Optimizing the sperm penetration assay with human follicular fluid

FERTILITY AND STERILITY

Vol. 53, No. 3, March 1990 Printed on acid·free paper in U.S.A.

Copyright <> 1990 The American Fertility Society

Optimizing the sperm penetration assay with human follicular fluid

R. Dale McClure, M.D.*t Raymond A. Tom, M.S.* Pramila V. Dandekar, M.S.:j: University of California School of Medicine, San Francisco, California

Major limitations of the conventional sperm penetration assay are the inability to assess several aspects of sperm function (zona binding and penetration) and the absence of human ovulatory products known to influence fertilization. We therefore modified the sperm penetration assay by the addition of human follicular fluid (FF) to induce the acrosome reaction in an attempt to improve the false-negative rate of the conventional technique. In 26 patients with negative results, results became positive in 20 with human FF and the acrosome reaction increased fourfold. In 19 different men, we compared the results of the conventional and modified assays with outcome of in vitro fertilization. The false-positive rate was the same, but the false-negative rate was reduced from 40% to 7% with the addition of human FF. Fertil Steril53:546, 1990

Although the sperm penetration assay (SPA) has been adopted by many reproductive laboratories as a clinical means of assessing the functional competence of human sperm, it has several shortcomings. The SPA assesses the fertilizing potential of human sperm, but does not assess other aspects of sperm function such as cervical mucus penetration, survival, migration to the site of fertilization, zona binding, and zona penetration. Additionallimitations of this test are the absence of female reproductive tract secretions and ovulatory products, which very likely play essential roles in the events leading to gamete fusion. 1 These inadequacies may result in false-negative or false-positive readings. Diagnostically, negative results in this assay are clinically more meaningful than posi-

Received June 21, 1989; revised and accepted November 2, 1989. * Department of Urology. t Reprint requests: R. Dale McClure, M.D., Department of Urology, University of California, San Francisco, California 94143-0738. :j: Department of Obstetrics and Gynecology and Reproductive Services.

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tive results. It is therefore very important to keep these false-negative findings to a minimum. Several comparative studies of the SPA carried out in parallel with in vitro fertilization (IVF) procedures suggest that, under the current assay conditions, a substantial number of false-negative results occur (10% to 20%).2-4 The heterogeneity of sperm populations, the individual variations in the rate of capacitation, the asynchrony of the acrosome reaction, and the lack of physiological in vivo stimulants are deficiencies of this bioassay that should also be addressed. This lack of correlation between SPA results and IVF outcome demonstrates that the SPA technique should be altered. One physiological approach to optimize the usefulness of this bioassay would be to use human ovulatory products known to influence fertilization. 5 •6 Preovulatory human follicular fluid (FF), present in the matrix of the cumulus cells surrounding the egg, is involved in capacitation and induction of the acrosome reaction. 7•8 In the present study, we assessed its effects on the acrosome reaction and penetration rates of sperm in men with abnormal results by the conventional SPA. We also compared results of this modified SPA with IVF outcome. Fertility and Sterility

MATERIALS AND METHODS Patients

Results of the SPA modified with human FF were compared with results of the conventional assay in 26 patients with a "negative" SPA from our infertility clinic (12 patients with 0% SPA; 14 patients with SPA 1% to 10%) and with IVF outcome in 19 different men whose wives were patients of our institution's IVF Program. An SPA score of :S;;10% in our laboratory is considered a negative result. Preparation of Sperm Suspensions

All semen samples were obtained by masturbation after 3 days of abstinence. Samples were assessed for initial concentration, motility, and sperm morphology. The swim-up procedure was used to separate the motile sperm for the SPA with sperm pelleted from two washings with modified Biggers, Whitten, and Whittingham (BWW) medium. BWW medium was modified as follows: NaCl was reduced to 84.07 mM, NaHC0 3 increased to 35.71 mM, 1. 71 mM calcium lactate replaced with 1.71 mM CaC12 -2H 2 0, and 6 mg/mL human serum albumin (fraction V, Sigma) added. At the end of the 2-hour swim-up period, motile sperm were recovered, the concentration was adjusted to between 15 and 20 X 106 motile sperm/mL and the sperm were incubated for an additional 18 hours for capacitation to take place. Gamete Interaction in Vitro

