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The occurrence of sperm antibodies in human reproduction
The occurrence of sperm antibodies in human reproduction I. Comparative new and improved antibody detection L.
METTLER
D.
SHIRWANI
T.
GRADL
test methods
for sperm
Kid. Ckmun?; The four essential methods for sperm antibody detection are considered and evaluated. The micro-sperm-agglutination and micro-sperm-immobilization tests have been improved and optimized. Quantitative estimation of sperm antibodies was made by an immunoelectrophoresis technique after separation of antibodies from the sera and genital tract secretions by affinity chromatography. Antibodies were typed by iii-rocket immunoelectrophoresis. The pathologic of the modified tests were found to be smafl and the biologic errors were tolerable. Satisfactory results were achieved for sperm antibodii belonging to tgA and IgM, but not for other immunogfobulins. (AM. J. OBSTET. GYNECOL. 136906, 1960.)
THE EARLY experiments of Metchnikoff’ and Metalnikoff’.’ many authors have shown that human spermatozoa can be agglutinated or immobilized by certain human sera.“+,’ Various test methods have been developed to measure the actilitv of sera to agglutinate or immobilize human spermatozoa, such as the macro-agglutination test of Kibrick. Belding, and Merrill,” the microagglutmatlon test of Franklin and Dukes,’ and the micro-sperm-immobilization test developed by Isqjima, Li, and Ashiraka.’ It can be srrn from recent critical reviews”. a. ‘-12 that such methods, often modified by different workers, har~e a low rate of reproducibility and make reliable comparison of results impossible. In male sera, for example, the macro-agglutination test” appears to have some correl,trion of‘ results but has proved unsuitable for female se~a. SINCE
From the Department oj Obstetrrcs and Gynecology, l’niwr$~ of Kiel. (Cnder the direction of Professor K. Sfvnr?! I Re(e/:vd
jar- pubiiratlon
.4ccepttd
Februa~
Januq
I!,
197X.
14, 1979
Reprint requests: Dr.med. Dr.habil. L. Mettkr. Department of Obstetrics and G,vnecology, 2300 GPt7ZOTl~.
106
Kiel,
Dr.
errors
The first World Health Organization (WHO) workshop on iso- and autoantibodies to human spermatozoa in Aarhus in 1974 tried to establish some unification in the techniques used.‘” A definite improvement resulted from incorporating the tray agglutination modification of the microsperm-agglutination test developed by Friberg” and the tray micro-sperm-immobilization test suggested by Husted and Hjort.‘” Boettcher and associatesI edited the results of an international comparative study of antibodies to spermatozoa and other antigens detected in sera of infertile patients deposited in the WHO reference bank of reproductive immunology. These had been presented at the Second WHO Workshop on the techniques for the detection of iso- and autoantibodies in the sera of infertile patients held at Aarhus, Denmark, in 1976. Initially, a definite improvement resulted in incorporating the tests.“, I5 However, details of reproducibility of the methods were not sufficiently maintained unless the technical requirements were strictly adhered to. This paper reviews the technical details inherent in the methods, with special emphasis on their reproducibility. Methods are also described for quantitation and typing of any antibodies found present.
0002-Y37Ri80/010106+1I$Ol.lb/O~
IWOThe
C.V.M<)~h~
Co.
Volume Number
136 1
Sperm
Table I. Results of 15 sperm antibody containing sera and three cervical investigations with five different donors on the same day Serum No.
Tray agglutination
Tray immobilization
antibodies
in human
reproduction.
I
107
mucus samples tested in three parallel RIE*
LRIE*
1
2
64 32
1x
32 64 128
2x 2x 1x
-
64 16
3x 2x
-
256 128
4x 1x
1,064 512 256
3x lx 1x
1X
-
6 7
256 512 1,064
1x 2x 2x
8
32
64 128
4x lx
256 512
3x 2x
+
*gM
+ +
w I&TM IgG
+
w
I@ 512
5x
512 1,064 2,128
lx lx 3x
I!&
-
5x
9
-
32 64
2x 3x
10
-
64 128
2x 3x
11
-
32 64 128
2x 2x lx
*gG +
*gG
+
I@
12
64 128
3x 2x
13
8 16 32
1x 3x 2x
-
+
wf
14
32 64
3x 2x
-
+
IF+
+
*gG
-
15 Cervical
132 256
2x 3x
mucus:
A
256 512
3x 2x
B
32 64
4x 1x
c
64
5x
*RIE, rocket
immunoelectrophoresis;
-
IgA %A
-
W
LRIE, line-rocket immunoelectrophoresis.
