- Email: [email protected]
Correlation of human spermatozoa heparin binding with the zona-free hamster egg in vitro penetration assay
Correlation of human spermatozoa heparin binding with the zona-free hamster egg in vitro penetration assay Jaime M. Vasquez, MD,. Martin A. Winer, PhD,",b Roy L. Ax, PhD,b and William R. Boone, PhD' Madison, WISconsin The purpose of our research was to determine whether heparin-binding characteristics of human spermatozoa are related to fertilizing potential, as determined by the hamster egg in vitro penetration assay, Penetration rates were standardized (hamster egg in vitro penetration assay index) by comparison with semen from fertile controls in each bioassay. Saturation of heparin-binding domains was achieved in 100% of raw ejaculates (prewash), but in only 53% of "swim-up" (postwash) samples. The dissociation constants ranged from 0.31 to 48.75 nmol/106 cells, and binding site concentrations from 0.47 to 20.82 x 1017 binding sites/cell. Heparin-binding affinity was significantly greater in prewash compared with postwash samples (p < 0.01). In prewash samples the number of binding sites differed significantly between subjects having low and high penetration indeces (5.67 ± 1.05 vs 2.01 ± 0.34 x 1017 binding sites/cell, p < 0.05). In prewash samples, binding affinity for heparin significantly correlated with hamster egg in vitro penetration assay indeces (R2 = 0.142, P < 0.05). In contrast, the number of binding sites in prewash samples was negatively correlated with hamster egg in vitro penetration assay indeces (R2 = 0.201, P < 0.05). These data indicate that the heparin-binding assay may prove to be a rapid, sensitive, and inexpensive means of assessing fertilizing potential of human spermatozoa. (AM J OBSTET GVNECOL 1989;160:20-6.)
Key words: Spermatozoa, glycosaminoglycans, fertilization, hamster egg in vitro penetration assay, computer-assisted semen analysis Capacitation of spermatozoa and the acrosome reaction are prerequisites for mammalian fertilization. 1.2 The hamster egg in vitro penetration assay has been used in many clinical and research laboratories to assess the potential of human spermatozoa to undergo capacitation, acrosome reaction, oocyte membrane fusion, and pronuclear formation.' Penetration rates have been used as a predictor of fertility in males on the basis of a significant correlation between hamster egg in vitro penetration assay rates and in vitro fertilization of human oocytes.' However, normal penetration of zona-free hamster eggs is not necessarily an indication that human spermatozoa can penetrate the zona pellucida of human oocytes.! From the DIvISIOn of ReproductIVe Endocrinology and Infertility, Department of Obstetrics and Gynecology,' University of Wisconsin Medical School, and the Department of Dazry Science and Endocrinology-Reproductive Physiology Program,' University of Wisconsin. Supported by a General Research Support Grant to the Unzverslty of WISconsin Medical School from the NatIOnal Institutes of Health, Division of Research Faclhtles and Resources, (J. M. V.) and a University of Wisconsin Graduate School Grant (j. M. V.J. Presented at the Thirty-fifth Annual Meeting of the SOCIety for Gynecologzc InvestigatIOn, Baltimore, Maryland, March 17-20,
1988.
Reprznt requests: J. M. Vasquez, MD, Department of Obstetrzcs and Gynecology, UniveTSlty of WISconsin-Madison, 600 Highland Ave., Rm. H4/630 MadISon, WI 53792
esc,
20
Previous studies suggest that glycosaminoglycans have a role in capacitation and the acrosome reaction.' Heparin, a highly sulfated glycosaminoglycan, has been found to facilitate sperm capacitation and the acrosome reaction in vitro in several mammalian species." 7 Tritiated heparin has been shown to bind to spermatozoa saturably, and the binding affinity and susceptibility to glycosaminoglycan-induced capacitation are related to fertility in bulls. s In displacement assays of heparin, Scatchard transformations of the data have indicated that the principal binding affinity (dissociation constants = 10- 7 mol! L) is biologically significant and comparable with hormone-receptor-binding affinities. Thermodynamic calculations have suggested that tritiated heparian binding to spermatozoa is a specific and exothermic process that has all of the classic characteristics of a receptor-ligand interaction." Tritiated heparin binds to human spermatozoa, and the binding affinity and the concentration of binding sites correlate with semen quality as determined by Cellsoft computer-assisted semen analysis. 'o In that study ejaculates with higher affinity and fewer binding sites had higher sperm concentrations, higher concentrations of motile spermatozoa, higher motility, and higher total cells per ejaculate compared with samples with low affinity and those in which saturation with heparin could not be reached. Those same spermatozoal traits
Volume 160 Number I
were subjected to computer analysis in a separate study and were positively correlated with penetration of zonafree hamster eggs and in vitro fertilization of human oocytes. II Heparin also binds to human sperm membranes and induces nuclear chromatin decondensation and deoxyribonucleic acid synthesis, processes required for pronuclear formation and the transcription of genetic information after fertilization.12 Thus, heparinbinding parameters may be related to fertility in humans as in other mammalian species. Binding may be indicative of the ability of human spermatozoa to undergo capacitation, acrosome reaction, and subsequent oocyte fertilization. These experiments correlate the binding characteristics of heparin to human spermatozoa with the ability of the cells to penetrate zona pellucida-free hamster eggs in vitro. Material and methods: Hamster egg in vitro penetration assays
Spermatozoa preparation. Twenty-six men requesting infertility evaluation and six men of proven fertility (controls) participated in this study. After obtaining the appropriate Human Subject's Committee clearance, semen samples were collected by masturbation after seventy-two hours of abstinence. After completion of liquefaction (prewash), aliquots were removed for computerized semen analysis and binding assays. The remaining portions of each sample were washed twice in Biggers, Whitten, and Whittingham medium (94.59 mmollL NaCI, 4.78 mmollL KCI, 1.71 mmollL CaCI", 1.19 mmollL KH 2 PO" 1.19 mmollL MgS04 , 25.07 mmollL NaHCO" 0.25 mmollL sodium pyruvate, 21.58 mmollL sodium lactate, 5.56 mmollL glucose, 125 IU /ml of penicillin, and 3 mg/ml of bovine serum albumin, pH 7.4) by centrifugation for 5 minutes at 300 x g to form a loose pellet. The supernatants were aspirated, and the pellets were overlayed with 0.3 to 1.0 ml of antibiotic-free Biggers, Whitten, and Whittingham modified to contain 84.1 mmollL NaCI, 35.7 mmollL NaHC0 3 , and 35 mg/ml of human serum albumin. Samples were incubated in 5% CO2 /95% air at 37° C for 75 to 120 minutes. Motile spermatozoa, which migrate from the pellet into the supernatant fluid, were aspirated (postwash samples), and aliquots were removed for semen analysis and binding assays. Preliminary data showed no significant difference in heparinbinding parameters when spermatozoa were frozen with or without glycerol. lo Thus samples were stored at approximately - 70° C without the addition of glycerol as a cryoprotectant until heparin-binding assays were performed. The remainder of each sample was adjusted to a concentration of 107 sperm/ml with modified Biggers, Whitten, and Whittingham for use in the zona-free hamster egg penetration assay.
