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Clinical significance of crossed in vitro sperm-cervical mucus penetration test in infertility investigation
·FERTILITY AND STERILITY
Vol. 52, No.6, December 1989 Printed on acid-free paper in U.S.A.
Copyright C> 1989 The American Fertility Society
Clinical significance of crossed in vitro sperm-cervical mucus penetration test in infertility investigation
Waltraud Eggert-Kruse, M.D. *t:j: Ingrid Gerhard, M.D. * Wolfgang Tilgen, M.D. § Benno Runnebaum, M.D. * University of Heidelberg, Heidelberg, Federal Republic of Germany
To evaluate the clinical significance of in vivo and in vitro testing of sperm ability to penetrate cervical mucus (CM), postcoital testing (PCT) and in vitro sperm-cervical mucus penetration testing were compared in a prospective study. Both in vivo and in vitro tests were standardized and performed after an oral course of estrogen therapy. Crossed in vitro sperm-cervical mucus penetration test, evaluated in 277 couples with CM ofpatients' wives and additionally with CM and semen of fertile donors, revealed that the male factor contributed to a significantly higher extent to deficient sperm-mucus interaction than the cervical factor. The overall pregnancy rate after 6 months was 23% (64/277). Whereas the outcome of PCT did not significantly predict subsequent fertility (PCT good pregnancy rate 24%/ PCT poor 20% ), significant differences were found for the sperm-cervical mucus penetration test with CM of patients' wives (pregnancy rate, 30.5% versus 8.5%) and for in vitro testing with donors' CM, but not for the mucus penetration test with donors' spermatozoa. Routine sperm analysis did not prove to be of prognostic value for a subsequent pregnancy. The results suggest that the in vitro sperm-cervical mucus penetration test is a good parameter of sperm function and, in particular, when performed as a cross-matching penetrability test, a valuable adjunct to PCT with regard to fertility prognosis. Fertil SteriI52:1032, 1989
For over a century, postcoital testing (PCT) has been considered valuable in infertility examination. Although the usefulness as an easy screening test remains unquestioned, the clinical significance of PCT for fertility prognosis is controversia1. 1,2 Sperm-mucus interaction is influenced by a multiReceived April 13, 1989; revised and accepted June 28, 1989.
* Division of Gynecological Endocrinology, Women's Hospital Infertility Unit. t Reprint requests: Waltraud Eggert-Kruse, M.D., Division of Gynecological Endocrinology, Women's Hospital Infertility Unit, University of Heidelberg, VoflstraJ3e 9, 6900 Heidelberg, Federal Republic of Germany. :/: Present address: Howard and Georgeanna Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia. § Department of Dermatology.
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plicity of different properties of semen and cervical mucus. Most important are hormonal,3 immuno10gical,4,5 and microbia16 ,7 factors, which might also influence each other. Furthermore, psychosexual problems may playa role in case of in vivo testing. The cervix is a major barrier regulating sperm transport to the site of fertilization, and interaction of male and female genital secretions can easily be examined in this area. In vitro sperm -cervical mucus penetration testing (SCMPT) is better to standardize, has a higher reproducibility, and allows observation of sperm-mucus interaction over a longer time period than PCT. It has been shown to be of prognostic value for subsequent fertility in a prospective study.8 In vitro testing is also advantageous in that it can be performed as a crossmatching penetrability test with material of fertile
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Fertility and Sterility
donors and thus offers additional information about possible immunological causes of infertility. The performance of cervical mucus (CM) penetration testing to evaluate sperm functional capacity is much easier and less time consuming than examination of sperm -egg interaction, for example, by means of the hamster-oocyte penetration test (HOP) or the recently introduced hemizona assay (HZA).9 The aim ofthe present investigation was to compare the clinical significance of in vivo and crossed in vitro testing of sperm-mucus penetration with regard to subsequent pregnancy in couples with long-standing infertility in a prospective study. MATERIALS AND METHODS
Couples with a minimum duration of infertility of 1 year and without symptoms of genital tract infection were randomly selected over a period of 2 years. Female patients were first checked for tubal patency by hysterosalpingography and/or laparoscopy. They were excluded when there was a severe tubal factor (both sides closed or one side closed and severe adhesions at the other side) or abnormalities of the uterus as a cause of infertility, so that the remaining 277 women offered at least one patent tube and a normal uterine configuration. Follicular growth and hormonal disorders were carefully examined by basal body temperature (BBT), ultrasound (US), endometrial biopsy, multiple determinations of gonadotropins, prolactin, estradiol, progesterone, testosterone, and the entire pattern of the adrenal gland hormones, including adrenocorticotropin hormone (ACTH) tests and thyroid function tests, and were treated accordingly. The male patients presented the entire variety of andrological findings excluding azoospermia. Andrological medication was administered to males whenever necessary after repeated sperm analyses. The median duration of infertility of the 277 couples studied was 5 years (range 1 to 18 years). The mean age of the male patients was 32.4 years; of their female partners, 29.7 years. For the routine sperm analysis,lO semen was obtained in the hospital after 5 days' sexual abstinence and was examined directly after liquefaction. Postcoital Testing
The PCT was timed according to BBT chart, US, and self observation of CM after a recomVol. 52, No.6, December 1989
mended period of 5 days of sexual abstinence. The cervix was exposed with an unlubricated speculum, cleaned with a large cotton swab of excess debris, and the endocervical mucus was carefully aspirated by means of a special device. l l The condition of the CM was classified according to Insler et al. 12 The cervical index ranged from 0 (poorest) to 12 (excellent). Endocervical mucus samples were placed on glass slides, covered, and examined under a light microscope using first the low-power field (LPF) magnification (100X) and then the high-power field (HPF) magnification (400X). The number of spermatozoa with forward progression in CM was counted; a mean of 20 fields was taken. When there were no spermatozoa found in preovulatory CM, a vaginal pool sample was additionally examined to ensure that semen had actually been deposited in the vagina; otherwise, testing was repeated in the next cycle. The routinely performed grading of PCT outcome has been described in detail elsewhere. 6 Briefly, PCT was regarded as good when at least two spermatozoa of highly progressive motility/HPF were counted in preovulatory CM 8 to 12 hours after intercourse; otherwise, it was classified as poor. For further evaluation, the PCT results were subdivided in four groups: PCT negative (no spermatozoa found in CM/LPF, but in vaginal secretions), PCT inadequate «2 motile sperm/HPF), PCT moderate (2 to 6 motile sperm/HPF), and PCT excellent (>7 motile sperm/HPF). A poor result was only accepted as valid if the mucus was in good condition and after the test had been repeated in at least one other cycle. An initial negative result was ignored if later positive. Positive results were accepted whatever the state of the CM. Samples contaminated with blood were discharged. In patients with anovulatory cycles, very late ovulation, or marked luteal insufficiency, PCT was performed after oral treatment with estrogens (80 /J-g ethinylestradiol per day, at least 7 days before PCT). The same procedure was applied in case of reduced mucus quality on the day of PCT. Performance of the In Vitro Sperm-Cervical Mucus Penetration Test
For in vitro testing of sperm penetration, the penetration meter according to Kremer13 was used. An oral course of estrogen therapy was compulsory before the sperm-CM penetration test was performed between the 9th and 14th days of the women's menstrual cycle.
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The CM of patients was obtained as described above and immediately sucked up into capillary tubes that were put into the reservoirs of the penetration meter filled with semen of the patients' partners directly after liquefaction. Care was taken to prevent air bubbles from disrupting the mucus column. The capillaries were fixed with modeling clay at one end with one drop of mucus protruding at the other end. The penetration meter was incubated in a moist chamber at 37°C before penetrated spermatozoa were read. Aliquots of semen, obtained at the hospital, were taken for the routine sperm analysis. lo For better comparison of results, the variables of sperm penetration into CM (penetration density, penetration distance, and motility grade [from immotile to highly propulsive motility]) were graded from 0 to 3 and summarized after 6 hours' observation time in a cumulative sperm penetration score, which has been reported in detail elsewhere.8 A score of;;:::6 was considered an adequate outcome of in vitro testing (sperm-CM penetration test good) and compared with poor results (score 0 to 5). In vitro testing was performed in parallel on the same penetration meter as a cross-matching penetrability test with the patient's semen sample and CM of a fertile woman (also hormonally standardized as described above) and a semen specimen of a fertile donor and patient's CM.
Table 1 Results of Semen Analysis With Respect to Subsequent Pregnancy4 Parameter No. of couples (%) Sperm volume (mL) pH Sperm count (X106/mL) Propulsive motility (%) Morphology (% normal forms) Viability (%)
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Eggert-Kruse et al.
