (0 to 23) 1 (0 to 8) 1 (0 to 12) 79 (41 to 98) 69 (36 to 94) Probability Probability:\: <0.001 <0.001, Cervical mucus obtained under standardized conditions and after pretreatment with Es. t Based on the cumulative sperm-cm penetration test score after 6 hours ohservation time (7). :\: Wilcoxon rank sum test., not significant. Vol. 63, No.3, March 1995 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction 619

9 Table 7 Relationship of Results of Sperm Morphology Assessment Using Strict Criteria* Before and After Passage of a Mucus Columnt Before After Morphological passage passage parameters through CM:j: through CM Probability 35 (9 to 56) 60 (36 to 82) Slightly amorphous 13 (7 to 20) 9 (5 to 22) <0.05 Amorphous 27 (16 to 57) 13 (3 to 32) Specific defects Small o (0 to 5) 1(lto1) <0.05 Large o (0 to 5) 2 (1 to 2) <0.05 Round 1 (0 to 12) 2 (1 to 5) Il Tapering 9 (0 to 38) 10 (1 to 43) Duplicate 0(0 to 8) 3 (2 to 5) <0.05 Cytoplasmic droplet 0(0 to 2) 1 (1 to 1) (total) 57 (37 to 84) 37 (12 to 62) Slight defects Thickening 4 (0 to 13) 1 (1 to 4) <0.001 Debris 0(0 to 5) 1(lto1) <0.05 Severe defects Bending 1 (0 to 4) 1(lto2) <0.001 Midpiece >30% 0(0 to 2) 1 (1 to 3) (total) 7 (0 to 16) 1 (0 to 6) Two tails 0(0 to 4) 1 (1 to 4) Coiled 0(0 to 0) 1 (1 to 1) (total) 0(0 to 4) 1 (1 to 4) Morphological index 54 (20 to 72) 70 (47 to 93) Total pathological forms 65 (43 to 89) 38 (13 to 64) Strict criteria (1 ~3). t Number of selected semen samples of very good quality was 30. :j: Cervical mucus of very good quality, obtained after hormonal standardization. Friedman-Wilcoxon test for dependent variables. II, not significant. mucus column (n = 30 selected semen samples of excellent quality). Although morphological properties before and after em migration offered a significant correlation (e.g., with r of for percent of pathological forms and for anomalies of the sperm head) (P < 0.001), a significant improvement for the rate of normal forms after em passage was found, as can be seen in Table 7. For example, the median percent of normal spermatozoa was 35% before and 60% after mucus passage (P < ). A particular reduction during em migration could be observed for anomalies of the sperm head, specifically for severely amorphous forms. In this group of semen samples, slightly amorphous forms, were also less frequent after em passage (median, 9% versus 13%, not significant), as well as malformations of the sperm neck (thickening of midpiece), (P < 0.001); no differences were found for defects of the sperm tail. Sperm Morphology and Fertilizing Ability Pregnancy rate could be determined in 159 couples; four couples were lost for follow-up. Within an observation period of 6 months, 17 of these couples achieved a pregnancy (11 %). In semen samples of patients with subsequent fertility, the percent normal forms offered a higher median (28%) and minimum (6%) than in the other men (median 20%, minimum 1 %), but this did not achieve statistical significance. However, the rate of anomalies of the sperm head, found with a median of 51 % (range, 34% to 74%) in ejaculates of fertile patients compared with 64% (range, 24% to 97%) in patients who did not achieve pregnancy, differed significantly (P < 0.01). In the later fertile patients, the percent of spermatozoa with severely amorphous heads did not exceed 38% (median, 23%; range, 8% to 38% versus median, 29%; range, 4% to 62% in the nonfertile patients, P < 0.01). The rate of spermatozoa with specific defects was not markedly different in both groups of patients and did not exceed 16% for anomalies of the sperm acrosome, 1 % of small heads, 5% of big heads, 4 % of round heads, 31 % of tapering forms, 4% of duplicate heads, and 7% of cytoplasmic droplets in the later fertile men. The morphological index was considerably better in patients with a subsequent pregnancy (median, 53%; range, 31 % to 71 % versus median, 45%; range, 7% to 80%) (P < 0.02, Wilcoxon tests). The rate of malformations of the sperm neck or tail based on the strict criteria was not related significantly to sperm fertilizing ability in vivo. The significance of sperm morphological properties for in vivo fertility (10) could be confirmed when 14% normal forms were taken as threshold with a PR of 14% (16 of 112), when semen samples offered ~14% of normal spermatozoa, compared with 2% (1 of 47) when the rate of normal forms was <14% according the strict classification (P < 0.03, X 2 analysis). Significant differences of subsequent PR were found also when further analyses were performed after exclusion of couples with infertility factors of the female partner, for example, hormonal disorders, tubal, or uterine pathology, in accordance with previous findings in a smaller and different group of couples who is not included in the population ofthe present investigation (10). For example, in patients of this study without severe tubal pathology, subsequent in vivo PR was 16% (16 of 101) when ejaculates offered ~14% normal forms based on strict criteria versus 2% (1 of 43) in case of reduced sperm morphology «14% normal) (P < 0.02). 620 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction Fertility and Sterility