Sperm heparin binding and fertilization
21
Preparation of zona-free hamster eggs. The estrous cycles of mature female golden hamsters (Harlan Sprague-Dawley Co., Indianapolis, Ind.) were monitored by vaginal smear cytologic studies. Only 4-day cycling hamsters, after two to three consecutive cycles, were used in the experiments. The hamsters were superovulated by an intraperitoneal injection of 25 I U of pregnant mare's serum gonadotropin (Organon, Inc., West Orange, N.J.) on the morning of estrus. Approximately 56 hours later, the animals received an intraperitoneal injection of 25 IU of human chorionic gonadotropin (Ayerst Laboratories, New York, N.Y.). Cumulus-enclosed oocytes were harvested from excised oviducts approximately 18 hours later. Eggs were freed from surrounding cumuli by exposure to 0.1 % hyaluronidase (Sigma Chemical Co., St. Louis, Mo.) in Biggers, Whitten, and Whittingham medium for 10 minutes. Cumulus-free eggs were then washed five times in Biggers, Whitten, and Whittingham and zona pellucidae were solubilized by brief exposure to 0.1 % trypsin (type II, Sigma). After zona removal, eggs were washed five times in modified Biggers, Whitten, and Whittingham. Fifty eggs in approximately 10 f.L1 were transferred by micropipette to plastic Petri dishes containing 200 f.LI of postwash spermatozoa (10 7 cells/ml) covered with paraffin oil. Penetration evaluation. After 3 hours of incubation at 37° C in 5% CO 2 /95% air, the inseminated eggs were removed, washed five times with Biggers, Whitten, and Whittingham modified to contain I mg/ml of polyvinyl alcohol but no serum albumin, CaCI 2 , or antibiotics to remove loosely attached spermatozoa, and fixed with 3% glutaraldehyde. Eggs were stained with 0.25% calcium-free lacmoid and 45% acetic acid. Sperm penetration was confirmed at x 400 by phase-contrast microscopy. The presence of swollen heads and the corresponding sperm tails in the ooplasm were the basis for establishing penetration. Fetilization l'esults were expressed as the percentage of total oocytes penetrated. These percentages were standardized by dividing them by the penetration percentage achieved by spermatozoa from fertile controls for each assay (hamster egg in vitro penetration assay index). Heparin-binding assays
Pre- and postwash semen samples were thawed in a 35° C water bath, and 50 f.LI aliquots were solubilized by boiling for 15 seconds in the presence of I moll L NaOH and 25 mmollL l3-mercaptoethanol. Protein concentrations were determined by the method of Bradford, IS which used bovine "{-globulin as a standard. Semen samples were diluted to a concentration of 50 f.Lg protein/ml with Tris-buffered saline solution (40 mmollL Tris hydroxymethyl aminomethane, 150
22
Vasquez et al.
mmollL NaCI, 5 mmollL benzamidine, 1 J-LmollL phenolmethylsulfonylftuoride, and 1 J-Lmoll L pepstatin A, pH 7.35). Ninety-six-well millititer HA filtration plates (Millipore Products, Bedford, Mass.) were placed on a vacuum filtrator and washed four times with assay buffer (40 mmol/L Tris, pH 7.35). One hundred microliters (approximately 5 J-Lg protein) were placed on nitrocellulose filters in each well and washed four times with assay buffer by vacuum filtration. Four serial dilutions of tritiated heparin (0.4 mCi/mg, New England Nuclear, Boston, Mass.) were added to duplicate samples, along with 150 J-LI of assay buffer for a total volume of 250 J-LI and concentrations ranging from 2.1 to 1067 nmoliL tritiated heparin/J-Lg of protein. Nonspecific binding was assessed by substituting 100 J-LI of 1 mg / ml of unlabeled heparin for an equivalent volume of assay buffer. The plates were covered and incubated at 37° C for 120 minutes to reach equilibrium. The contents of the plates were filtered, and the cells were washed three times with assay buffer at 5° C to remove the unbound fraction. The nitrocellulose filters were punched from the plates into scintillation mini-vials and mixed with 5 ml of Ecoscint scintillation cocktail (National Diagnostics, Manville, N.J.). The vials were shaken overnight and counted in a Packard liquid scintillation spectrophotometer (Downers Grove, Ill.). The dissociation constants and the number of binding sites were determined by Scatchard analysis." Data were expressed as nmoles tritiated heparin bound/ lOb cells, and number of binding sites (x 10 17 ) / cell, respectively. Sperm cell concentrations were determined by Cellsoft computer-assisted analysis in pre- and postwash samples.!O Computer-assisted semen analysis. Sperm concentrations were evaluated with a Cellsoft automated semen analyzer (CRYO Resources, Ltd., New York, N.Y.). Standard equipment (Olympus BH2 microscope, lOX objective, 6.7X photo eyepiece, MTV-3 video camera adaptor, 1O-unit Makler Chamber) for operating a Cellsoft automated semen analyzer was used. The general parameters for the semen analysis were as follows: 20 frames, 30 Hz, one minimum sampling for motility, three minimum samplings for velocity, 200 maximum for velocity, eight threshold velocities, 115 threshold gray, 0.688 Pixel scale, one dilution factor, 5- to 25-cell size range.