Notpregnant b
64 (23%)
213 (77%)
3.3 7.5 45 40
(1 to 7) (7.2 to 7.7) (8 to 92) (1 to 70)
3.2 7.5 41 40
(1 to 7.5) (6.7 to 8) (1 to 234) (1 to 70)
60 65
(47 to 69) (50 to SO)
60 65
(32 to 70) (38 to SO)
4 The P values were not significant. b Median (range).
4 hours of coincubation was considered to be adequate. Statistical Analysis
The pregnancy rate was determined after 6 months. Results of PCT and in vitro sperm-cervical mucus penetration tests with patients' spermatozoa and CM and material of fertile donors were correlated with standard parameters of sperm and mucus quality and analyzed for their clinical significance for subsequent fertility. For statistical evaluation, Wilcoxon's rank sum, Kruskal-Wallis, Friedman, and x2 tests were used; data were processed using the Statistical Analysis System (SAS).
Performance of the Hemizona Assay
The ability of spermatozoa to bind to zona pellucida of human oocytes was evaluated in a smaller number of semen samples (n = 16) by means of the HZA, which has been described in detail by Burkman et a1. 9 Salt-stored, nonliving human eggs were used. After preparation, they were cut into halves with a micromanipulation system (Narishige, Tokyo, Japan). Semen aliquots were washed twice with Ham's F-10, supplemented with 7.5% heat-inactivated human fetal cord serum, and after a "swim-up" separation, used for coincubation with the zona pellucida halves (under oil, 37°C, 5% CO2). The number of tightly bound spermatozoa to the outer surface of the zona halves was counted after 4 hours. The results of the HZA were compared in a parallel test setting with the outcome of in vitro sperm-cervical mucus penetration testing of the same ejaculate. For the penetration meter test, donors' CM of excellent quality (cervical index 12) was used. A zona binding of >20 spermatozoa after
Pregnant b
RESULTS
The overall pregnancy rate after 6 months was 23% (64/277). The results of sperm analysis were compared in couples who later achieved a pregnancy and those who did not and are shown in Table 1. No significant differences were found for ejaculate volume, pH, fructose concentration, viability, number of round cells, sperm count, percent of progressive motility, percent of normal forms (World Health Organization [WHO] criterialO ) (Wilcoxon's rank sum test), and after classifying of results at prospectively determined cutoffs (40 X 106/mL and 40% for sperm count and motility, respectively [x 2 analysis] ). However, a clear differentiation was obtained by the comparison of the penetration density in cervical mucus of patients' wives with a median of 100 spermatozoa in the later pregnant group compared with a median of 40 in the nonpregnant group (P < 0.01) (Wilcoxon's test). An excellent motility grade of spermatozoa in
In vivo and in vitro sperm penetration
Fertility and Sterility
Table 2
Influence of Semen Quality on Results of Postcoital Tests
Definition of PCTa.b (No. of spermatozoa) No. of couples (%) Sperm volume <1.5mL ~4.5mL
Median (range) Sperm count/mL <20 X 106 ~40 X 106 Median (range) Progressive motility <20% ~40%
Median (range) Morphology (% normal g ) ~60%
Median (range)
Negative (O/LPF') 67
(26.7)
Inadequate «2/HPF d ) 51
(20.3)
90
(35.9)
Positive (~7/HPF)
45
p
(17.1)
6 9 15 22 2.9 (1 to 8.7)
2 4 14 28 3.5 (1.2 to 7.5)
3 3 19 21 3.2 (1 to 6.4)
1 2 12 27 3.6 (1 to 9.5)
12 28 33
31 42 (0 to 234)
10 23 38
20 46 (6 to 186)
4 63 46
4 69 (13 to 137)
0 37 52
0 84 (29 to 161)
20 19 25
35 33 (1 to 60)
5 19 30
10 38 (1 to 60)
3 51 40
3 56 (10 to 70)
0 43 50
0 98 (25 to 70)
19 53
34 (34 to 69)
21 58
43 (47 to 69)
55 60
63 (32 to 70)
38 64
95 (59 to 69)
a PCT, postcoital test. b X 2 analysis. e LPF, low-power field. d HPF, high-power field.
eNS, not significant. ' Kruskal-Wallis test. g % normal forms (WHO criteria).
wives' eM after 6 hours' incubation was significantly more frequent in the fertile than in the infertile group of patients (44% versus 24%; P < 0.005 [X 2 analysis]). Results of Postcoital Testing
The peT could be evaluated in 253 couples. The results are presented in Table 2. The outcome of peT was poor in 46.6% and good in 53.4% of couples. With regard to subsequent fertility, no significant difference was found between both groups. Pregnancy rate was 19.5% (23/118) when peT was poor and 24.4% (33/135) when peT was good. For further analysis, both groups were subdivided so that four groups resulted: peT negative, inadequate, fair, and excellent. The peT groups did not differ in duration of infertility; female and male ages; distribution of tubal, uterine, ovarian, and other hormonal infertility factors; cervical index; and number of patients with oral treatment with estrogens before peT. Statistical analysis revealed that peT results were strongly influenced by the quality of semen with significant differences for sperm count (P < 0.001), progressive motility (P < 0.001), percent normal forms (P < 0.001), and viability (P < 0.001). No significant differences were found for the fructose concentration, pH, number of round cells, and the ejaculate volume, although semen specimens < 1.5 mL were more frequent in the negative than in the other groups. Positive peT results were Vol. 52, No.6, December 1989
Moderate (2 to 6/HPF)
found in none of the patients with marked asthenozoospermia «20% progressive motility) or oligozoospermia «20 million/mL). Although the pregnancy rate was lower in the negative peT group (15%, 10/67) than in the other groups (peT inadequate pregnancy rate, 26% [13/ 51]; peT moderate, 24% [22/90]; and peT excellent,24% [11/45], respectively), this difference did not achieve statistical significance (P > 0.05). When the latter three groups were tested against the negative group, no significant difference was found either. Results of the Cross-Matching Penetrability Test
When the above-described hormonal approach was applied to standardize the mucus quality before the in vitro sperm penetration test, the male factor contributed to a significantly higher extent to deficient sperm-mucus interaction than the cervical factor. Sperm density, penetration distance, and motility grade after 6 hours were significantly higher when in vitro testing was performed with donors' spermatozoa than with donors' cervical mucus (P < 0.001) (Friedman test). Analysis of the whole study population revealed that the state of the mucus (Insler index) did not significantly influence the outcome of the penetration meter test with wives' eM, as well as with donors' eM, when the above-described hormonal approach was applied to standardize the mucus qual-
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Table 3 Influence of the Mucus Quality on the Results of Crossed In Vitro Sperm-Cervical Mucus Penetration Tests Results of SCMPT4
With wives' cervical mucus Cervical index d ~10 ~8
~6
With donors' spermatozoa Cervical index ~10
~8 ~6
With donors' cervical mucus
Poor b
Goode
p
n
%
n
%
104
38
173
62
80 91 95 32
77
88 91 12
130 156 164 238
76 91 96 88
NS' NS NS
20 24 26
63 75 81
188 220 230
80 93 97
<0.03 <0.001 <0.001
88
32
187
68
4 SCMPT, sperm-cervical mucus penetration test. b SCMPT score, 0 to 5. e SCMPT score, ~6. d According to Insler et al. 11 eNS, not significant.