Material and methods Fifteen serum samples of female patients with sperm-agglutinating (n = 10) and sperm-immobilizing (n = 8) activity present, as ascertained by the microsperm-agglutinating? and micro-sperm-immobilizing* tests, were used to evaluate the reproducibility of the individual methods. Three sera had both agglutinating and immobilizing activity present. Three cervical mucus samples with sperm-agglutinating activity were examined as well.
The samples were selected from a group of 343 female patients with unknown causes of sterility. Samples from five different sperm donors had been tested in parallel on the same day and with the same donor on four different days to ensure quality control. Donor spermatozoa with motility under 75% and morphological abnormalities exceeding 5%, as well as samples containing leukocytes and cell debris, were discarded. Laboratory. The following methods were applied and optimal technical conditions established: (1) the
108
tvlettler,
Shitwani,
and Grad1
January 1, 1980 Am. J. Obstet. Gynecol.
Table II. Kesults of 15 sperm antibody containing sera and three cervical investigations with the same sperm donor on 4 different days Tray agglutination 64 128
lx 4x
32 128
1x IX
16 32
4x lx
256 128
3x 2x
256 1.004
3x 2x
256 512 1,064
3x lx lx
16 32
lx 4x
saline
(PBS):
. 2 Hz0
64
5x
256 512
4x lx -
RIE
LRIE
+
W
+
IgG
+
*@f IgG
+
IgG
+
IgM
256 512
2x 3x
+
I&
128 511 1,064
2x 2x lx
+
*gG W
+
w
32 64
lx 6x
+
W
128 256
4x lx
+
IgG
32 64 128
3x lx lx
+
I?5
+
I@
4x lx
8 16
3x 2x
+
W’
32 128
3x 2X
+
W’
+
IgG
+
*iit*
+
*Is*
+
*cc*
-
132
128 256
3x 2x
32 64
2x 3x
64
5x
micro-sperm-agglutination test as described by Friberg.’ ’ (2) the micro-sperm-immobilization test described as a rube-slide test by Isojima and associates’ and in tissue culture chambers by Husted and Hjort,‘” (3) affinity chromatography and rocket immunoelectrophoresis (RIE) (Axelsen, Kroll, and Weeke),” and (4) line-rocket immunoelectrophoresis (LRIE) (Kroll).” A number of buffers and solutions are required in the methodology. To avoid repetition they are described her<:: 4 X2 gm of’ Na,HPO,
immobilization
64 128
-
Phosphatr-hujjerrd
Tray
mucus samples tested in three parallel
5x
-
-
1.24 gm of KH,PO, 4.50 gm of NaCl in 1,000 ml of distilled Fructose
27.3 gm of 1.7 gm of 2.0 gm of 0.1 gm of Baker
water
bujjrr:
fructose Na,HPO, . 2 Hz0 NaCl KH*PO, in 1,000 ml of distilled
buffer:
30.0 gm of
D (+) glucose Hz0 3.06 gm of Na,HPO, . 2 Hz0 2.00 gm of NaCl 0.10 gm of KH2P04
water
Volume Number
136 1
Hank’s salt solution: commercial balanced salt solution, Serva, Heidelberg, Germany, and Gibco-Biocult, Glasgow, Great Britain TC 199 with Earle’s salts and L-glutamine: GibcoBiocult, Glasgow, Great Britain Fetal cay serum: Behring-Werke Marburg, Serva, Heidelberg, Germany Brinster medium: Gibco-Biocult, Glasgow, Great Britain, and Biological Company, New York Alsever solution, pH 6. I: 4.72 gm of N+HPO, 9 2 H,O 1.42 gm of citric acid . Hz0 20.5 gm of D (+) glucose . HZ0 4.2 gm of NaCl in 1,000 ml of distilled water I. Tray agglutination test. I4 Ejaculated spermatozoa from healthy donors (22 to 27 years of age) were incubated in glass tubes (inner diameter 1 cm) for 30 minutes at 37” C. Dead and immotile spermatozoa sedimented in the liquified ejaculate were discarded. In the supernatant fluid the sperm count was adjusted to 10 x lo6 spermatozoa/ml with glucose buffer. Then 2 ~1 of serum dilution (geometrical dilution series starting with 1: 4 dilution) were incubated with 1 ~1 of sperm dilution