Statistical analysis Subjects were grouped into the following three categories on the basis of fertility history and hamster egg in vitro penetration assay indexes: (1) controls (hamster egg in vitro penetration assay index = 1.0, n = 6), (2) infertility patients with low penetration indeces (hamster egg in vitro penetration assay index < 1.0, n = 19), and (3) patients with high penetration indeces (hamster
January 1989 Am J Obstet Gyneco1
egg in vitro penetration assay index> 1.0, n = 7). Samples that failed to achieve saturation were excluded from statistical analysis, since accurate heparin-bindin~ parameters could not be determined. Linear regression models were used to compare hamster egg in vitro penetration assay indeces with heparin-binding data from pre- and pmtwash samples. Data were analyzed with a minimum confidence level of 95%. Analysis of variance was used to compare the differences in heparin-binding parameters between pre- and postwash samples and between subjects with low, high, or control hamster egg in vitro penetration assay indeces. Specific differences between the groups were evaluated by the use of Fisher's least significant difference post-hoc procedures. 15
Results Hamster egg in vitro penetration assay rates. The average hamster egg in vitro penetration assay rate (percentage of ova penetrated) for all the subjects including patients and controls was 26.4% ± 4.8% (± SE), with a range of 0% to 90%. Hamster egg in vitro penetration assay rate data were clustered either above 31 % or below 12%. No values were found between those two limits. Hamster egg in vitro penetration assay rates were below 10% in 14 samples and between 10% and 12% in four samples. The mean hamster egg in vitro penetration assay rate in the control group was significantly higher than that in the low-rate patients, but lower than in patients with a high hamster egg in vitro penetration assay rate (48.5% ± 9.4% vs 5.2% ± 0.9% vs 57.9% ± 5.5%, respectively). In all cases except one, the classification of a subject into a high or low penetration group on the basis of hamster egg in vitro penetration assay rate corresponded with the classification based on the hamster egg in vitro penetration assay index. Consequently, data were grouped for statistical analysis according to classifications obtained from the standardized hamster egg in vitro penetration assay indeces. Heparin-binding data. Heparin-binding parameters and hamster egg in vitro penetration assay indeces were compared in patients with low and high hamster egg in vitro penetration assay indeces, as well as in control subjects. Heparin-binding assays were performed on 32 prewash and 32 postwash samples. All prewash samples bound saturating levels of tritiated heparin, but only 17 (53%) of the postwash samples did so. In the prewash samples the mean numbers of binding sites were 5.67 ± 1.05, 2.01 ± 0.34, and 2.49 ± 0.56 x 1017 binding sites/cell in the low (n = 19), high (n = 7), and control (n = 6) hamster egg in vitro penetration assay index categories, respectively (Fig. 1, A). In the postwash samples, the mean numbers of binding sites were 5.43 ± 1.02,
Sperm heparin binding and fertilization
Volume 160 Number I
23
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3.43 ± 1.55, and 0.89 ± 0.43 X 10 17 binding sites/cell in the low (n = 9), high (n = 6), and control (n = 2) hamster egg in vitro penetration assay index categories, respectively. In the prewash samples, the mean dissociation constants were 3.15 ± 0.54, 1.48 ± 0.52, and 1.51 ± 0.16 nmol/ 106 cells in the low, high, and control hamster egg in vitro penetration assay index categories, respectively (Fig. 1, B). In the postwash samples, the mean dissociation constants were 16.42 ± 5.53, 5.39 ± 2.12, and 2.76 ± 0.77 nmol/106 cells, in the low, high, and control hamster egg in vitro peneH'ation assay index categories, respectively, In prewash samples, subjects with low hamster egg in vitro penetration assay index had significantly more binding sites per cell than patients with a high hamster egg in vitro penetration assay index (Fig. 1, A). In the postwash samples,
there was no significant difference between heparinbinding affinity 01' the number of binding domains of the different groups (Fig. 1, A and B). Regression analysis of hamster in vitro penetration assay data and heparin-binding parameters. The percen tage of hamster ova penetrated was inversely related to both the dissociation constants and the number of heparin-binding sites in the prewash samples (data not shown). The dissociation constants and the number of binding sites in the raw ejaculates (prewash) were also inversely related to the standardized hamster egg in vitro penetration assay indeces. When the number of binding sites in the raw ejaculates was plotted against the hamster egg in vitro penetration assay indeces, a negative linear relationship was found (Fig. 2, A) with an
24
Vasquez et al.
January 1989 Am J Obstet Gynecol
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Penetration Indices Fig. 2. Composite linear regression with 95% confidence limits for (A) number of binding sites in prewash spermatozoa vs hamster egg in vitro penetration assay (HIPA) indeces (p < 0.05) and (B) dissociation constants (Kds) of prewash spermatozoa vs hamster egg in vitro penetration assay indeces (p < 0.05).
R2 of 0.201 (P < 0.05). When the raw ejaculate dissociation constants were plotted against the hamster egg in vitro penetration assay indeces, a negative linear relationship was found (Fig. 2, B) with an R2 of 0.142 (P < 0.05). Thus in prewash samples with higher hamster egg in vitro penetration assay indeces, spermatozoa had fewer binding sites but a greater affinity for heparin. There was no significant correlation between the number of binding sites or the dissociation constants of the postwash samples and the hamster egg in vitro penetration assay indeces (data not shown).
Comment The objective of our research was to compare the heparin-binding parameters of human spermatozoa with hamster egg in vitro penetration assay data standardized by the use of semen from control subjects of proven fertility. Data presented herein indicate that heparin binding was related to the ability of human spermatozoa to penetrate zona-free hamster ova. Heparin-binding affinity was positively correlated with the relative percentage of zona-free hamster ova pen-
etrated. In contrast, the number of binding sites per cell was negatively correlated with penetration indeces. There was no significant correlation between heparinbinding parameters in post-wash samples and hamster in vitro penetration data, which indicates alterations in heparin-binding characteristics during semen processing for insemination. The "swim-up" has been used routinely in in vitro fertilization of human and zona-free hamster oocytes to enhance penetration when compared with "unrisen samples."3. II 16 I7 This enhanced penetration may be explained in part by the migration of motile spermatozoa into the supernatant fluid." Other investigators lO have indicated that the binding affinity of human spermatozoa for heparin significantly correlated with the percentage of motile sperm and the concentration of motile sperm in the raw ejaculates. Those motility traits have been associated with higher fertility in other studies. 3. II However, there is no experimental evidence to indicate a direct relationship between heparin binding and motility in subpopulations of spermatozoa from an ejaculate. In our study, postwash samples had lower
Volume 160 Number I