ity before in vitro testing (see Table 3). In individual couples, however, penetration of spermatozoa in eM of patients' partners and donors differed considerably, in particular, when the cervical index was markedly reduced. Significant differences were found for the results of sperm penetration testing with donors' spermatozoa and eM of patients' wives (P < 0.001) (x2 test). Poor in vitro sperm penetration was less frequent when donors' eM, instead of patients' eM, was used (32% versus 38%) and was found in only 12% when spermatozoa of fertile donors were allowed to penetrate the eM of the infertile women. Results of the Hemizona Assay (HZA)
When the ability of spermatozoa to penetrate hormonally standardized human eM and to bind to prepared zona pellucida halves of human oocytes was compared in a pilot study, a good correlation was found. The specimens of proven fertile men (n = 8) showed excellent in vitro sperm-eM penetration, as well as adequate zona binding (ZB). These ejaculates reached the maximum possible spermeM penetration score with a penetration distance of >45 mm, a sperm density of> 100, and a highly progressive motility in eM after 6 hours' incubation. In vitro sperm penetration was very good in, additionally, 3 of the other samples. The zona binding of these 11 samples ranged from 23 to 105, with a median of 59. Three specimens offered slightly reduced mucus penetration ability (score 7 to 8) and a ZB of 42 to 63; 1 specimen with a borderline 1036
score of 6 showed a ZB of 22; and 1 sample with a poor penetration meter outcome, a ZB of <20 spermatozoa (inadequate). The testing of semen samples with very poor in vitro sperm-eM penetration was limited by the number of motile spermatozoa necessary for the HZA. Comparison of In Vivo and In Vitro Sperm Penetration Tests With Regard to Subsequent Fertility
Pregnancy rate of the 277 couples differed significantly according to the results of in vitro sperm penetration tests with eM of patients' wives (P < 0.001), as well as with eM of fertile donors (P < 0.01). No significant differences of subsequent fertility were seen when a poor and good outcome of in vitro testing using donors' spermatozoa and patients' eM was compared. Table 4 shows the results of in vivo (peT) and in vitro (sperm-eM penetration test) sperm penetration and subsequent fertility. This analysis includes only couples with a standardized hormonal treatment with oral estrogens in female patients for both tests. Postcoital testing did not discriminate between patients who later achieved a pregnancy and those who did not. However, significantly more patients achieved a pregnancy within 6 months when results of the in vitro sperm-mucus penetration test (with wives' eM) were good (30.5% versus 8.5% when in vitro testing was poor [P < 0.001]). Significant differences of pregnancy rate were also found when donors' eM was used (28.3% versus 9.8% [P < 0.01]), but not for the in vitro spermeM penetration test with donors' spermatozoa. Further analyses of the couples who achieved a subsequent pregnancy in spite of poor results of an in vitro sperm-mucus penetration test revealed a poor cervical index (3 to 5) in five of the patients. The partners had excellent results of sperm analysis and in vitro penetration of donors' eM was good. Prognostic Value of Sperm Penetration Tests With Respect to Sperm Analysis
Table 5 shows the results of in vitro sperm-eM penetration tests and peT and subsequent pregnancy after selection of patients with 40 X 106/mL as a cutoff point for sperm count and 40% as a cutoff for sperm progressive motility. With regard to peT, pregnancy rate was twofold higher in case of good peT in the group of patients with reduced sperm count (25% versus 12.5% [P
Eggert-Kruse et aI. In vivo and in vitro sperm penetration
Fertility and Sterility
Table 4 Comparison ofIn Vitro (SCMPTa) and In Vivo (PCT b ) Sperm Penetration Into Hormonally Standardized Cervical Mucus C With Regard to Subsequent Fertility Results of sperm penetration tests Goode
SCMPT with wives' cervical mucus (WCM) SCMPT with donor's cervical mucus (DCM) SCMPT with WCM and donors' spermatozoa PCT .
p
Total
n
%
n
%
n
%
6/71
8.5 9.8 22.2 20.9
39/128 39/138 37/169 27/113
30.5 28.3 21.9 24.1
45/199 45/199 43/196 45/199
22.6 22.6 21.6 22.6
6/61 6/27 18/86
SCMPT, sperm-cervical mucus penetration test. PCT, postcoital test. , Treatment with ethinylestradiol, 80 ,.,.g/d, for 7 d before testing.
d SCMPT score, 0 to 5. e SCMPT score, ;;,:6. 1 NS, not significant.
a
b
< 0.05]). Again, significantly more pregnancies were achieved when in vitro sperm penetration with eM of patients' wives offered a good result (33% versus 7% in the group of patients with a reduced sperm count [P < 0.005]) and, in particular, in the subgroup of patients with reduced progressive motility (in vitro penetration testing good pregnancy rate, 39%; in vitro penetration testing poor pregnancy rate, 7% [P < 0.001]). In patients with normal sperm counts and/or motility, pregnancy rate was also markedly higher when in vitro sperm-eM penetration testing (with
<0.001 <0.01 NS' NS
wives' eM as well as with donors' eM) showed a good result. No such differences were found with regard to the results of peT. DISCUSSION
The results of the present investigation clearly indicate that sperm-mucus penetration tests are a useful adjunct to semen analysis. Particularly, in vitro sperm penetration meter testing, using hormonally standardized eM of patients' wives, was a valuable means of evaluating the functional capac-
Table 5 Results of In Vivo and In Vitro Sperm-Cervical Mucus Penetration Tests With Regard to Sperm Analysis and Subsequent Pregnancy Pregnancies per group (sperm count < 40 X 106/ m L) Results of penetration tests Poor a
Good b
Pregnancies per group (sperm count;;,: 40 X 106/m L) Total
Poor a
Good b
no. (%)
SCMPT' With wives' CM d With donors' CM With donors' sperm PCT"
no. (%)
3/46 (7)e
10/30 (33)e
5/39 (13) 2/10 (20) 6/48 (13)
8/37 (22) 10/65 (15) 7/28 (25)
13/ 76 (17)1
3/24 (13) 1/22 (5) 4/16 (25) 11/36 (31)
Pregnancies per group (progressive motility < 40% ) Results of penetration tests Poor
Good
Vol. 52, No.6, December 1989
(29) (31) (26) (24)
31/120 (26) 1
Total
Poor
Good
Total
no. (%)
3/43 (7)h 3/38 (8)e
13/33 (39)h 13/38 (34)e
3/10 (30) 11/47 (23)
13/66 (20) 5/29 (17)
SCMPT score, 0 to 5. SCMPT score, ;;,:6. 'SCMPT, sperm-cervical mucus penetration test. d CM, cervical mucus. a
b
28/96 30/98 26/102 20/84
Pregnancies per group (progressive motility;;,: 40%)
no. (%)
SCMPTc With wives' CM With donors' CM With donors' sperm PCT
Total
16/ 76 (21) 1
3/19 3/17 3/15 6/29
(16) (18) (20) (21)
25/93 25/95 23/94 22/83
(27) (26) (25) (27)
28/112 (25) 1
e p < 0.005. not significant. "PCT, postcoital test.
1 NS,
h P< 0.001.
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In vivo and in vitro sperm penetration
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ity of spermatozoa with prognostic value for subsequent fertility. It could be demonstrated that a better selection of couples with and without a subsequent pregnancy was achieved by in vitro testing of sperm penetration than with the PCT. Several studies have shown that motility characteristics of spermatozoa and the velocity are of the utmost importance for male fertilityY In the present investigation, consistent with the results of others,15 parameters of routine semen analysis (sperm count, motility, volume, morphology, viability, pH, and number of round cells) did not predict future fertility. However, if semen morphology is evaluated with other methods than the standard WHO criteria, more information might be obtained. Semen samples with a high percentage of morphologically abnormal spermatozoa (P pattern) had significantly reduced chances to fertilize in an in vitro fertilization (lVF) program. 16 The postcoital test has been introduced > 100 years ago and is widely accepted. Infertilityevaluation is frequently begun with PCT intended to elucidate sperm deficiencies or mucus hostility. However, although generally known and easy to perform, there is no common agreement on the standardization of the PCT .1,2 It is well known that the hormonal influence is of paramount importance. 17-19 In other studies on PCT, as prerequisites for the test were often inadequate, this may apply particularly to the definition of ovulation on which the outcome so critically depends. Because the mucus may be receptive to spermatozoa only when it is in its best state and for only 2 to 3 days of the cycle in the periovulatory phase,3 it is essential to base the negative results on tests with good mucus and in more than one cycle. The usual term "cervical infertility" is often inappropriate, except after cervical surgery, anatomical abnormalities, or an inherent inability to produce mucus, factors that are very uncommon. Inadequate mucus quality, more often than cervical mucus hostility, reflects severe hormonal disorders. Furthermore, other investigators included patients with clomiphene citrate (CC) treatment. There is evidence that the administration of CC changes the mucus quality significantly by its antiestrogenic action. High levels of androgens may also exert a negative effect on the properties of CM. Without hormonal control of the mucus quality, the information obtained by PCT might reflect a better ovulatory function in the later pregnant woman and, therefore, better chances of pregnancy. 1038
Eggert-Kruse et aI.