Human follicular fluid was provided by our In Vitro Fertilization/Embryo Transfer Program during laparoscopic or ultrasound-guided follicular aspiration from preovulatory follicles. The pooled human FF was centrifuged at 500 X g for 10 minutes to remove extraneous cellular materials and heat-deactivated at 56°C for 30 minutes before being frozen at -120oC in 200A samples. A 150A sample of human FF-treated or -untreated sperm suspension with a motile concentration of 5 to 10 X 106 /mL was placed under 5% C0 2/95% air, 37°C, equilibrated Silicon fluid (Dow Corning Corp., Midland, MI) in a plastic Petri dish (10 X 30 mm) and 30 to 40 zona-free hamster eggs were introduced into this suspension. The SPA was performed by methods previously described. 9 •10 The eggs were considered fertilized when decondensed sperm heads with associated sperm tails Vol. 53, No.3, March 1990

were visualized with phase-contrast microscopy (400X). The number of decondensed sperm heads/ number of fertilized eggs was recorded as the fertility index (FI). Induction and Detection of the Acrosome Reaction

For induction of the acrosome reaction, suspensions of sperm that had undergone the 18-hour incubation for capacitation were divided into two samples (350A.) centrifuged for 5 minutes at 500 X g, and resuspended in 0.4 mL modified BWW (untreated) or 0.4 mL of human FF diluted 1:4 with modified BWW.8 These two suspensions were incubated for 0.5 hours at 37oC followed by the addition of 3 mL of modified BWW and centrifugation. This procedure was repeated twice (for a total of 3 times). One hundred cells were assessed for motility, and the concentration was adjusted to between 5 to 10 X 106 motile sperm. Equal concentrations of motile sperm per milliliter were used in the paired comparison between treated and untreated samples. After the SPA, 100A. of sperm suspension (after removal of eggs for evaluation from the 150A droplet) was mixed with 6 mL of prewarmed phosphatebuffered saline (PBS), pH 7.4. The sperm were pelleted at 500 X g for 5 minutes; the addition of 50A. of95% (v/v) ETOH for45 minutes at4oC rendered the sperm membrane permeable. The acrosome status and morphology of 100 sperm for each paired suspension were assessed by fluoresceinated pisum sativum agglutinin mounted in a medium consisting of 1.5% (wt/v) 1,4 diazobicyclo (2.2.2) octane (Sigma Chemical Co., St. Louis, MO) in a 9:1 ratio of glycerol:PBS with an adjusted final pH of 8.6. 11 Human sperm may not lose acrosomal content completely upon demise. Sperm considered nonviable, with vital staining and the hypo-osmotic swelling test/ 2 still retain sufficient glycoconjugates in the acrosome region to react with fluoresceinated pisum sativum agglutinin labeling. This population of sperm had essentially background levels of the spontaneous acrosome reaction (3% to 6%). HumaniVF

The preparation of sperm for human IVF was similar to that for the SPA. Hams F -10 medium (GIBCO, Grand Island, NY), supplemented with 7% heat-deactivated maternal serum, was used to McClure et al.

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Table 1

Effect of the Addition of Human FF to Samples Previously Negative on the SPA a SPA results Negative

Positive

Acrosome reaction

32.3 ± 8.2 (3) 35.2 ± 6.4 b (20)

6±4 (26) 26 ±lOb (26)

%

NohumanFF HumanFF

4.7 ± 3.4 (23) 3.3 ± 1.8b (6)

%

a Values are mean ± SEM; values in parentheses are no. of samples.