affinity (higher dissociation constants) compared with prewash samples. The significance of the higher dissociation constants observed in postwash samples in the present experiments is unknown, but may be related to the procedures involved in sperm preparation of "swim-up" fractions. Seminal plasma has been found to alter tritiated heparin binding of epididymal sperm and inhibit follicular-fluid, proteoglycan-induced acrosome reactions. '8 -2o Thus defective adsorption of seminal plasma heparin-binding proteins added to sperm at ejaculation could be responsible for impaired capacitation and decreased sperm fertilizing ability in vitro or in vivo. Heparin-binding polypeptides isolated from sperm cell membranes and seminal plasma have been partially characterized!1. 22 Whether they playa role in modulating sperm capacitation and fetilization in vivo remains to be elucidated. However, the well-established techniques for hamster in vitro penetration assays and in vitro fertilization 16. 17. 2j can be used as experimental models to study the physiologic role of glycosaminoglycans on sperm capacitation and fertilization. Heparin has been shown to facilitate sperm capacitation,7 accelerate conversion of proacrosin to acrosin;4.25 and induce decondensation of human sperm nuclei. 26 27 Previous studies also indicated that heparinbinding affinity was related to fertility in bulls! In humans, this has not been established directly, although it has been correlated with semen parameters thought to be indicative of fertility.1O Our data show a correlation between hamster egg penetration rates, heparinbinding affinity, and number of binding sites. Further research is warranted to determine whether heparin binding is necessary for human spermatozoa to undergo capacitation and subsequent oocyte fertilization and the role, if any, of genital tract glycosaminoglycans during fertilization. However, the results of the current study suggest that glycosaminoglycans may be useful probes to evaluate human sperm-fertilizing ability. Other investigators2B . 29 have shown that bovine sperm incubated with heparin acrosome react and fertilize an' increased percentage of bovine oocytes in vitro compared with sperm incubated without heparin. In these studies the frequency of acrosome reactions induced by the fusogenic lipid lysophosphatidylcholine under capacitating conditions with heparin correlated highly with the ability of sperm to fertilize oocytes in vitro."9 The effects of heparin on sperm capacitation, as judged by in vitro fertilization, and on sperm sensitization to lysophosphatidylcholine-induced acrosome reaction were time and dose dependent, with maximum responses occurring at 5 to 10 /-Lg/ml of heparin. In humans, the ability of heparin to enhance in vitro fertilization of human oocytes has not been established. However (Hensleigh He, Wheeler PA. Unpublished
Sperm heparin binding and fertilization
25
observations), have demonstrated that heparin increases zona-free hamster egg in vitro penetration rates by human spermatozoa, and also increases the number of sperm undergoing the acrosome reaction. Further research is warranted to determine whether the addition of heparin in vitro may lead to significant increases in the efficiency rates of human in vitro fertilization or gamete intra fallopian tube transfer. REFERENCES I. Yanagimachi R Mechanisms of fertilization in mammals. In: Mastroianni L Jr, Biggers JD, eds. Fertilization and embryonic development in vitro. New York: Plenum Press, 1981 :82. 2. Talbot P. Events leading to fertilization in mammals. In: Rolland R. ed. Proceedings of the XI World Congress on Fertility and Sterility, Dublin, Ireland, 1983:121. 3. Hall JL. Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 1981 ;35:457. 4. Gould ]E. Overstreet JW, Yanagimachi H, Yanagimachi R, Katz DF, Hanson FW. What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs? Fertil Steril 1983;40:344. 5. Meizel S, Turner KO. Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm.] Exp Zool 1986;237:137. 6. Handrow RR, Lenz RW, Ax RL. Structural comparisons among glycosaminoglycans to promote an acrosome reaction in bovine spermatozoa. Biochem Biophys Res Commun 1982;107:1376. 7. Parrish ]J. Susko-Parrish ]L. First NL. Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro. Theriogenology 1985; 24:537. 8. Marks ]L, Ax RL. Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm. J Dairy Sci 1985;68:2078. 9. Handrow RR, Boehm SK. Lenz RW, Robinson JA, Ax RL. Specific binding of the glycosaminoglycan 3H-heparin to bull, monkey and rabbit spermatozoa in vitro.] Androl 1984;5:551. 10. Miller D], Agnew G, Boone WR, Ax RL, Vasquez JM. Relationship of heparin binding with computer analyzed physical traits of human sperm. Fertil Steril 1988;49: 145. 11. Holt WV, Moore HDM, Hillier SG. Computer-assisted measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays. Fertil Steril 1985;44:112. 12. Huret]L. Nuclear chromatin decondensation of human sperm: a review. Arch Androl 1986;16:97. 13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248. 14. Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949;51:660. 15. Daniel WN, ed. Biostatistics: a foundation for analysis in the health sciences. New York: John Wiley & Sons. 1978. 16. Rogers BJ. The sperm penetration assay: its usefulness reevaluated. Fertil Steril 1985;43:821. 17. YovlCh ]L, Stanger JD, Yovich ]M. The management of oligospermic infertility by in vitro fertilization. Ann NY Acad Sci 1985;442:195. 18. Lenz RW. Ax RL, Grimek H], First NL. Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovme spermatozoa. Blochem Biophys Res Commun 1982;106:1092. 19. Lee CN, Handrow RR, Lenz RW, Ax RL. Interactions of seminal plasma and glycosaminoglycans on acrosome re-
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20.
21.
22.
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actions in bovine spermatozoa in vitro. Gamete Res 1985;12:345. Russell LD, Peterson RN, Hunt W, Strack LE. Posttesticular surface modifications and contributions of reproductive tract fluids to the surface polypeptide composition of boar spermatozoa. Bioi Reprod 1984;30:959. Lavin CA, Robinson JA, Ax RL. Characterization of heparin binding domains from monkey and bull spermatozoa. In: Proceedings of the forty-second annual meeting of the American Fertility Society, Toronto, Canada, 1986: (suppl)32. Miller DJ, First NL, Ax RL. Isolation and characterization of seminal fluid proteins that bind heparin. Proceedings on regulation of ovarian and testicular function. vol 219. In: Mahesh VB, Dhindsa DS, Anderson E, Kalra SP, eds. Advances in experimental medicine and biology. New York: Plenum Press, 1987. Quinn P, Warner GM, Kerin JF, Kirby C. Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann NY Acad Sci 1985;442: 195.
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24. Parrish RF, Wincek TJ, Polakoski KL. Glycosaminoglycan stimulation of the in vitro conversion of boar proacrosin into acrosin. J Androl 1980; 1:89. 25. Wincek TJ, Parrish RF, Polakoski KL. Fertilization: a uterine glycosaminoglycan stimulates the conversion of sperm proacrosin to acrosin. Science 1979;203:553. 26. Delgado NM, Magdaleno VM, Merchant H, Rosado A, Reyes R. Heparin-induced release of DNA template restrictions in human sperm zinc-depleted nuclei. Arch AndroI1984;12:211. 27. Delgado NM. Huacuja L, Merchant H, Reyes R, Rosado A. Species specific decondensation of human spermatozoa nuclei by heparin. Arch Androl 1980;4:305. 28. Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986;25:591. 29. Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Bioi Reprod 1988;38: 1171.
Interferon--y in the diagnosis and pathogenesis of pelvic inflammatory disease Jamie A. Grifo, MD, PhD, Jan Jeremias, BS, William J. Ledger, MD, and Steven S. Witkin, PhD New York, New York Serologic markers were evaluated to determine if they could aid in the differential diagnosis of pelvic inflammatory disease in 48 consecutive women seeking evaluation for pelvic pain. On the basis of clinical and microbiologic parameters, 29 patients (60.4%) were diagnosed as having pelvic inflammatory disease. Neisseria gonorrhoeae only was isolated from the cervix of eight (27.6%) patients with pelvic inflammatory disease, five (17.2%) had only Chlamydia, and two (6.9%) had Neisseria and Chlamydia, whereas in 15 (48.3%) patients no pathogen was isolated. Interferon--y was present in significantly more sera (p < 0.025) from patients with pelvic inflammatory ~isease (65.5%) than from women without pelvic inflammatory disease (15.8%). Sera from 10 healthy women lacked detectable interferon--y. In patients with only Neisseria, seven (87.5%) had circulating interferon--y; three (60%) of the women with only Chlamydia, one (50%) woman with Neisseria and Chlamydia, and eight (57.1%) with no identified pathogens were also positive for interferon--y. Sera from 11 of 28 patients with pelvic inflammatory disease (39%) but only one of 19 sera from women without pelvic inflammatory disease (5%) also inhibited the Candida-induced proliferation of control lymphocytes. This immunosuppressive activity was prevented by immunoprecipitation of interferon--y by anti-interferon--y antibody but not by treatment with anti-interferon-a antibody. The persistence of interferon--y in the sera of patients with pelvic inflammatory disease may aid in the differential diagnosis of this disease and increase our understanding of the pathogenesis of microbial-mediated tubal damage. (AM J OasTET GYNECOL 1989;160:26-31.)
Key words: Pelvic inflammatory disease, interferon--y, Chlamydia, Neisseria gonorrhoeae
From the DivlSZon of Immunology, Department of Obstetrics and Gynecology, Cornell Unzverszty Medzcal College. Presented at the Thirty-fifth Annual meetzng of the SOCIety for G.~ necologic Investigation, Baltzmore, Maryland, March 17-20,
1988.