The stimulating effect of estrogens on mucus production by the epithelial cells of the cervix results in a quantitatively measured and clinically observed increase of cervical mucus. 3,l1 In the present investigation, to our knowledge for the first time, the attempt was made to standardize the mucus quality for both in vivo and in vitro testing of sperm penetration by obligatory oral treatment with ethinylestradiol for at least 7 days before CM was taken. With this approach, PCT did not prove to be a consistent predictor of pregnancy. In couples with a poor and good PCT, pregnancy rate after 6 months was nearly the same (20% versus 24%). Only the subgroup of patients with a negative PCT had a slightly, but not significantly, reduced chance of fertility. Fifteen percent of couples with negative test results achieved a pregnancy. Compared with in vitro testing of sperm penetration, PCT provides information about sexual function. The psychological pressure associated with an exactly timed PCT resulting in temporal male impotence is a common cause of initial failure to obtain positive results. The oral administration of estrogens gives couples more days for PCT and might lower the pressure of exactly timed intercourse. However, even after repetition under these criteria, PCT was not able to clearly predict fertility. The data suggest that in all couples not exhibiting an excellent outcome of PCT, the in vitro sperm-cervical mucus penetration test is a useful adjunct, regardless of results of sperm analysis. In vitro capillary tube tests to evaluate spermmucus interaction have been the subject of interest for >20 years. 13 Sperm penetration meter testing provides objective, quantitative, and reproducible data about sperm functional capacity. In vitro sperm-CM penetration allows cross-testing with donors' spermatozoa and mucus to clearly define the cervical or the male factor responsible for deficient local compatibility without any sophisticated methods. Serum antisperm antibodies do not reflect properly the immunological situation in secretions of the genital tract in most instances, and the clinical significance of circulating antisperm antibodies is questionable. 4,5,17 Detection of antisperm antibodies in serum samples of male and/or female patients during infertility investigation did neither impair the results of PCT nor the fertility prognosis in a prospective study. 20 The difference of 6% between good in vitro sperm penetration with use of patients' and do-
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Fertility and Sterility
nors' cervical mucus might reflect local antisperm antibodies as cause of deficient sperm-mucus interaction. This parameter of mucus quality cannot be detected by classification of the CM with different indices.lO,n For example, the CM of two patients was not penetrated by spermatozoa of seven different donors, although the mucus offered a cervical index of 12 according to Insler12 on clinical examination. The pH of the CM may also influence sperm penetration and surviva1. 21 Another explanation for poor mucus quality might be the noncompliance of patients concerning the short-term estrogen medication. A study to further clarify these points is in progress. It has been hypothesized that microbial colonization might influence the ability of spermatozoa to penetrate CM. Cervix and vagina are colonized by a large variety of microorganisms, some of which are potentially pathogenic. However, in asymptomatic couples, this did not markedly influence sperm-mucus interaction in vivo and in vitro.6 The effects of antibiotic treatment according to antimicrobial susceptibility tests after an extensive microbial screening on the results of cross-matching penetrability testing of spermatozoa have been reported in detail previously. 7 The present study demonstrates that impaired sperm-mucus interaction was significantly more often related to the male than the cervical factor (P < 0.001) when the hormonal influence was controlled. Impaired sperm function might express male autoimmunity or inherent defects of spermatozoa not detected by routine sperm analysis. Antisperm antibodies were found nearly 25 times more often in semen than in CM.17 Poor sperm penetration in both partners' and donors' CM reflects reduced sperm fertilizing capacity, which is shown by the poor pregnancy rate in case of inadequate in vitro sperm penetration. In vitro sperm-cervical mucus penetration testing includes not only the evaluation ofthe penetration distance of spermatozoa, but also the number of spermatozoa being able to penetrate the individual mucus sample, the quality of motility (motility pattern), and the duration of forward progressive motility. Each of these factors, necessary to reach the upper female genital tract on physiological conditions, contributes to the sperm-CM penetration test (SCMPT) result. 8 Significantly more couples with a good, compared with a poor, in vitro sperm penetration test achieved a pregnancy (P < 0.001). However, six couples conceived in spite of a negative in vitro sperm penetration test. When looking Vol. 52, No.6, December 1989
at the quality of CM, all of these women had a reduced cervical index. Sperm quality was good, but spermatozoa were not able at the time of examination to penetrate the mucus barrier. This could be detected by donors' CM testing. Therefore, it is recommended that the sperm-CM penetration test should be repeated in case of poor mucus quality, in spite of oral administration of ethinylestradiol. On the other hand, a normal in vitro sperm-CM penetration test may not indicate adequate sperm transport into the upper genital tract and may not accurately reflect sperm capacity to ascend and to penetrate the egg. 22 Interference of antisperm antibodies with reproductive processes may occur by impairment of sperm migration not only through the cervix, but also in the uterine cavity, the fallopian tubes, and by a blocking adherence of spermatozoa to the surface of the zona pellucida of the oocyte. As a new test in infertility investigation, the HZA has recently been introduced to evaluate sperm ability to bind to the zona pellucida of human donor eggs as a parameter of sperm function. The HZA has been shown to significantly correlate with the IVF outcome.9 Compared with sperm-mucus testing, the preparation of eggs and semen is much more time consuming and needs a highly trained technician and expensive equipment, whereas the SCMPT is a simple, inexpensive laboratory test. Although the limited number of samples, compared in HZA and in vitro sperm-CM penetration test in a pilot study, does not allow drawing definite conclusions, the results might suggest a good correlation of sperm-mucus and sperm-egg interaction. Future efforts are necessary to clearly define the role of both tests in infertility investigation and the group of patients, who will benefit from a combined testing procedure. Maybe the cervical mucus acts as a filter, allowing only the functionally best spermatozoa to penetrate. It has been reported that the population of spermatozoa found in the internal os of the cervix showed a significantly higher proportion of normal cells than those in the external OS.23 Furthermore, the immunological properties of CM might reflect the immunological status of secretions of the upper genital tract. Antibodies secreted in the cervix could possibly indicate those in the endometrium, which might impair implantation. A vigorous, straightforward progression might enable spermatozoa not only to traverse viscous media, but also to pass through the uteral tubal junction and to
Eggert-Kruse et al.
In vivo and in vitro sperm penetration
1039
penetrate the corona radiata and to fertilize the egg. Hull et al. 24 have shown that spermatozoa unable to penetrate preovulatory CM are generally also unable to fertilize human oocytes. Schats and Aitken25 demonstrated a correlation between impaired sperm -mucus penetration in vitro and fertilization of hamster eggs. In conclusion, the results suggest that the intrinsic motility, crucial in enabling the spermatozoa to penetrate cervical mucus, is a good parameter of sperm function. In vitro sperm-cervical mucus penetration is of prognostic value for subsequent fertility, and in vitro SCMPT provides, in particular, when performed as a cross-test, valuable information not obtained by PCT. Therefore, it can be recommended as an essential part of the investigation of the infertile couple. Acknowledgments. The technical assistance of Ms. Deborah Johnson, Jones' Institute, Norfolk, VA, in performing the HZA, and the expert statistical advice of Peter Roebruck, Ph.D., Institute of Medical Biometry and Statistics, University of Heidelberg, are gratefully acknowledged.
REFERENCES 1. Overstreet JW: Evaluation of sperm-cervical mucus interaction. Fertil Steril45:324, 1986 2. Hull MGR, Savage PE, Bromham DR: Prognostic value of the postcoital test: prospective study based on time-specific conception rates. Br J Obstet Gynaeco189:299, 1982 3. Moghissi KS: The function of the cervix in fertility. Fertil Steril 23:295, 1972 4. Schumacher GFB: Immunology of spermatozoa and cervical mucus. Hum Reprod (OxO 3:289, 1988 5. Alexander NJ, Anderson DJ: Immunology of semen. Fertil Steril47:192,1987 6. Eggert-Kruse W, Gerhard I, Hofmann H, Runnebaum B, Petzoldt D: Influence of microbial colonization on spermmucus interaction in vivo and in vitro. Hum Reprod (OxO 2:301,1987 7. Eggert-Kruse W, Hofmann H, Gerhard I, Bilke A, Runnebaum B, Petzoldt D: Effects of antimicrobial therapy on sperm-mucus interaction. Hum Reprod (OxO 3:861, 1988 8. 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 Steril51:317,1989 9. Burkman LJ, Coddington CC, Franken DR, Kruger TF,
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10.
11. 12.
13. 14.
15.
16.
17.
18.
19. 20.
21. 22.
23.
24.
25.