10 DISCUSSION The definition of normal sperm morphology and its clinical relevance are controversial. Very strict criteria for definition of normal spermatozoa have been proposed by Kruger and co-workers (1-3), the so-called Tygerberg or Norfolk criteria and have been correlated with the success of IVF. However, under the natural conditions of conception, one of the essential preliminaries to fertilization is the process of sperm transport in the female genital tract. Therefore, in the present study the differentiated morphological pattern, based on these criteria, was analyzed with regard to sperm functional capacity and sperm-mucus interaction in vivo and in vitro as important parameter for sperm fertilizing ability. Not only were semen samples assessed using strict criteria (1-3) for sperm morphology, aliquots from the same ejaculates were submitted also to routine sperm analysis (6) to correlate results. For both methods of sperm morphology evaluation, rapid staining procedures were used (DiffQuick-Stain and Testsimplets) that proved to be practical and suitable for routine use (11, 12). Comparative studies on Papanicolaou-stained smears and ready-touse slides (Testsimplets) showed coinciding results (11). Significant correlations for morphological sperm properties were found, although the percent of normal forms using the WHO classification (6) was much higher compared with strict criteria (1-3), of the pathological forms; particularly anomalies of the sperm head correlated strongly but not malformations of the sperm neck or tail. This might, on the one hand, be related to the low prevalence of these defects and, on the other hand, to the principles of the strict classification in which anomalies of the sperm head are considered superior to other defects, and therefore spermatozoa with combined malformations are put in the category of head pathology. It is supposed that the assessment of sperm morphology varies widely between laboratories. In the present investigation, the median rate of normal oval forms as well as the morphological index are in accordance with reports in men referred for IVF treatment (2). There was no intertechnician variability for sperm morphology evaluation because all slides were read by the same observer. The intraobserver variability was low (r = for duplicate readings with regard to head anomalies, r = for percent of normal forms, P < ) and thus comparable with the variability described by others (2). Apart from the significant correlation of morphological properties of spermatozoa when determined with both strict and standard criteria, a significant association was found with other parameters ofroutine sperm analysis, in particular sperm count, motility, and viability. The significant interrelationship of standard parameters of microscopical sperm analysis (16) underlines the importance of the inclusion criteria for studies on sperm morphology, for example, when patients with semen samples with reduced motility or sperm sperm count are excluded (1, 3), this will influence certainly the distribution of data with regard to the rate of normal and pathological forms and possibly other findings. The factors involved in the production of malformed spermatozoa are not yet well known in men. One factor of clinical interest might be infection of the genital glands. The potential influence on sperm functional capacity, for example, of mycoplasmas, C. trachomatis, or E. coli is a matter of debate (13, 14). Therefore, all semen samples were screened for colonization with potentially pathogenic microrganisms, to evaluate a possible relationship with morphological pathology as potential marker for microbially induced spermatogenic defects. No relationship with the strictly determined morphological pattern could be found. However, subclinical infection of the male genital tract with influence on spermatogenesis is not reflected sufficiently by the mere presence of bacteria in semen and has to be confirmed by adaequate markers. Few investigators have studied specifically those spermatozoa that are capable of penetrating the CM. Postcoital testing is used widely during infertility investigation and is considered a useful screening procedure for local compatibility (9). However, although generally accepted and easy to perform, there is no common agreement on the standardization of the PCT. Postcoital testing outcome is determined multifactorially and a significant correlation with, for example, sperm count and progressive motility have been shown previously (7, 17). It has to be regarded that the hormonal influence is of paramount importance for the outcome of PCT (4, 9). The stimulating effect of Es on mucus production by the epithelial cells of the cervix results in a quantitatively measured and clinically observed increase of CM (4,8, 15). Therefore, a standardized hormonal approach before PCT was used to analyze the relationship of the morphological pattern determined with strict criteria and sperm migration ability in vivo. Other parameters known to interfere with PCT results also were controlled, such as sexual abstinence before testing, the time postcoitus, and the ph of the CM (9). Vol. 63, No.3, March 1995 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction 621