Reprznt requests: Steven S. WItkin, PhD, Department of Obstetrzcs and Gynecology, Cornell Unzverszty Medzcal College, 515 E. 71st St., New York, NY 10021.
26
The difficulty in diagnosing pelvic inflammatory disease has been well documented. The routine use of laparoscopy by Jacobsen and Westrom' demonstrated the great inaccuracy of clinical diagnosis. Cervical cultures, while helpful when positive, have a relatively high false-negative rate, which makes culture-based diag-
Key words: Spermatozoa, glycosaminoglycans, fertilization, hamster egg in vitro penetration assay, computer-assisted semen analysis Capacitation of spermatozoa and the acrosome reaction are prerequisites for mammalian fertilization. 1.2 The hamster egg in vitro penetration assay has been used in many clinical and research laboratories to assess the potential of human spermatozoa to undergo capacitation, acrosome reaction, oocyte membrane fusion, and pronuclear formation.' Penetration rates have been used as a predictor of fertility in males on the basis of a significant correlation between hamster egg in vitro penetration assay rates and in vitro fertilization of human oocytes.' However, normal penetration of zona-free hamster eggs is not necessarily an indication that human spermatozoa can penetrate the zona pellucida of human oocytes.! From the DIvISIOn of ReproductIVe Endocrinology and Infertility, Department of Obstetrics and Gynecology,' University of Wisconsin Medical School, and the Department of Dazry Science and Endocrinology-Reproductive Physiology Program,' University of Wisconsin. Supported by a General Research Support Grant to the Unzverslty of WISconsin Medical School from the NatIOnal Institutes of Health, Division of Research Faclhtles and Resources, (J. M. V.) and a University of Wisconsin Graduate School Grant (j. M. V.J. Presented at the Thirty-fifth Annual Meeting of the SOCIety for Gynecologzc InvestigatIOn, Baltimore, Maryland, March 17-20,
1988.
Reprznt requests: J. M. Vasquez, MD, Department of Obstetrzcs and Gynecology, UniveTSlty of WISconsin-Madison, 600 Highland Ave., Rm. H4/630 MadISon, WI 53792
esc,
20
Previous studies suggest that glycosaminoglycans have a role in capacitation and the acrosome reaction.' Heparin, a highly sulfated glycosaminoglycan, has been found to facilitate sperm capacitation and the acrosome reaction in vitro in several mammalian species." 7 Tritiated heparin has been shown to bind to spermatozoa saturably, and the binding affinity and susceptibility to glycosaminoglycan-induced capacitation are related to fertility in bulls. s In displacement assays of heparin, Scatchard transformations of the data have indicated that the principal binding affinity (dissociation constants = 10- 7 mol! L) is biologically significant and comparable with hormone-receptor-binding affinities. Thermodynamic calculations have suggested that tritiated heparian binding to spermatozoa is a specific and exothermic process that has all of the classic characteristics of a receptor-ligand interaction." Tritiated heparin binds to human spermatozoa, and the binding affinity and the concentration of binding sites correlate with semen quality as determined by Cellsoft computer-assisted semen analysis. 'o In that study ejaculates with higher affinity and fewer binding sites had higher sperm concentrations, higher concentrations of motile spermatozoa, higher motility, and higher total cells per ejaculate compared with samples with low affinity and those in which saturation with heparin could not be reached. Those same spermatozoal traits
Volume 160 Number I
were subjected to computer analysis in a separate study and were positively correlated with penetration of zonafree hamster eggs and in vitro fertilization of human oocytes. II Heparin also binds to human sperm membranes and induces nuclear chromatin decondensation and deoxyribonucleic acid synthesis, processes required for pronuclear formation and the transcription of genetic information after fertilization.12 Thus, heparinbinding parameters may be related to fertility in humans as in other mammalian species. Binding may be indicative of the ability of human spermatozoa to undergo capacitation, acrosome reaction, and subsequent oocyte fertilization. These experiments correlate the binding characteristics of heparin to human spermatozoa with the ability of the cells to penetrate zona pellucida-free hamster eggs in vitro. Material and methods: Hamster egg in vitro penetration assays
Spermatozoa preparation. Twenty-six men requesting infertility evaluation and six men of proven fertility (controls) participated in this study. After obtaining the appropriate Human Subject's Committee clearance, semen samples were collected by masturbation after seventy-two hours of abstinence. After completion of liquefaction (prewash), aliquots were removed for computerized semen analysis and binding assays. The remaining portions of each sample were washed twice in Biggers, Whitten, and Whittingham medium (94.59 mmollL NaCI, 4.78 mmollL KCI, 1.71 mmollL CaCI", 1.19 mmollL KH 2 PO" 1.19 mmollL MgS04 , 25.07 mmollL NaHCO" 0.25 mmollL sodium pyruvate, 21.58 mmollL sodium lactate, 5.56 mmollL glucose, 125 IU /ml of penicillin, and 3 mg/ml of bovine serum albumin, pH 7.4) by centrifugation for 5 minutes at 300 x g to form a loose pellet. The supernatants were aspirated, and the pellets were overlayed with 0.3 to 1.0 ml of antibiotic-free Biggers, Whitten, and Whittingham modified to contain 84.1 mmollL NaCI, 35.7 mmollL NaHC0 3 , and 35 mg/ml of human serum albumin. Samples were incubated in 5% CO2 /95% air at 37° C for 75 to 120 minutes. Motile spermatozoa, which migrate from the pellet into the supernatant fluid, were aspirated (postwash samples), and aliquots were removed for semen analysis and binding assays. Preliminary data showed no significant difference in heparinbinding parameters when spermatozoa were frozen with or without glycerol. lo Thus samples were stored at approximately - 70° C without the addition of glycerol as a cryoprotectant until heparin-binding assays were performed. The remainder of each sample was adjusted to a concentration of 107 sperm/ml with modified Biggers, Whitten, and Whittingham for use in the zona-free hamster egg penetration assay.