Eggert-Kruse et al. In vivo and in vitro sperm penetration
Rosenwaks Z, Hodgen GD: The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril49:688, 1988 World Health Organization: WHO Laboratory Manual for the Examination of Human Semen and Semen-Cervical Mucus Interaction. Cambridge, The Press Syndicate ofthe University of Cambridge, 1987 Jondet M, Scholler R: A simple device for collecting human cervical mucus. Fertil Steril 34:72, 1980 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 10:223, 1972 Kremer J: A simple sperm penetration test. Int J Fertil10: 209,1965 Hinting A, Comhaire F, Schoonjans F: Capacity of objectively assessed sperm motility characteristics in differentiating between semen of fertile and subfertile men. Fertil Steril 50:635, 1988 Polansky FF, Lamb EJ: Do the results of semen analysis predict future fertility? A survival analysis study. Fertil SteriI49:1059,1988 Kruger TF, Acosta AA, Simmons KF, Swanson RS, Matta JF, Oehninger S: Predictive value of abnormal sperm morphology in in vitro fertilization. Fertil Steril49:112, 1988 Kremer J, Jager S: Sperm-cervical mucus interaction, in particular in the presence of antispermatozoal antibodies. Hum Reprod (OxO 3:69, 1988 Insler V, Bernstein D, Glezerman M, Misgav N: Correlation of seminal fluid analysis with mucus-penetrating ability of spermatozoa. Fertil Steril 32:316, 1979 Moghissi KS: Postcoital test: physiological basis, technique, and interpretation. Fertil Steril27:117, 1976 Eggert-Kruse W, Christmann M, Gerhard I, Pohl S, Klinga K, Runnebaum B: Circulating antisperm-antibodies and fertility prognosis-a prospective study. Hum Reprod (OxO 4:513, 1989 Zavos PM, Cohen MR: The pH of cervical mucus and the postcoital test. Fertil Steril 34:234, 1980 Stone SC: Peritoneal recovery of sperm in patients with infertility associated with inadequate cervical mucus. Fertil Steri140:802, 1983 Fredricsson B, Bjork G: Morphology of postcoital spermatozoa in the cervical secretion and its clinical significance. Fertil SteriI28:841, 1977 Hull MGR, McLeod FN, Joyce DN, Ray BD, McDermott A: Human in-vitro fertilization, in-vivo sperm penetration of cervical mucus, and unexplained infertility. Lancet 1: 245,1984 Schats R, Aitken RJ: The role of cervical mucus-semen interaction in infertility of unknown aetiology. Br J Obstet Gynaecol 91:371, 1984
Fertility and Sterility
Vol. 52, No.6, December 1989 Printed on acid-free paper in U.S.A.
Copyright C> 1989 The American Fertility Society
Clinical significance of crossed in vitro sperm-cervical mucus penetration test in infertility investigation
Waltraud Eggert-Kruse, M.D. *t:j: Ingrid Gerhard, M.D. * Wolfgang Tilgen, M.D. § Benno Runnebaum, M.D. * University of Heidelberg, Heidelberg, Federal Republic of Germany
To evaluate the clinical significance of in vivo and in vitro testing of sperm ability to penetrate cervical mucus (CM), postcoital testing (PCT) and in vitro sperm-cervical mucus penetration testing were compared in a prospective study. Both in vivo and in vitro tests were standardized and performed after an oral course of estrogen therapy. Crossed in vitro sperm-cervical mucus penetration test, evaluated in 277 couples with CM ofpatients' wives and additionally with CM and semen of fertile donors, revealed that the male factor contributed to a significantly higher extent to deficient sperm-mucus interaction than the cervical factor. The overall pregnancy rate after 6 months was 23% (64/277). Whereas the outcome of PCT did not significantly predict subsequent fertility (PCT good pregnancy rate 24%/ PCT poor 20% ), significant differences were found for the sperm-cervical mucus penetration test with CM of patients' wives (pregnancy rate, 30.5% versus 8.5%) and for in vitro testing with donors' CM, but not for the mucus penetration test with donors' spermatozoa. Routine sperm analysis did not prove to be of prognostic value for a subsequent pregnancy. The results suggest that the in vitro sperm-cervical mucus penetration test is a good parameter of sperm function and, in particular, when performed as a cross-matching penetrability test, a valuable adjunct to PCT with regard to fertility prognosis. Fertil SteriI52:1032, 1989
For over a century, postcoital testing (PCT) has been considered valuable in infertility examination. Although the usefulness as an easy screening test remains unquestioned, the clinical significance of PCT for fertility prognosis is controversia1. 1,2 Sperm-mucus interaction is influenced by a multiReceived April 13, 1989; revised and accepted June 28, 1989.
* Division of Gynecological Endocrinology, Women's Hospital Infertility Unit. t Reprint requests: Waltraud Eggert-Kruse, M.D., Division of Gynecological Endocrinology, Women's Hospital Infertility Unit, University of Heidelberg, VoflstraJ3e 9, 6900 Heidelberg, Federal Republic of Germany. :/: Present address: Howard and Georgeanna Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, Virginia. § Department of Dermatology.
1032
plicity of different properties of semen and cervical mucus. Most important are hormonal,3 immuno10gical,4,5 and microbia16 ,7 factors, which might also influence each other. Furthermore, psychosexual problems may playa role in case of in vivo testing. The cervix is a major barrier regulating sperm transport to the site of fertilization, and interaction of male and female genital secretions can easily be examined in this area. In vitro sperm -cervical mucus penetration testing (SCMPT) is better to standardize, has a higher reproducibility, and allows observation of sperm-mucus interaction over a longer time period than PCT. It has been shown to be of prognostic value for subsequent fertility in a prospective study.8 In vitro testing is also advantageous in that it can be performed as a crossmatching penetrability test with material of fertile
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Fertility and Sterility
donors and thus offers additional information about possible immunological causes of infertility. The performance of cervical mucus (CM) penetration testing to evaluate sperm functional capacity is much easier and less time consuming than examination of sperm -egg interaction, for example, by means of the hamster-oocyte penetration test (HOP) or the recently introduced hemizona assay (HZA).9 The aim ofthe present investigation was to compare the clinical significance of in vivo and crossed in vitro testing of sperm-mucus penetration with regard to subsequent pregnancy in couples with long-standing infertility in a prospective study. MATERIALS AND METHODS
Couples with a minimum duration of infertility of 1 year and without symptoms of genital tract infection were randomly selected over a period of 2 years. Female patients were first checked for tubal patency by hysterosalpingography and/or laparoscopy. They were excluded when there was a severe tubal factor (both sides closed or one side closed and severe adhesions at the other side) or abnormalities of the uterus as a cause of infertility, so that the remaining 277 women offered at least one patent tube and a normal uterine configuration. Follicular growth and hormonal disorders were carefully examined by basal body temperature (BBT), ultrasound (US), endometrial biopsy, multiple determinations of gonadotropins, prolactin, estradiol, progesterone, testosterone, and the entire pattern of the adrenal gland hormones, including adrenocorticotropin hormone (ACTH) tests and thyroid function tests, and were treated accordingly. The male patients presented the entire variety of andrological findings excluding azoospermia. Andrological medication was administered to males whenever necessary after repeated sperm analyses. The median duration of infertility of the 277 couples studied was 5 years (range 1 to 18 years). The mean age of the male patients was 32.4 years; of their female partners, 29.7 years. For the routine sperm analysis,lO semen was obtained in the hospital after 5 days' sexual abstinence and was examined directly after liquefaction. Postcoital Testing
The PCT was timed according to BBT chart, US, and self observation of CM after a recomVol. 52, No.6, December 1989
mended period of 5 days of sexual abstinence. The cervix was exposed with an unlubricated speculum, cleaned with a large cotton swab of excess debris, and the endocervical mucus was carefully aspirated by means of a special device. l l The condition of the CM was classified according to Insler et al. 12 The cervical index ranged from 0 (poorest) to 12 (excellent). Endocervical mucus samples were placed on glass slides, covered, and examined under a light microscope using first the low-power field (LPF) magnification (100X) and then the high-power field (HPF) magnification (400X). The number of spermatozoa with forward progression in CM was counted; a mean of 20 fields was taken. When there were no spermatozoa found in preovulatory CM, a vaginal pool sample was additionally examined to ensure that semen had actually been deposited in the vagina; otherwise, testing was repeated in the next cycle. The routinely performed grading of PCT outcome has been described in detail elsewhere. 6 Briefly, PCT was regarded as good when at least two spermatozoa of highly progressive motility/HPF were counted in preovulatory CM 8 to 12 hours after intercourse; otherwise, it was classified as poor. For further evaluation, the PCT results were subdivided in four groups: PCT negative (no spermatozoa found in CM/LPF, but in vaginal secretions), PCT inadequate «2 motile sperm/HPF), PCT moderate (2 to 6 motile sperm/HPF), and PCT excellent (>7 motile sperm/HPF). A poor result was only accepted as valid if the mucus was in good condition and after the test had been repeated in at least one other cycle. An initial negative result was ignored if later positive. Positive results were accepted whatever the state of the CM. Samples contaminated with blood were discharged. In patients with anovulatory cycles, very late ovulation, or marked luteal insufficiency, PCT was performed after oral treatment with estrogens (80 /J-g ethinylestradiol per day, at least 7 days before PCT). The same procedure was applied in case of reduced mucus quality on the day of PCT. Performance of the In Vitro Sperm-Cervical Mucus Penetration Test
For in vitro testing of sperm penetration, the penetration meter according to Kremer13 was used. An oral course of estrogen therapy was compulsory before the sperm-CM penetration test was performed between the 9th and 14th days of the women's menstrual cycle.