11 The percent of normal forms according to the strict classification increased significantly in parallel with a better outcome of pet. Although a median of 17% normal was found in couples with negative pet, the median was 39% when pet was excellent. A significantly lower rate of head malformations was found in case of good sperm-mucus interaction, in particular severely amorphous forms. This is in accordance with other reports, in which using conventional criteria, a significantly higher rate of normal spermatozoa in the em after coitus (17) or after insemination using cervical caps (18) was found than in the semen samples. On the other hand, the present findings demonstrate that a considerable rate of pathological forms in semen samples, determined with strict criteria, was compatible with good pet outcome and that normal morphology of spermatozoa must not mean adequate migration ability. The relatively weak relationship of morphological properties and pet also suggests that sperm morphology is only one factor in the complex process of sperm-mucus interaction in vivo that involves a multiplicity of different parameters, for example, sperm functional properties, mucus quality, biochemical, microbiological, immunological, and psychosexual factors (4-9, 13, 17, 19). Sperm penetration meter testing in vitro provides objective, quantitative, and reliable data about sperm functional capacity (5) and can be better standardized than pet. In the present study, sperm-em penetration test was performed by one consistent observer and offered a high reproducibility (r > 0.9). The evaluation of in vitro sperm-em penetration allows cross-testing with donor spermatozoa and mucus to clearly define the cervical or the male factor responsible for deficient test results without any sophisticated method. It could be demonstrated that impaired sperm-mucus interaction is related significantly more often to the male than the cervical factor, provided the hormonal influence is controlled (7) and is associated significantly with subsequent fertility (8). Related to strictly determined sperm morphological properties, the present study suggests that the em acts as a filter, allowing preferentially the best spermatozoa to penetrate. The rate of spermatozoa with head malformations and pathological forms in general was reduced in the case of adaequate sperm-em penetration test, whereas no marked barrier effect was found specifically for neck or tail defects. The selection capacity of em for morphological abnormal spermatozoa could be confirmed when em of fertile donors instead of em obtained from patients' female partners was used in the crossed sperm-em penetration test. This could be further substantiated in the present study when an in vitro model was used. Pathologically configurated cells in individual semen sampies were reduced selectively during passage through em columns of excellent quality, and only a low percentage of malformated spermatozoa reached the upper parts of the capillary. Analyzing the differentiated morphology pattern in this biological model, em offered a barrier effect particularly for severely amorphous sperm heads and also for slightly amorphous forms but not for small, big, round heads, or tapering forms. The morphological index also was influenced significantly after em migration. From the neck abnormalities, a thickening of the midpiece was the main parameter that offered differences before and after passage through em. In this in vitro experiment, only ejaculates with very good quality according to WHO criteria were used. Because of the interrelationship of seminal parameters, an even more pronounced filtering effect of em might be hypothesized in semen samples with reduced quality. Previous reports, based on standard criteria of sperm morphology evaluation, are inconclusive with regard to the relevance of different types of sperm anomalies for penetration ability. Although some authors reported that tail defects would have a stronger impact on efficient mucus penetration than malformations of the sperm head (20), this could not be confirmed by others (5, 17, 18). Perry et al. (21) found the selective filtration of abnormal spermatozoa by the em in vitro equally efficient for different types of pathological configurated spermatozoa. The sperm head is in intimate contact with the mucus macromolecules during penetration and em of good quality may act as a sperm selector, but the reduction of abnormal sperm cells also may be a feature of the semen itself. Based on the sperm properties rather than on a specific active or passive filtering capacity of the em, is, for example, the relationship of poor morphology and reduced motility. It could be demonstrated that these important semen parameters were interrelated significantly, with r of when standard methods and with r of when strict criteria for morphology assessment were applied. In using videomicrography to simultaneously analyze the motions and morphology of individual human sperm, Katz et al. (22) demonstrated that morphological normal spermatozoa (determined with standard criteria) swam faster and straighter and exhibited higher beat fre- 622 Eggert-Kruse et al. Relationship of sperm morphology and sperm-mucus interaction Fertility and Sterility