Sperm heparin binding and fertilization
21
Preparation of zona-free hamster eggs. The estrous cycles of mature female golden hamsters (Harlan Sprague-Dawley Co., Indianapolis, Ind.) were monitored by vaginal smear cytologic studies. Only 4-day cycling hamsters, after two to three consecutive cycles, were used in the experiments. The hamsters were superovulated by an intraperitoneal injection of 25 I U of pregnant mare's serum gonadotropin (Organon, Inc., West Orange, N.J.) on the morning of estrus. Approximately 56 hours later, the animals received an intraperitoneal injection of 25 IU of human chorionic gonadotropin (Ayerst Laboratories, New York, N.Y.). Cumulus-enclosed oocytes were harvested from excised oviducts approximately 18 hours later. Eggs were freed from surrounding cumuli by exposure to 0.1 % hyaluronidase (Sigma Chemical Co., St. Louis, Mo.) in Biggers, Whitten, and Whittingham medium for 10 minutes. Cumulus-free eggs were then washed five times in Biggers, Whitten, and Whittingham and zona pellucidae were solubilized by brief exposure to 0.1 % trypsin (type II, Sigma). After zona removal, eggs were washed five times in modified Biggers, Whitten, and Whittingham. Fifty eggs in approximately 10 f.L1 were transferred by micropipette to plastic Petri dishes containing 200 f.LI of postwash spermatozoa (10 7 cells/ml) covered with paraffin oil. Penetration evaluation. After 3 hours of incubation at 37° C in 5% CO 2 /95% air, the inseminated eggs were removed, washed five times with Biggers, Whitten, and Whittingham modified to contain I mg/ml of polyvinyl alcohol but no serum albumin, CaCI 2 , or antibiotics to remove loosely attached spermatozoa, and fixed with 3% glutaraldehyde. Eggs were stained with 0.25% calcium-free lacmoid and 45% acetic acid. Sperm penetration was confirmed at x 400 by phase-contrast microscopy. The presence of swollen heads and the corresponding sperm tails in the ooplasm were the basis for establishing penetration. Fetilization l'esults were expressed as the percentage of total oocytes penetrated. These percentages were standardized by dividing them by the penetration percentage achieved by spermatozoa from fertile controls for each assay (hamster egg in vitro penetration assay index). Heparin-binding assays
Pre- and postwash semen samples were thawed in a 35° C water bath, and 50 f.LI aliquots were solubilized by boiling for 15 seconds in the presence of I moll L NaOH and 25 mmollL l3-mercaptoethanol. Protein concentrations were determined by the method of Bradford, IS which used bovine "{-globulin as a standard. Semen samples were diluted to a concentration of 50 f.Lg protein/ml with Tris-buffered saline solution (40 mmollL Tris hydroxymethyl aminomethane, 150
22
Vasquez et al.
mmollL NaCI, 5 mmollL benzamidine, 1 J-LmollL phenolmethylsulfonylftuoride, and 1 J-Lmoll L pepstatin A, pH 7.35). Ninety-six-well millititer HA filtration plates (Millipore Products, Bedford, Mass.) were placed on a vacuum filtrator and washed four times with assay buffer (40 mmol/L Tris, pH 7.35). One hundred microliters (approximately 5 J-Lg protein) were placed on nitrocellulose filters in each well and washed four times with assay buffer by vacuum filtration. Four serial dilutions of tritiated heparin (0.4 mCi/mg, New England Nuclear, Boston, Mass.) were added to duplicate samples, along with 150 J-LI of assay buffer for a total volume of 250 J-LI and concentrations ranging from 2.1 to 1067 nmoliL tritiated heparin/J-Lg of protein. Nonspecific binding was assessed by substituting 100 J-LI of 1 mg / ml of unlabeled heparin for an equivalent volume of assay buffer. The plates were covered and incubated at 37° C for 120 minutes to reach equilibrium. The contents of the plates were filtered, and the cells were washed three times with assay buffer at 5° C to remove the unbound fraction. The nitrocellulose filters were punched from the plates into scintillation mini-vials and mixed with 5 ml of Ecoscint scintillation cocktail (National Diagnostics, Manville, N.J.). The vials were shaken overnight and counted in a Packard liquid scintillation spectrophotometer (Downers Grove, Ill.). The dissociation constants and the number of binding sites were determined by Scatchard analysis." Data were expressed as nmoles tritiated heparin bound/ lOb cells, and number of binding sites (x 10 17 ) / cell, respectively. Sperm cell concentrations were determined by Cellsoft computer-assisted analysis in pre- and postwash samples.!O Computer-assisted semen analysis. Sperm concentrations were evaluated with a Cellsoft automated semen analyzer (CRYO Resources, Ltd., New York, N.Y.). Standard equipment (Olympus BH2 microscope, lOX objective, 6.7X photo eyepiece, MTV-3 video camera adaptor, 1O-unit Makler Chamber) for operating a Cellsoft automated semen analyzer was used. The general parameters for the semen analysis were as follows: 20 frames, 30 Hz, one minimum sampling for motility, three minimum samplings for velocity, 200 maximum for velocity, eight threshold velocities, 115 threshold gray, 0.688 Pixel scale, one dilution factor, 5- to 25-cell size range.
Statistical analysis Subjects were grouped into the following three categories on the basis of fertility history and hamster egg in vitro penetration assay indexes: (1) controls (hamster egg in vitro penetration assay index = 1.0, n = 6), (2) infertility patients with low penetration indeces (hamster egg in vitro penetration assay index < 1.0, n = 19), and (3) patients with high penetration indeces (hamster
January 1989 Am J Obstet Gyneco1
egg in vitro penetration assay index> 1.0, n = 7). Samples that failed to achieve saturation were excluded from statistical analysis, since accurate heparin-bindin~ parameters could not be determined. Linear regression models were used to compare hamster egg in vitro penetration assay indeces with heparin-binding data from pre- and pmtwash samples. Data were analyzed with a minimum confidence level of 95%. Analysis of variance was used to compare the differences in heparin-binding parameters between pre- and postwash samples and between subjects with low, high, or control hamster egg in vitro penetration assay indeces. Specific differences between the groups were evaluated by the use of Fisher's least significant difference post-hoc procedures. 15
Results Hamster egg in vitro penetration assay rates. The average hamster egg in vitro penetration assay rate (percentage of ova penetrated) for all the subjects including patients and controls was 26.4% ± 4.8% (± SE), with a range of 0% to 90%. Hamster egg in vitro penetration assay rate data were clustered either above 31 % or below 12%. No values were found between those two limits. Hamster egg in vitro penetration assay rates were below 10% in 14 samples and between 10% and 12% in four samples. The mean hamster egg in vitro penetration assay rate in the control group was significantly higher than that in the low-rate patients, but lower than in patients with a high hamster egg in vitro penetration assay rate (48.5% ± 9.4% vs 5.2% ± 0.9% vs 57.9% ± 5.5%, respectively). In all cases except one, the classification of a subject into a high or low penetration group on the basis of hamster egg in vitro penetration assay rate corresponded with the classification based on the hamster egg in vitro penetration assay index. Consequently, data were grouped for statistical analysis according to classifications obtained from the standardized hamster egg in vitro penetration assay indeces. Heparin-binding data. Heparin-binding parameters and hamster egg in vitro penetration assay indeces were compared in patients with low and high hamster egg in vitro penetration assay indeces, as well as in control subjects. Heparin-binding assays were performed on 32 prewash and 32 postwash samples. All prewash samples bound saturating levels of tritiated heparin, but only 17 (53%) of the postwash samples did so. In the prewash samples the mean numbers of binding sites were 5.67 ± 1.05, 2.01 ± 0.34, and 2.49 ± 0.56 x 1017 binding sites/cell in the low (n = 19), high (n = 7), and control (n = 6) hamster egg in vitro penetration assay index categories, respectively (Fig. 1, A). In the postwash samples, the mean numbers of binding sites were 5.43 ± 1.02,
Sperm heparin binding and fertilization
Volume 160 Number I
23
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3.43 ± 1.55, and 0.89 ± 0.43 X 10 17 binding sites/cell in the low (n = 9), high (n = 6), and control (n = 2) hamster egg in vitro penetration assay index categories, respectively. In the prewash samples, the mean dissociation constants were 3.15 ± 0.54, 1.48 ± 0.52, and 1.51 ± 0.16 nmol/ 106 cells in the low, high, and control hamster egg in vitro penetration assay index categories, respectively (Fig. 1, B). In the postwash samples, the mean dissociation constants were 16.42 ± 5.53, 5.39 ± 2.12, and 2.76 ± 0.77 nmol/106 cells, in the low, high, and control hamster egg in vitro peneH'ation assay index categories, respectively, In prewash samples, subjects with low hamster egg in vitro penetration assay index had significantly more binding sites per cell than patients with a high hamster egg in vitro penetration assay index (Fig. 1, A). In the postwash samples,
there was no significant difference between heparinbinding affinity 01' the number of binding domains of the different groups (Fig. 1, A and B). Regression analysis of hamster in vitro penetration assay data and heparin-binding parameters. The percen tage of hamster ova penetrated was inversely related to both the dissociation constants and the number of heparin-binding sites in the prewash samples (data not shown). The dissociation constants and the number of binding sites in the raw ejaculates (prewash) were also inversely related to the standardized hamster egg in vitro penetration assay indeces. When the number of binding sites in the raw ejaculates was plotted against the hamster egg in vitro penetration assay indeces, a negative linear relationship was found (Fig. 2, A) with an
24
Vasquez et al.