Eggert-Kruse et a1.
In vivo and in vitro sperm penetration
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The CM of patients was obtained as described above and immediately sucked up into capillary tubes that were put into the reservoirs of the penetration meter filled with semen of the patients' partners directly after liquefaction. Care was taken to prevent air bubbles from disrupting the mucus column. The capillaries were fixed with modeling clay at one end with one drop of mucus protruding at the other end. The penetration meter was incubated in a moist chamber at 37°C before penetrated spermatozoa were read. Aliquots of semen, obtained at the hospital, were taken for the routine sperm analysis. lo For better comparison of results, the variables of sperm penetration into CM (penetration density, penetration distance, and motility grade [from immotile to highly propulsive motility]) were graded from 0 to 3 and summarized after 6 hours' observation time in a cumulative sperm penetration score, which has been reported in detail elsewhere.8 A score of;;:::6 was considered an adequate outcome of in vitro testing (sperm-CM penetration test good) and compared with poor results (score 0 to 5). In vitro testing was performed in parallel on the same penetration meter as a cross-matching penetrability test with the patient's semen sample and CM of a fertile woman (also hormonally standardized as described above) and a semen specimen of a fertile donor and patient's CM.
Table 1 Results of Semen Analysis With Respect to Subsequent Pregnancy4 Parameter No. of couples (%) Sperm volume (mL) pH Sperm count (X106/mL) Propulsive motility (%) Morphology (% normal forms) Viability (%)
1034
Eggert-Kruse et al.
Notpregnant b
64 (23%)
213 (77%)
3.3 7.5 45 40
(1 to 7) (7.2 to 7.7) (8 to 92) (1 to 70)
3.2 7.5 41 40
(1 to 7.5) (6.7 to 8) (1 to 234) (1 to 70)
60 65
(47 to 69) (50 to SO)
60 65
(32 to 70) (38 to SO)
4 The P values were not significant. b Median (range).
4 hours of coincubation was considered to be adequate. Statistical Analysis
The pregnancy rate was determined after 6 months. Results of PCT and in vitro sperm-cervical mucus penetration tests with patients' spermatozoa and CM and material of fertile donors were correlated with standard parameters of sperm and mucus quality and analyzed for their clinical significance for subsequent fertility. For statistical evaluation, Wilcoxon's rank sum, Kruskal-Wallis, Friedman, and x2 tests were used; data were processed using the Statistical Analysis System (SAS).
Performance of the Hemizona Assay
The ability of spermatozoa to bind to zona pellucida of human oocytes was evaluated in a smaller number of semen samples (n = 16) by means of the HZA, which has been described in detail by Burkman et a1. 9 Salt-stored, nonliving human eggs were used. After preparation, they were cut into halves with a micromanipulation system (Narishige, Tokyo, Japan). Semen aliquots were washed twice with Ham's F-10, supplemented with 7.5% heat-inactivated human fetal cord serum, and after a "swim-up" separation, used for coincubation with the zona pellucida halves (under oil, 37°C, 5% CO2). The number of tightly bound spermatozoa to the outer surface of the zona halves was counted after 4 hours. The results of the HZA were compared in a parallel test setting with the outcome of in vitro sperm-cervical mucus penetration testing of the same ejaculate. For the penetration meter test, donors' CM of excellent quality (cervical index 12) was used. A zona binding of >20 spermatozoa after
Pregnant b
RESULTS
The overall pregnancy rate after 6 months was 23% (64/277). The results of sperm analysis were compared in couples who later achieved a pregnancy and those who did not and are shown in Table 1. No significant differences were found for ejaculate volume, pH, fructose concentration, viability, number of round cells, sperm count, percent of progressive motility, percent of normal forms (World Health Organization [WHO] criterialO ) (Wilcoxon's rank sum test), and after classifying of results at prospectively determined cutoffs (40 X 106/mL and 40% for sperm count and motility, respectively [x 2 analysis] ). However, a clear differentiation was obtained by the comparison of the penetration density in cervical mucus of patients' wives with a median of 100 spermatozoa in the later pregnant group compared with a median of 40 in the nonpregnant group (P < 0.01) (Wilcoxon's test). An excellent motility grade of spermatozoa in
In vivo and in vitro sperm penetration
Fertility and Sterility
Table 2
Influence of Semen Quality on Results of Postcoital Tests
Definition of PCTa.b (No. of spermatozoa) No. of couples (%) Sperm volume <1.5mL ~4.5mL
Median (range) Sperm count/mL <20 X 106 ~40 X 106 Median (range) Progressive motility <20% ~40%
Median (range) Morphology (% normal g ) ~60%
Median (range)
Negative (O/LPF') 67
(26.7)
Inadequate «2/HPF d ) 51
(20.3)
90
(35.9)
Positive (~7/HPF)
45
p
(17.1)
6 9 15 22 2.9 (1 to 8.7)
2 4 14 28 3.5 (1.2 to 7.5)
3 3 19 21 3.2 (1 to 6.4)
1 2 12 27 3.6 (1 to 9.5)
12 28 33
31 42 (0 to 234)
10 23 38
20 46 (6 to 186)
4 63 46
4 69 (13 to 137)
0 37 52
0 84 (29 to 161)
20 19 25
35 33 (1 to 60)
5 19 30
10 38 (1 to 60)
3 51 40
3 56 (10 to 70)
0 43 50
0 98 (25 to 70)
19 53
34 (34 to 69)
21 58
43 (47 to 69)
55 60
63 (32 to 70)
38 64
95 (59 to 69)
a PCT, postcoital test. b X 2 analysis. e LPF, low-power field. d HPF, high-power field.
eNS, not significant. ' Kruskal-Wallis test. g % normal forms (WHO criteria).
wives' eM after 6 hours' incubation was significantly more frequent in the fertile than in the infertile group of patients (44% versus 24%; P < 0.005 [X 2 analysis]). Results of Postcoital Testing
The peT could be evaluated in 253 couples. The results are presented in Table 2. The outcome of peT was poor in 46.6% and good in 53.4% of couples. With regard to subsequent fertility, no significant difference was found between both groups. Pregnancy rate was 19.5% (23/118) when peT was poor and 24.4% (33/135) when peT was good. For further analysis, both groups were subdivided so that four groups resulted: peT negative, inadequate, fair, and excellent. The peT groups did not differ in duration of infertility; female and male ages; distribution of tubal, uterine, ovarian, and other hormonal infertility factors; cervical index; and number of patients with oral treatment with estrogens before peT. Statistical analysis revealed that peT results were strongly influenced by the quality of semen with significant differences for sperm count (P < 0.001), progressive motility (P < 0.001), percent normal forms (P < 0.001), and viability (P < 0.001). No significant differences were found for the fructose concentration, pH, number of round cells, and the ejaculate volume, although semen specimens < 1.5 mL were more frequent in the negative than in the other groups. Positive peT results were Vol. 52, No.6, December 1989
Moderate (2 to 6/HPF)
found in none of the patients with marked asthenozoospermia «20% progressive motility) or oligozoospermia «20 million/mL). Although the pregnancy rate was lower in the negative peT group (15%, 10/67) than in the other groups (peT inadequate pregnancy rate, 26% [13/ 51]; peT moderate, 24% [22/90]; and peT excellent,24% [11/45], respectively), this difference did not achieve statistical significance (P > 0.05). When the latter three groups were tested against the negative group, no significant difference was found either. Results of the Cross-Matching Penetrability Test
When the above-described hormonal approach was applied to standardize the mucus quality before the in vitro sperm penetration test, the male factor contributed to a significantly higher extent to deficient sperm-mucus interaction than the cervical factor. Sperm density, penetration distance, and motility grade after 6 hours were significantly higher when in vitro testing was performed with donors' spermatozoa than with donors' cervical mucus (P < 0.001) (Friedman test). Analysis of the whole study population revealed that the state of the mucus (Insler index) did not significantly influence the outcome of the penetration meter test with wives' eM, as well as with donors' eM, when the above-described hormonal approach was applied to standardize the mucus qual-
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In vivo and in vitro sperm penetration
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Table 3 Influence of the Mucus Quality on the Results of Crossed In Vitro Sperm-Cervical Mucus Penetration Tests Results of SCMPT4
With wives' cervical mucus Cervical index d ~10 ~8
~6
With donors' spermatozoa Cervical index ~10
~8 ~6
With donors' cervical mucus
Poor b
Goode
p
n
%
n
%
104
38
173
62
80 91 95 32
77
88 91 12
130 156 164 238
76 91 96 88
NS' NS NS
20 24 26
63 75 81
188 220 230
80 93 97
<0.03 <0.001 <0.001
88
32
187
68
4 SCMPT, sperm-cervical mucus penetration test. b SCMPT score, 0 to 5. e SCMPT score, ~6. d According to Insler et al. 11 eNS, not significant.