on tissue culture plates under paraffin oil in three parallel determinations (37” C, 90 minutes). The plates (M@ller coated, Moss, Norway) were incubated at 37” C for 90 minutes and examined under an inverted microscope at a magnification of x 100. Four steps were estimated: ali sperm agglutinated, +3; large sperm aggregates, few normal individually moving sperm, +2; small sperm aggregates, many individually moving sperm, + 1; no agglutinations, 0. The highest dilution revealing an agglutination of + 1 was considered to be the titer. At a magnification of x 600 the type of agglutination was determined (head-to-head, tail-to-tail, head-to-tail, tip-of-tail-to-tip-of-tail). In every experiment one known positive and one known negative serum were tested as reference points. The method was optimized for the time of sperm liquefication, sperm concentration, ratio of serum to sperm, incubation time, and dilution media for serum and sperm. The best conditions of each step were used to determine the methodologic and biologic variations. Ten positive agglutinating sera and three cervical mucus samples were tested with the sperm of five donors. All five donors showed good sperm motility (more than 75%) and a concentration of 50 to 80 . lo6 cells/ml. Sera were tested with the sperm of one donor on four different days and the sperm of five different donors on the same day. 2. Tray immobilization test. Is Sperm and serum dilu-
Sperm
antibodies
in human
reproduction.
I
109
1128" 1 64 '. 1 32
.
1 16 . 1 B .. I L .. 1 2 .' 1
2
3
Fig. 1. Effect of incubation
L
5
6
> t13ecrc
JiSi
time on sperm agglutination.
tions were prepared as described in above. Serum was inactivated at 57” C for 30 minutes. On tissue culture plates under paraffin oil, 1 ~1 of serum dilutions were mixed with 1 ~1 of sperm dilutions with a microsyringe (geometrical dilution steps in three parallels). The plates were incubated at 37” C for 1 hour. After this time 1~1 of guinea pig complement was added (guinea pig serum, Behring-Werke, Marburg, Germany). Following an additional hour of incubation at 37” C the degrees of immobilized spermatozoa were estimated under the inverted microscope at a magnification of x100. Four steps were estimated: 50% immotile sperm, 0; 50% immotile sperm, +; 75% immotile sperm, ++; 100% immotile sperm, + + + The highest dilution which revealed an immobilization of + in comparison to a negative control serum was considered to be the titer. Thus in every test one known positive and one known negative serum were tested in three parallel determinations. For optimization guinea pig complement was diluted in PBS and mixed in three parallel determinations with serum dilutions of a known sperm-immobilizing serum. In a one-step assay sperm, serum, and complement were mixed and incubated for 2 hours at 37” C. In a two-step assay serum and sperm were mixed and incubated for 1 hour at 37” C. Consecutively complement was added and the plates were incubated for an additional hour at 37” C. Finally the degree of immobilization was determined. Eight known sperm-immobilizing sera were tested with the sperm of five different donors. The sperm of one donor was used on 4 different days. 3. Affinity chromatography and RIE. AFFINITY
CHROMATOGRAPHY
(EXTRACTION
OF ANTI-
Spermatozoa were washed three times in PBS (centrifugation at 150 x g for 10 minutes at 15” C). At least 10’ spermatozoa were resuspended in 100 ~1 of serum and incubated at 37” C for 1 hour, followed by 2 BODIES).
110
Mettler,
Shitwani.
and Grad1 Am.