12 quencies than did morphological abnormal sperm and suggested that the heads of the abnormal sperm experienced greater resistance from the em than did normal sperm heads. While the diminished motile vigor ofthe abnormal sperm is likely to contribute to their reduced ability of mucus migration, the biological characteristics of sperm that succeed in penetrating em have not been defined. Although slightly amorphous forms may still have their normal function, spermatozoa with severely abnormal head morphology may have concomitant abnormalities in the intracellular events involved in the production and transduction of energy within the cell, but only little is known about the cell biology of sperm energetics and of the nature of these specific lesions or their origins during spermatogenesis. The Tygerberg or Norfolk criteria are based partly on the appearance of sperm cells seen in the endocervical mucus (2). However, a significant reduction, but nevertheless a considerable rate of pathological forms was found after passage of capillaries filled with high quality em, suggesting that normal spermatozoa, as defined with the strict classification, are not those found exclusively within the em. On the other hand, not all samples with normal morphology offered adequate sperm-em penetration test outcome. A considerable overlap of morphological properties with regard to migration ability was found, indicating that both characteristics describe different sperm features. The mechanisms of sperm restriction from entering the cervical canal are not well understood. One determinant is immunological factors that may interfere with sperm penetration ability, for example, sperm antibodies of the IgA class in semen samples (19). Antisperm antibodies in em in unselected sub fertile populations are a rare cause that also is indicated by very similar results using patients' and donors' em in parallel on the same penetration meter for migration testing in the present study. Furthermore, the penetrability of each mucus sample was confirmed by cross-testing with donors' spermatozoa. Future studies are necessary to characterize biochemical and physicochemical factors in genital secretions of both partners determining the complex interaction of spermatozoa and em at the cervical level. The strong relationship of sperm morphology with em migration found in the present study suggests that morphological abnormal sperm in general are nonfunctional. It has not only been demonstrated that morphological abnormal cells swam with less vigor than do normal oval forms (22) but also that they were less likely to undergo motility hyperactivation under capacitation conditions (23), to show an acrosome reaction and to fuse with zona-free hamster eggs (24), to exhibit adequate binding to the human zona (25), and to fertilize human oocytes in vitro (1, 3). The end point of interest for patients is a subsequent pregnancy. The present study did not only reveal impaired functional capacity in spermmucus interaction tests in vivo and in vitro of spermatozoa with poor morphology but also confirmed their reduced fertilizing capacity under the natural conditions of conception (10). The morphological pattern was significantly different in semen samples of patients who did achieve subsequent pregnancy compared with those who did not. Thus sperm morphological qualities according to the strict classification are important not only for fertilization in vitro where the sperm cells are placed in the immediate vicinity of the oocyte but also for conception in vivo. The findings suggest that one function of the uterine cervix as a major barrier regulating sperm transport to the site of fertilization is being an effective filter that restricts the entrance of morphological abnormal spermatozoa. However, the specific selection capacity of em and sperm-mucus interaction is a much more complex phenomenon that warrants further investigation. REFERENCES 1. Kruger TF, Menkveld R, Stander FSH, Lombard CJ, van der Merve JP, van Zyl JA, et al. Sperm morphologic features as a prognostic factor in in vitro fertilization. Fertil Steril 1986;46: Menkveld R, Stander FSH, Kotze T JW, Kruger TF, van Zyl JA. The evaluation of morphological characteristics of human spermatozoa according to stricter criteria. Hum Reprod 1990;5: Kruger TF, Acosta AA, Simmons KF, Swanson RJ, Matta JF, Oehninger S. Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril1988;49: Bergman P. Sperm migration and cyclic changes in cervical mucus. Fertil Steril 1953;4: Kremer J. The in vitro spermatozoal penetration test in fertility investigations [thesis]. Groningen: University of Groningen, 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: 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: Eggert-Kruse W, Leinhos G, Gerhard I, Tilgen W, Runnebaum B. Prognostic value of in vitro sperm penetration into Vol. 63, No.3, March 1995 Eggert-Kruse et al. 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