ity before in vitro testing (see Table 3). In individual couples, however, penetration of spermatozoa in eM of patients' partners and donors differed considerably, in particular, when the cervical index was markedly reduced. Significant differences were found for the results of sperm penetration testing with donors' spermatozoa and eM of patients' wives (P < 0.001) (x2 test). Poor in vitro sperm penetration was less frequent when donors' eM, instead of patients' eM, was used (32% versus 38%) and was found in only 12% when spermatozoa of fertile donors were allowed to penetrate the eM of the infertile women. Results of the Hemizona Assay (HZA)
When the ability of spermatozoa to penetrate hormonally standardized human eM and to bind to prepared zona pellucida halves of human oocytes was compared in a pilot study, a good correlation was found. The specimens of proven fertile men (n = 8) showed excellent in vitro sperm-eM penetration, as well as adequate zona binding (ZB). These ejaculates reached the maximum possible spermeM penetration score with a penetration distance of >45 mm, a sperm density of> 100, and a highly progressive motility in eM after 6 hours' incubation. In vitro sperm penetration was very good in, additionally, 3 of the other samples. The zona binding of these 11 samples ranged from 23 to 105, with a median of 59. Three specimens offered slightly reduced mucus penetration ability (score 7 to 8) and a ZB of 42 to 63; 1 specimen with a borderline 1036
score of 6 showed a ZB of 22; and 1 sample with a poor penetration meter outcome, a ZB of <20 spermatozoa (inadequate). The testing of semen samples with very poor in vitro sperm-eM penetration was limited by the number of motile spermatozoa necessary for the HZA. Comparison of In Vivo and In Vitro Sperm Penetration Tests With Regard to Subsequent Fertility
Pregnancy rate of the 277 couples differed significantly according to the results of in vitro sperm penetration tests with eM of patients' wives (P < 0.001), as well as with eM of fertile donors (P < 0.01). No significant differences of subsequent fertility were seen when a poor and good outcome of in vitro testing using donors' spermatozoa and patients' eM was compared. Table 4 shows the results of in vivo (peT) and in vitro (sperm-eM penetration test) sperm penetration and subsequent fertility. This analysis includes only couples with a standardized hormonal treatment with oral estrogens in female patients for both tests. Postcoital testing did not discriminate between patients who later achieved a pregnancy and those who did not. However, significantly more patients achieved a pregnancy within 6 months when results of the in vitro sperm-mucus penetration test (with wives' eM) were good (30.5% versus 8.5% when in vitro testing was poor [P < 0.001]). Significant differences of pregnancy rate were also found when donors' eM was used (28.3% versus 9.8% [P < 0.01]), but not for the in vitro spermeM penetration test with donors' spermatozoa. Further analyses of the couples who achieved a subsequent pregnancy in spite of poor results of an in vitro sperm-mucus penetration test revealed a poor cervical index (3 to 5) in five of the patients. The partners had excellent results of sperm analysis and in vitro penetration of donors' eM was good. Prognostic Value of Sperm Penetration Tests With Respect to Sperm Analysis
Table 5 shows the results of in vitro sperm-eM penetration tests and peT and subsequent pregnancy after selection of patients with 40 X 106/mL as a cutoff point for sperm count and 40% as a cutoff for sperm progressive motility. With regard to peT, pregnancy rate was twofold higher in case of good peT in the group of patients with reduced sperm count (25% versus 12.5% [P
Eggert-Kruse et aI. In vivo and in vitro sperm penetration
Fertility and Sterility
Table 4 Comparison ofIn Vitro (SCMPTa) and In Vivo (PCT b ) Sperm Penetration Into Hormonally Standardized Cervical Mucus C With Regard to Subsequent Fertility Results of sperm penetration tests Goode
SCMPT with wives' cervical mucus (WCM) SCMPT with donor's cervical mucus (DCM) SCMPT with WCM and donors' spermatozoa PCT .
p
Total
n
%
n
%
n
%
6/71
8.5 9.8 22.2 20.9
39/128 39/138 37/169 27/113
30.5 28.3 21.9 24.1
45/199 45/199 43/196 45/199
22.6 22.6 21.6 22.6
6/61 6/27 18/86
SCMPT, sperm-cervical mucus penetration test. PCT, postcoital test. , Treatment with ethinylestradiol, 80 ,.,.g/d, for 7 d before testing.
d SCMPT score, 0 to 5. e SCMPT score, ;;,:6. 1 NS, not significant.
a
b
< 0.05]). Again, significantly more pregnancies were achieved when in vitro sperm penetration with eM of patients' wives offered a good result (33% versus 7% in the group of patients with a reduced sperm count [P < 0.005]) and, in particular, in the subgroup of patients with reduced progressive motility (in vitro penetration testing good pregnancy rate, 39%; in vitro penetration testing poor pregnancy rate, 7% [P < 0.001]). In patients with normal sperm counts and/or motility, pregnancy rate was also markedly higher when in vitro sperm-eM penetration testing (with
<0.001 <0.01 NS' NS
wives' eM as well as with donors' eM) showed a good result. No such differences were found with regard to the results of peT. DISCUSSION
The results of the present investigation clearly indicate that sperm-mucus penetration tests are a useful adjunct to semen analysis. Particularly, in vitro sperm penetration meter testing, using hormonally standardized eM of patients' wives, was a valuable means of evaluating the functional capac-
Table 5 Results of In Vivo and In Vitro Sperm-Cervical Mucus Penetration Tests With Regard to Sperm Analysis and Subsequent Pregnancy Pregnancies per group (sperm count < 40 X 106/ m L) Results of penetration tests Poor a
Good b
Pregnancies per group (sperm count;;,: 40 X 106/m L) Total
Poor a
Good b
no. (%)
SCMPT' With wives' CM d With donors' CM With donors' sperm PCT"
no. (%)
3/46 (7)e
10/30 (33)e
5/39 (13) 2/10 (20) 6/48 (13)
8/37 (22) 10/65 (15) 7/28 (25)
13/ 76 (17)1
3/24 (13) 1/22 (5) 4/16 (25) 11/36 (31)
Pregnancies per group (progressive motility < 40% ) Results of penetration tests Poor
Good
Vol. 52, No.6, December 1989
(29) (31) (26) (24)
31/120 (26) 1
Total
Poor
Good
Total
no. (%)
3/43 (7)h 3/38 (8)e
13/33 (39)h 13/38 (34)e
3/10 (30) 11/47 (23)
13/66 (20) 5/29 (17)
SCMPT score, 0 to 5. SCMPT score, ;;,:6. 'SCMPT, sperm-cervical mucus penetration test. d CM, cervical mucus. a
b
28/96 30/98 26/102 20/84
Pregnancies per group (progressive motility;;,: 40%)
no. (%)
SCMPTc With wives' CM With donors' CM With donors' sperm PCT
Total
16/ 76 (21) 1
3/19 3/17 3/15 6/29
(16) (18) (20) (21)
25/93 25/95 23/94 22/83
(27) (26) (25) (27)
28/112 (25) 1
e p < 0.005. not significant. "PCT, postcoital test.
1 NS,
h P< 0.001.
Eggert-Kruse et al.