Titer
+
PBS
BAKER 1 -7; .>’
BRINSTER medium
TC 199 + /: -
._:
_’
Fig. 2. Effect i)f different media used to dilute serum in the \l~el-m-agglutrnarion test. hours at 4” (;. The sediment was washed twice with Baker buffer (150 x g, 10 minutes, 15” C) and resuspended in 100 ~1 of a dissociation solution for the elution of’ sperm-bound antibodies (10.0 gm of NaCl; 9.0 gm ol KH,PO,; 0.1 gm of Na,HPO., . 2Hz0 in 1,000 ml of distilled water) (pH 3.1). The mixture was incubated at 37” (; for I hour. After centrifugation the supernatant fluid containing the antibodies was quantified by RIF.. RIE (QUASTIFICATION OF ANTIBODIES). A 1% agarose gel Has prepared in Tris-citrate buffer, pH 9.4, at 95” C (!).2 gm of 7.ris-hydroxymethylaminomethan; 1.05 gm of’ citric. acicl-HZ0 in 1,000 ml of distilled water). After being cooled to 40 or 45” C, 8 ml of gel were mixed with 0.16 ml of rabbit antihuman serum (rabbit antiserum 1 s. human serum, Behring-Werke, Marburg, Germany). Glass plates, 10 by 10 cm, were covcred with this mixture. After solidification wells (2 mm in diameter) were cut 2 cm away from one edge. Then 3 ~1 of the &ted antibody solution were placed into thy wells with a microsyringe. Twenty minutes after filling the wells were sealed with one drop of agarose. Consecutively the plates were put into the electrophorests apparatus, where the buffer chambers contained T‘ris-citrate buffer. The connection between buffer and elec trode was maintained by a four-layered paper strip. The paper strips were sealed to the plates with liquid agarose. Electrophoresis was performed at 4 v/cm for S.5 hours. Then these plates were squeezed, washed. and stained with Coomassie blue.” The test results were multiplied by a iatio of the determined effective and theoretical values. A calibration curve reveals the antibody content. Purification of antibodies (affinity chromatography) was optimized with respect to the washing solutions for spermatozoa and to precipitate, the number of used spermatozoa for absorption, temperature for forming
January J. Oket.
1, 1980 Gvnecol.
the precipitate, and time and temperature for dissociation as well as composition of the dissociation solution. For immunoelectrophoresis the buffer system and the time were optimized. The recovery ratio for the purification step was determined. Optimal conditions of each step were used for the establishment of the calibration curves for IgA, IgM, and IgG. In seven or eight dilution steps the heights of the precipitation peaks for standard immunoglobulins, run in five parallel determinations, were determined (IgA, 50 IUiml = 0.84 mg/ml; IgM, 400 lU/ml = 3.5 mgiml; IgC, 22 IU/ml = 191 mg/ml; Behring-Werke, Marburg, Germany). In order to determine the methodologic and biologic standard deviations, 15 sera were examined with spermatozoa from four different donors for each. Spermatozoa of one donor were examined on 4 different days, of one donor on 3 different days, and of two donors only once. Every purification was performed in five parallel determinations. 4. Ident@&on ofantibody epe b LRIE. In eiectrophoresis IgG antibodies migrate toward the cathode, IgA and IgM antibodies to the anode. The differentiation between IgA and IgM antibodies was performed by LRIE.‘* From a 1% agarose gel in Tris-citrate buffer, pH 9.4, a strip 2 cm (wide) was cut along one side of the plate and filled with the eluted antibody solution (10%). In wells along the bottom line standard immunoglobulins were added (3 ~1). Electrophoresis was performed with 4 v/cm for 35 hours. Fusion of peak and line indicate the identity of the eluted antibody. The rest of the plate was covered with 6 ml of gel containing 2% antihuman rabbit serum.
Results The 15 selected sperm antibodies containing sera and cervical mucus samples revealed under the described optimal experimental conditions the results described in Tables I and II. In RIE all resulting precipitation peaks higher than 2 mm of migration were regarded as positive. lmmunoglobulins were not evaluated quantitatively. The three parallel investigations performed on the same day with one particular serum sample and one sperm donor always revealed identical results. Tray agglutination test. Timt, C$ liqu+ction. Aliquot sperm samples of one donor examined 5, 10, 20, 30, 45, 60, 120, and 180 minutes after ejaculation revealed no difference in sperm count of the supernate. In respect to sperm motility, an incubation period of 1 hour proved to be . optimal.