January 1989 Am J Obstet Gynecol
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Penetration Indices Fig. 2. Composite linear regression with 95% confidence limits for (A) number of binding sites in prewash spermatozoa vs hamster egg in vitro penetration assay (HIPA) indeces (p < 0.05) and (B) dissociation constants (Kds) of prewash spermatozoa vs hamster egg in vitro penetration assay indeces (p < 0.05).
R2 of 0.201 (P < 0.05). When the raw ejaculate dissociation constants were plotted against the hamster egg in vitro penetration assay indeces, a negative linear relationship was found (Fig. 2, B) with an R2 of 0.142 (P < 0.05). Thus in prewash samples with higher hamster egg in vitro penetration assay indeces, spermatozoa had fewer binding sites but a greater affinity for heparin. There was no significant correlation between the number of binding sites or the dissociation constants of the postwash samples and the hamster egg in vitro penetration assay indeces (data not shown).
Comment The objective of our research was to compare the heparin-binding parameters of human spermatozoa with hamster egg in vitro penetration assay data standardized by the use of semen from control subjects of proven fertility. Data presented herein indicate that heparin binding was related to the ability of human spermatozoa to penetrate zona-free hamster ova. Heparin-binding affinity was positively correlated with the relative percentage of zona-free hamster ova pen-
etrated. In contrast, the number of binding sites per cell was negatively correlated with penetration indeces. There was no significant correlation between heparinbinding parameters in post-wash samples and hamster in vitro penetration data, which indicates alterations in heparin-binding characteristics during semen processing for insemination. The "swim-up" has been used routinely in in vitro fertilization of human and zona-free hamster oocytes to enhance penetration when compared with "unrisen samples."3. II 16 I7 This enhanced penetration may be explained in part by the migration of motile spermatozoa into the supernatant fluid." Other investigators lO have indicated that the binding affinity of human spermatozoa for heparin significantly correlated with the percentage of motile sperm and the concentration of motile sperm in the raw ejaculates. Those motility traits have been associated with higher fertility in other studies. 3. II However, there is no experimental evidence to indicate a direct relationship between heparin binding and motility in subpopulations of spermatozoa from an ejaculate. In our study, postwash samples had lower
Volume 160 Number I
affinity (higher dissociation constants) compared with prewash samples. The significance of the higher dissociation constants observed in postwash samples in the present experiments is unknown, but may be related to the procedures involved in sperm preparation of "swim-up" fractions. Seminal plasma has been found to alter tritiated heparin binding of epididymal sperm and inhibit follicular-fluid, proteoglycan-induced acrosome reactions. '8 -2o Thus defective adsorption of seminal plasma heparin-binding proteins added to sperm at ejaculation could be responsible for impaired capacitation and decreased sperm fertilizing ability in vitro or in vivo. Heparin-binding polypeptides isolated from sperm cell membranes and seminal plasma have been partially characterized!1. 22 Whether they playa role in modulating sperm capacitation and fetilization in vivo remains to be elucidated. However, the well-established techniques for hamster in vitro penetration assays and in vitro fertilization 16. 17. 2j can be used as experimental models to study the physiologic role of glycosaminoglycans on sperm capacitation and fertilization. Heparin has been shown to facilitate sperm capacitation,7 accelerate conversion of proacrosin to acrosin;4.25 and induce decondensation of human sperm nuclei. 26 27 Previous studies also indicated that heparinbinding affinity was related to fertility in bulls! In humans, this has not been established directly, although it has been correlated with semen parameters thought to be indicative of fertility.1O Our data show a correlation between hamster egg penetration rates, heparinbinding affinity, and number of binding sites. Further research is warranted to determine whether heparin binding is necessary for human spermatozoa to undergo capacitation and subsequent oocyte fertilization and the role, if any, of genital tract glycosaminoglycans during fertilization. However, the results of the current study suggest that glycosaminoglycans may be useful probes to evaluate human sperm-fertilizing ability. Other investigators2B . 29 have shown that bovine sperm incubated with heparin acrosome react and fertilize an' increased percentage of bovine oocytes in vitro compared with sperm incubated without heparin. In these studies the frequency of acrosome reactions induced by the fusogenic lipid lysophosphatidylcholine under capacitating conditions with heparin correlated highly with the ability of sperm to fertilize oocytes in vitro."9 The effects of heparin on sperm capacitation, as judged by in vitro fertilization, and on sperm sensitization to lysophosphatidylcholine-induced acrosome reaction were time and dose dependent, with maximum responses occurring at 5 to 10 /-Lg/ml of heparin. In humans, the ability of heparin to enhance in vitro fertilization of human oocytes has not been established. However (Hensleigh He, Wheeler PA. Unpublished
Sperm heparin binding and fertilization
25
observations), have demonstrated that heparin increases zona-free hamster egg in vitro penetration rates by human spermatozoa, and also increases the number of sperm undergoing the acrosome reaction. Further research is warranted to determine whether the addition of heparin in vitro may lead to significant increases in the efficiency rates of human in vitro fertilization or gamete intra fallopian tube transfer. REFERENCES I. Yanagimachi R Mechanisms of fertilization in mammals. In: Mastroianni L Jr, Biggers JD, eds. Fertilization and embryonic development in vitro. New York: Plenum Press, 1981 :82. 2. Talbot P. Events leading to fertilization in mammals. In: Rolland R. ed. Proceedings of the XI World Congress on Fertility and Sterility, Dublin, Ireland, 1983:121. 3. Hall JL. Relationship between semen quality and human sperm penetration of zona-free hamster ova. Fertil Steril 1981 ;35:457. 4. Gould ]E. Overstreet JW, Yanagimachi H, Yanagimachi R, Katz DF, Hanson FW. What functions of the sperm cell are measured by in vitro fertilization of zona-free hamster eggs? Fertil Steril 1983;40:344. 5. Meizel S, Turner KO. Glycosaminoglycans stimulate the acrosome reaction of previously capacitated hamster sperm.] Exp Zool 1986;237:137. 6. Handrow RR, Lenz RW, Ax RL. Structural comparisons among glycosaminoglycans to promote an acrosome reaction in bovine spermatozoa. Biochem Biophys Res Commun 1982;107:1376. 7. Parrish ]J. Susko-Parrish ]L. First NL. Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro. Theriogenology 1985; 24:537. 8. Marks ]L, Ax RL. Relationship of nonreturn rates of dairy bulls to binding affinity of heparin to sperm. J Dairy Sci 1985;68:2078. 9. Handrow RR, Boehm SK. Lenz RW, Robinson JA, Ax RL. Specific binding of the glycosaminoglycan 3H-heparin to bull, monkey and rabbit spermatozoa in vitro.] Androl 1984;5:551. 10. Miller D], Agnew G, Boone WR, Ax RL, Vasquez JM. Relationship of heparin binding with computer analyzed physical traits of human sperm. Fertil Steril 1988;49: 145. 11. Holt WV, Moore HDM, Hillier SG. Computer-assisted measurement of sperm swimming speed in human semen: correlation of results with in vitro fertilization assays. Fertil Steril 1985;44:112. 12. Huret]L. Nuclear chromatin decondensation of human sperm: a review. Arch Androl 1986;16:97. 13. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72: 248. 14. Scatchard G. The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949;51:660. 15. Daniel WN, ed. Biostatistics: a foundation for analysis in the health sciences. New York: John Wiley & Sons. 1978. 16. Rogers BJ. The sperm penetration assay: its usefulness reevaluated. Fertil Steril 1985;43:821. 17. YovlCh ]L, Stanger JD, Yovich ]M. The management of oligospermic infertility by in vitro fertilization. Ann NY Acad Sci 1985;442:195. 18. Lenz RW. Ax RL, Grimek H], First NL. Proteoglycan from bovine follicular fluid enhances an acrosome reaction in bovme spermatozoa. Blochem Biophys Res Commun 1982;106:1092. 19. Lee CN, Handrow RR, Lenz RW, Ax RL. Interactions of seminal plasma and glycosaminoglycans on acrosome re-
Vasquez et al.