In vivo and in vitro sperm penetration
1037
ity of spermatozoa with prognostic value for subsequent fertility. It could be demonstrated that a better selection of couples with and without a subsequent pregnancy was achieved by in vitro testing of sperm penetration than with the PCT. Several studies have shown that motility characteristics of spermatozoa and the velocity are of the utmost importance for male fertilityY In the present investigation, consistent with the results of others,15 parameters of routine semen analysis (sperm count, motility, volume, morphology, viability, pH, and number of round cells) did not predict future fertility. However, if semen morphology is evaluated with other methods than the standard WHO criteria, more information might be obtained. Semen samples with a high percentage of morphologically abnormal spermatozoa (P pattern) had significantly reduced chances to fertilize in an in vitro fertilization (lVF) program. 16 The postcoital test has been introduced > 100 years ago and is widely accepted. Infertilityevaluation is frequently begun with PCT intended to elucidate sperm deficiencies or mucus hostility. However, although generally known and easy to perform, there is no common agreement on the standardization of the PCT .1,2 It is well known that the hormonal influence is of paramount importance. 17-19 In other studies on PCT, as prerequisites for the test were often inadequate, this may apply particularly to the definition of ovulation on which the outcome so critically depends. Because the mucus may be receptive to spermatozoa only when it is in its best state and for only 2 to 3 days of the cycle in the periovulatory phase,3 it is essential to base the negative results on tests with good mucus and in more than one cycle. The usual term "cervical infertility" is often inappropriate, except after cervical surgery, anatomical abnormalities, or an inherent inability to produce mucus, factors that are very uncommon. Inadequate mucus quality, more often than cervical mucus hostility, reflects severe hormonal disorders. Furthermore, other investigators included patients with clomiphene citrate (CC) treatment. There is evidence that the administration of CC changes the mucus quality significantly by its antiestrogenic action. High levels of androgens may also exert a negative effect on the properties of CM. Without hormonal control of the mucus quality, the information obtained by PCT might reflect a better ovulatory function in the later pregnant woman and, therefore, better chances of pregnancy. 1038
Eggert-Kruse et aI.
The stimulating effect of estrogens on mucus production by the epithelial cells of the cervix results in a quantitatively measured and clinically observed increase of cervical mucus. 3,l1 In the present investigation, to our knowledge for the first time, the attempt was made to standardize the mucus quality for both in vivo and in vitro testing of sperm penetration by obligatory oral treatment with ethinylestradiol for at least 7 days before CM was taken. With this approach, PCT did not prove to be a consistent predictor of pregnancy. In couples with a poor and good PCT, pregnancy rate after 6 months was nearly the same (20% versus 24%). Only the subgroup of patients with a negative PCT had a slightly, but not significantly, reduced chance of fertility. Fifteen percent of couples with negative test results achieved a pregnancy. Compared with in vitro testing of sperm penetration, PCT provides information about sexual function. The psychological pressure associated with an exactly timed PCT resulting in temporal male impotence is a common cause of initial failure to obtain positive results. The oral administration of estrogens gives couples more days for PCT and might lower the pressure of exactly timed intercourse. However, even after repetition under these criteria, PCT was not able to clearly predict fertility. The data suggest that in all couples not exhibiting an excellent outcome of PCT, the in vitro sperm-cervical mucus penetration test is a useful adjunct, regardless of results of sperm analysis. In vitro capillary tube tests to evaluate spermmucus interaction have been the subject of interest for >20 years. 13 Sperm penetration meter testing provides objective, quantitative, and reproducible data about sperm functional capacity. In vitro sperm-CM penetration allows cross-testing with donors' spermatozoa and mucus to clearly define the cervical or the male factor responsible for deficient local compatibility without any sophisticated methods. Serum antisperm antibodies do not reflect properly the immunological situation in secretions of the genital tract in most instances, and the clinical significance of circulating antisperm antibodies is questionable. 4,5,17 Detection of antisperm antibodies in serum samples of male and/or female patients during infertility investigation did neither impair the results of PCT nor the fertility prognosis in a prospective study. 20 The difference of 6% between good in vitro sperm penetration with use of patients' and do-
In vivo and in vitro sperm penetration
Fertility and Sterility
nors' cervical mucus might reflect local antisperm antibodies as cause of deficient sperm-mucus interaction. This parameter of mucus quality cannot be detected by classification of the CM with different indices.lO,n For example, the CM of two patients was not penetrated by spermatozoa of seven different donors, although the mucus offered a cervical index of 12 according to Insler12 on clinical examination. The pH of the CM may also influence sperm penetration and surviva1. 21 Another explanation for poor mucus quality might be the noncompliance of patients concerning the short-term estrogen medication. A study to further clarify these points is in progress. It has been hypothesized that microbial colonization might influence the ability of spermatozoa to penetrate CM. Cervix and vagina are colonized by a large variety of microorganisms, some of which are potentially pathogenic. However, in asymptomatic couples, this did not markedly influence sperm-mucus interaction in vivo and in vitro.6 The effects of antibiotic treatment according to antimicrobial susceptibility tests after an extensive microbial screening on the results of cross-matching penetrability testing of spermatozoa have been reported in detail previously. 7 The present study demonstrates that impaired sperm-mucus interaction was significantly more often related to the male than the cervical factor (P < 0.001) when the hormonal influence was controlled. Impaired sperm function might express male autoimmunity or inherent defects of spermatozoa not detected by routine sperm analysis. Antisperm antibodies were found nearly 25 times more often in semen than in CM.17 Poor sperm penetration in both partners' and donors' CM reflects reduced sperm fertilizing capacity, which is shown by the poor pregnancy rate in case of inadequate in vitro sperm penetration. In vitro sperm-cervical mucus penetration testing includes not only the evaluation ofthe penetration distance of spermatozoa, but also the number of spermatozoa being able to penetrate the individual mucus sample, the quality of motility (motility pattern), and the duration of forward progressive motility. Each of these factors, necessary to reach the upper female genital tract on physiological conditions, contributes to the sperm-CM penetration test (SCMPT) result. 8 Significantly more couples with a good, compared with a poor, in vitro sperm penetration test achieved a pregnancy (P < 0.001). However, six couples conceived in spite of a negative in vitro sperm penetration test. When looking Vol. 52, No.6, December 1989
at the quality of CM, all of these women had a reduced cervical index. Sperm quality was good, but spermatozoa were not able at the time of examination to penetrate the mucus barrier. This could be detected by donors' CM testing. Therefore, it is recommended that the sperm-CM penetration test should be repeated in case of poor mucus quality, in spite of oral administration of ethinylestradiol. On the other hand, a normal in vitro sperm-CM penetration test may not indicate adequate sperm transport into the upper genital tract and may not accurately reflect sperm capacity to ascend and to penetrate the egg. 22 Interference of antisperm antibodies with reproductive processes may occur by impairment of sperm migration not only through the cervix, but also in the uterine cavity, the fallopian tubes, and by a blocking adherence of spermatozoa to the surface of the zona pellucida of the oocyte. As a new test in infertility investigation, the HZA has recently been introduced to evaluate sperm ability to bind to the zona pellucida of human donor eggs as a parameter of sperm function. The HZA has been shown to significantly correlate with the IVF outcome.9 Compared with sperm-mucus testing, the preparation of eggs and semen is much more time consuming and needs a highly trained technician and expensive equipment, whereas the SCMPT is a simple, inexpensive laboratory test. Although the limited number of samples, compared in HZA and in vitro sperm-CM penetration test in a pilot study, does not allow drawing definite conclusions, the results might suggest a good correlation of sperm-mucus and sperm-egg interaction. Future efforts are necessary to clearly define the role of both tests in infertility investigation and the group of patients, who will benefit from a combined testing procedure. Maybe the cervical mucus acts as a filter, allowing only the functionally best spermatozoa to penetrate. It has been reported that the population of spermatozoa found in the internal os of the cervix showed a significantly higher proportion of normal cells than those in the external OS.23 Furthermore, the immunological properties of CM might reflect the immunological status of secretions of the upper genital tract. Antibodies secreted in the cervix could possibly indicate those in the endometrium, which might impair implantation. A vigorous, straightforward progression might enable spermatozoa not only to traverse viscous media, but also to pass through the uteral tubal junction and to
Eggert-Kruse et al.
In vivo and in vitro sperm penetration
1039
penetrate the corona radiata and to fertilize the egg. Hull et al. 24 have shown that spermatozoa unable to penetrate preovulatory CM are generally also unable to fertilize human oocytes. Schats and Aitken25 demonstrated a correlation between impaired sperm -mucus penetration in vitro and fertilization of hamster eggs. In conclusion, the results suggest that the intrinsic motility, crucial in enabling the spermatozoa to penetrate cervical mucus, is a good parameter of sperm function. In vitro sperm-cervical mucus penetration is of prognostic value for subsequent fertility, and in vitro SCMPT provides, in particular, when performed as a cross-test, valuable information not obtained by PCT. Therefore, it can be recommended as an essential part of the investigation of the infertile couple. Acknowledgments. The technical assistance of Ms. Deborah Johnson, Jones' Institute, Norfolk, VA, in performing the HZA, and the expert statistical advice of Peter Roebruck, Ph.D., Institute of Medical Biometry and Statistics, University of Heidelberg, are gratefully acknowledged.
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