wash the sperm twice, with centrifugation for 10 minutes at 300 X g. At the end of a 5- to 6-hour incubation, motile sperm in the overlying supernatant were recovered and 50,000 motile sperm/egg were used to inseminate oocytes. All reagents used were of analytical grade or of a quality acceptable for human in vitro fertilization. Human oocytes were scored 17 to 20 hours after insemination for fertilization as evidenced by visualization of the male and female pronuclei in the ooplasm. Pregnancy Outcome In Vivo

In six men who had fathered children within the previous 12 months but whose results on the conventional SPA were repeatedly negative over a 2month period, the SPA was repeated with the addition of human FF. The percentage of penetration and the acrosome status were examined. Fisher's Exact Test (2-tail) and Logistic Regression Analysis were used to evaluate the relationship of IVF outcome with semen parameters, the acrosome reaction, and results of the SPA with and without human FF. RESULTS

In 26 patients with negative results on the SPA penetration), results of retesting were positive in 3 when human FF was not added to the sperm suspension and positive in 20 when human FF was added (Table 1). With human FF, the acrosome reaction increased approximately fourfold, but neither the number of eggs penetrated nor the FI matched this in degree. Of the 26 samples, 3 showed normal semen parameters, 17 had reduced motility (one-half of these had abnormal morphology, one-third reduced concentration), and 6 had reduced concentration and motility and abnormal morphology. The ability of human FF to induce the acrosome (~10%

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b

1 ± 0.6 (26) 2.56 ± 1.1 b (26)

P < 0.05 (paired t-test between treatments).

reaction and increase the percentage of penetration of the SPA was demonstrably repeatable. In eight SPAs, performed over 3 months in a donor with normal semen parameters, human FF consistently gave significantly improved penetration results. Without human FF the mean percentage penetration was 40% (range 30% to 56%), the mean percent acrosome reaction was 6% (range 4% to 8%) and the mean FI was 1.16 (range 1 to 1.3). With the addition of human FF the mean percent penetration increased to 75% (range 61% to 100% ), the mean acrosome reaction increased to 31% (range 27% to 37%), and the mean FI increased to 2 (range 1 to 4). The percent penetration, acrosome reaction and fertility index were significantly different with the addition of human FF (P = 0.001, P = 0.000, andP = 0.009, respectively). In the 19 husbands of IVF patients, semen samples were evaluated by the SPA with and without human FF 1 week before IVF. Semen parameters were normal in 10 men; reduced motility was found in 2 and abnormal morphology in 7. Two or more oocytes were recovered in all IVF cycles. The routine SPA, without human FF, yielded false-negative results in 6; with human FF, the false-negative incidence lessened to 1 (Fig. 1). The addition of human FF to the 19 sperm samples confirmed the pattern described earlier: i.e., an increase in penetration, the acrosome reaction, and the FI of the sperm population, regardless of morphology. Six fertile men (husbands of women who were either pregnant or had just delivered) with normal conventional semen parameters had had negative results on two separate SPAs. Their mean percent penetration was 1.5 (range 0 to 6) and their mean acrosome reaction was 4% (range 2% to 8%). With the addition of human FF the mean percent penetration increased to 43% (range 18% to 100%) and the acrosome reaction increased to a mean of 22% (range 18% to 33%). Without human FF, the SPA would have indicated a negative pregnancy outFertility and Sterility

SPAinoHFF)

+ +

6

9

RDSitivity = 60 'JCo specificity = 75 "'

JVF

3

1

SPA(HFF)

+ +

1

14

IVF

sensitivity = 93.3 " specificity = 75 " 3

1

Figure 1. Relationship between the SPA with and without HFF and IVF outcome in 19 husbands of IVF patients.

come. With the addition of human FF there was a 100% correlation with in vivo results. Statistical analysis revealed only one correlation sufficient for prediction of IVF outcome: for human FF-SPA versus IVF-outcome, logistic regression analysis yielded P = 0.0159 and the Fisher Exact Test P = 0.016. All other parameters bore no sufficient correlation (P > 0.2). DISCUSSION