20.
21.
22.
23.
actions in bovine spermatozoa in vitro. Gamete Res 1985;12:345. Russell LD, Peterson RN, Hunt W, Strack LE. Posttesticular surface modifications and contributions of reproductive tract fluids to the surface polypeptide composition of boar spermatozoa. Bioi Reprod 1984;30:959. Lavin CA, Robinson JA, Ax RL. Characterization of heparin binding domains from monkey and bull spermatozoa. In: Proceedings of the forty-second annual meeting of the American Fertility Society, Toronto, Canada, 1986: (suppl)32. Miller DJ, First NL, Ax RL. Isolation and characterization of seminal fluid proteins that bind heparin. Proceedings on regulation of ovarian and testicular function. vol 219. In: Mahesh VB, Dhindsa DS, Anderson E, Kalra SP, eds. Advances in experimental medicine and biology. New York: Plenum Press, 1987. Quinn P, Warner GM, Kerin JF, Kirby C. Culture factors affecting the success rate of in vitro fertilization and embryo transfer. Ann NY Acad Sci 1985;442: 195.
January 1989 Am J Obstet Gynecol
24. Parrish RF, Wincek TJ, Polakoski KL. Glycosaminoglycan stimulation of the in vitro conversion of boar proacrosin into acrosin. J Androl 1980; 1:89. 25. Wincek TJ, Parrish RF, Polakoski KL. Fertilization: a uterine glycosaminoglycan stimulates the conversion of sperm proacrosin to acrosin. Science 1979;203:553. 26. Delgado NM, Magdaleno VM, Merchant H, Rosado A, Reyes R. Heparin-induced release of DNA template restrictions in human sperm zinc-depleted nuclei. Arch AndroI1984;12:211. 27. Delgado NM. Huacuja L, Merchant H, Reyes R, Rosado A. Species specific decondensation of human spermatozoa nuclei by heparin. Arch Androl 1980;4:305. 28. Parrish JJ, Susko-Parrish JL, Leibfried-Rutledge ML, Critser ES, Eyestone WH, First NL. Bovine in vitro fertilization with frozen-thawed semen. Theriogenology 1986;25:591. 29. Parrish JJ, Susko-Parrish JL, Winer MA, First NL. Capacitation of bovine sperm by heparin. Bioi Reprod 1988;38: 1171.
Interferon--y in the diagnosis and pathogenesis of pelvic inflammatory disease Jamie A. Grifo, MD, PhD, Jan Jeremias, BS, William J. Ledger, MD, and Steven S. Witkin, PhD New York, New York Serologic markers were evaluated to determine if they could aid in the differential diagnosis of pelvic inflammatory disease in 48 consecutive women seeking evaluation for pelvic pain. On the basis of clinical and microbiologic parameters, 29 patients (60.4%) were diagnosed as having pelvic inflammatory disease. Neisseria gonorrhoeae only was isolated from the cervix of eight (27.6%) patients with pelvic inflammatory disease, five (17.2%) had only Chlamydia, and two (6.9%) had Neisseria and Chlamydia, whereas in 15 (48.3%) patients no pathogen was isolated. Interferon--y was present in significantly more sera (p < 0.025) from patients with pelvic inflammatory ~isease (65.5%) than from women without pelvic inflammatory disease (15.8%). Sera from 10 healthy women lacked detectable interferon--y. In patients with only Neisseria, seven (87.5%) had circulating interferon--y; three (60%) of the women with only Chlamydia, one (50%) woman with Neisseria and Chlamydia, and eight (57.1%) with no identified pathogens were also positive for interferon--y. Sera from 11 of 28 patients with pelvic inflammatory disease (39%) but only one of 19 sera from women without pelvic inflammatory disease (5%) also inhibited the Candida-induced proliferation of control lymphocytes. This immunosuppressive activity was prevented by immunoprecipitation of interferon--y by anti-interferon--y antibody but not by treatment with anti-interferon-a antibody. The persistence of interferon--y in the sera of patients with pelvic inflammatory disease may aid in the differential diagnosis of this disease and increase our understanding of the pathogenesis of microbial-mediated tubal damage. (AM J OasTET GYNECOL 1989;160:26-31.)
Key words: Pelvic inflammatory disease, interferon--y, Chlamydia, Neisseria gonorrhoeae
From the DivlSZon of Immunology, Department of Obstetrics and Gynecology, Cornell Unzverszty Medzcal College. Presented at the Thirty-fifth Annual meetzng of the SOCIety for G.~ necologic Investigation, Baltzmore, Maryland, March 17-20,
1988.
Reprznt requests: Steven S. WItkin, PhD, Department of Obstetrzcs and Gynecology, Cornell Unzverszty Medzcal College, 515 E. 71st St., New York, NY 10021.
26
The difficulty in diagnosing pelvic inflammatory disease has been well documented. The routine use of laparoscopy by Jacobsen and Westrom' demonstrated the great inaccuracy of clinical diagnosis. Cervical cultures, while helpful when positive, have a relatively high false-negative rate, which makes culture-based diag-