In the SPA, the events before and after gamete membrane fusion are similar to those observed in homologous fertilization. 9·10 The SPA therefore should be capable of detecting fertilization-related abnormalities and as such has been used by many reproductive laboratories to differentiate between potentially fertile and infertile men. Initial reports of the predictive value of SPA results vis a vis results of IVF were favorable. 3·13 '14 Unfortunately, more recent studies, particularly in couples with male-factor infertility, have shown a poorer correlation.2'15 Under experimental conditions, the "spontaneous" acrosome reaction rate is low. Aitken and coworkers have used the calcium ionophore A23187 to compensate for this lack. 16·17 This ionophore, which synchronizes the induction of the acrosome reaction and thus obviates individualized incubation periods, lowered this group's false-negative Vol. 53, No.3, March 1990

rate from 60% to 4.3% in samples screened before IVF. 17 Unfortunately, with this technique the biological competence of sperm to undergo capacitation and the acrosome reaction is not addressed. The storage of spermatozoa in a Tes-Tris egg yolk buffer at 4 oc similarly circumvents the problems created by individual differences in capacitation rates. 18 The low-temperature storage technique achieves the same synchrony as does the calcium ionophore. When the spermatozoa are cooled, the calcium levels in the cytoplasm rise to induce membrane fusion upon reheating to 37°C. This system has a better predictive value than the routine SPA with IVF programs. 19 Although both these techniques increase the sensitivity of the SPA, they are not physiological and therefore may not be relevant to the events that occur during IVF. Discussions of the physiological site and initiators of the acrosome reaction have focused on two egg investments, the cumulus oophorus and the zona pellucida,5 which, in humans, have been shown to stimulate the acrosome reaction in vitro. Fertilized sperm must pass through the extracellular matrix of the cumulus that encloses the recently ovulated oocyte. 20 During this passage, they are bathed in molecules that initiate the acrosome reaction and which either are secreted by the cumulus or are entrapped by the cumulus from the antral FF. Preovulatory ovarian FF has been shown to increase the acrosome reaction rate in vitro in capacitated human and nonhuman sperm.8·20 The acrosome-inducing activity of human FF was found to be in the 50,000 molecular weight fraction on a Sephadex G-75 column, and it appears to be secreted by both the cumulus and granulosa cells obtained from human preovulatory follicles. 21 •22 Thomas and Meizel 23 have demonstrated that human FF can cause the requisite influx of calcium (Ca++), and Yudin et al. 24 concluded that its addition to human sperm initiates a physiologically normal acrosome reaction. One of the deficiencies of the routine SPA may be the lack of a stimulus for the acrosome reaction in vivo. As demonstrated in Table 1, human FF increases the acrosome reaction rate and improves the percentage of penetration of the SPA. In six men, SPA results remained negative after human FF, although the acrosome reaction increased, and their sperm dysfunction appears to be related to other factors. The predominately abnormal morphology of the acrosome-reacted sperm from these six men suggests that this modified assay can serve as a gross indicator of concealed defect(s). Human McClure et al.

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FF appears to trigger capacitated sperm to undergo the acrosome reaction independent of morphology; however, it is more likely that a positive SPA will result if the majority of the sperm also have normal morphology. The "acid test" for evaluating the accuracy of the SPA is the comparison with results of IVF. With the addition of human FF, the specificity and predictive value of positive results remained unchanged, but the sensitivity increased from 60% to 93.3% (Fig. 1). The false-positive rate (25%) is to be expected and may be related to the shortcomings of the assay, e.g. the absence of female reproductive tract secretions and zona pellucida. However, the clinically significant false-negative rate has been reduced from 40% to 7%. With the routine SPA, many couples may have been discouraged from participating in an IVF program. In 1988, Yee and Cummings6 showed that negative results on SPA could be converted to positive after exposure of sperm to human FF and theorized that, by more realistically approximating IVF conditions, one could eliminate a majority of the falsenegative readings. Our data confirm that preovulatory human FF has optimized the SPA. It is not clear whether human FF promotes capacitation or is involved in the induction and/or completion of the acrosome reaction. This approach appears to be more physiological than others used to induce these events.

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