Volume 136 Number1
sperm concmtration. Comparing five different sperm concentrations ( 10S, 106. 10’. 2 X 107, and lo8 cells/ml) with respect to the titer of agglutination after incubation of 1 ~1 of sperm dilutions with 2 /*I of serum dilutions 10’ cells/ml revealed optimal results. Ratio of swum to spcrr~ The sperm agglutination titer was found to be substantially influenced by the ratio of serum to sperm. Beginning with ratios of 1: 100, 1 : 50, and 1: 10 (serum-sperm v/v) insignificant agglutination titers were registered. With ratios of 1: 5 and 1: 2 higher titers were achieved. The optimal titer resulted within a dilution range of 1: 1 and 5: 1 (serum-sperm v/v). Further reduction of sperm portions exerted a depressive effect on the agglutination reaction. Incubation time. The influence of incubation time on the sperm agglutination reaction was tested by studying agglutination titers of sperm antibody containing sera for time periods between 15 minutes and 6 hours (Fig. 1). The highest titers resulted after 4 hours. At this time, however, sperm motility is decreasing. Therefore an incubation time of 90 to 120 minutes was regarded as sufficient for proper evaluation. Dilution media for serum and sperm. With the use of different media for serum dilution (Fig. 2) Baker buffer revealed the highest agglutination titers. For sperm dilutions (Fig. 3) PBS, glucose buffer, fructose buffer, pyruvate buffer, Brinster medium, and TC 199 were compared. Optimal results were achieved by fructose buffer. With this buffer the motility of spermatozoa was, however, reduced. Therefore Baker buffer (glucose buffer) was selected for the test. The methodologic error (same serum, same sperm sample) amounted to * 1.6% (related to 2 log of titers). The biologic errors under different experimental conditions were: same serum, different days, different donors, 58.3%; same serum, different days, same donor, t 14.6%; same serum, same day, different donors, +- 12.5%; different sera, same day, same donor, -C2 1.2%. The differences in sperm quality (biologic error) from different donors and the differences in sperm quality from one donor on different days were similar. Due to these errors only sera with titers equal to or higher than 1: 8 were considered to be positive. Tray immobilization test. As indicated in Table III, in the one-step assay the highest titers were found at high concentrations of complement and serum. The two-step assay revealed a distribution pattern with two peaks, suggestive of a competitive reaction. The sperm immobilization reaction could be determined better in the two-step assay. The optimal ratio of serum to complement proved to be 1: 1 v/v. The methodologic error of this test examined in all eight positive sera with aliquot serum sam-
Sperm
PBS
antibodies
glucose Stiffer
in human
fructose buffer
Fig. 3. Effect of different agglutination reaction.
pyruva:e buiier
reproduction.
BRINSTER medium
I
111
TC 199 . ilet ;5 .,iFLP
media used to dilute sperm in the
ples and sperm samples of one donor on 1 day was less than 0.5% and thus is negligible. The biologic error, however, amounted to 28 to 35%: same serum, different days, different sperm donors, 229%; same serum, different days, same sperm donor, ?287&; same serum. same day, different sperm donors, ~35%. Titer differences between different sperm donors were more or less the same as between sperm samples from one donor on different days. Due to the rather high biologic error a serum, to be called positive, must show at least a positive reaction at a dilution of 1: 16. RIE.
Extraction EFFECT
of antibodies. OF DIFFERENT
WASHING
SOLUTIONS
ON
THE
The heights of the precipitation peaks (Fig. 4) were considered in relation to the different applied washing solutions. In all cases a total lo6 spermatozoa were used and washed twice with different media: PBS, Baker buffer, Hank’s solution, and distilled water. No immunoglobulins were absorbed with distilled water. Baker buffer, Hank’s solution, and PBS revealed no statistically significant heights of precipitation peaks. EXTENT
TOTAL
OF IMMUNOPRECIPITATION.
SPERM
COUNT
NECESSARY
FOR EXTRACTION
OF
In five parallel determinations 100 ~1 of serum samples with known sperm agglutinating activity were absorbed with a total of 103, 104, 105. 106, lo’, lOs, and 2 . 10s spermatozoa. The antibodies eluted were quantified by RIE. A total of lo4 spermatozoa was the lowest number giving a clear measurable precipitation peak (Fig. 5). The best reproducibility was found at a sperm count of lo6 spermatozoa. Temperature for absorption. The incubation of sperSPERM
ANTIBODIES.
112
Mettler,
Shirwani,
and Grad1
Table III. Effect of dilution immobilization test* Serum dilutions I: 128 I: 64 1 : 32 1 : 16 1. 8 I: 4 Compkm&
dilutions
January 1. 1980 Am. J. Obstet. Gynecol.
of serum or complement
on the one- and two-step
assay of the micro-sperm-
Two-step assay
I 1:64 1: 64 1:64 1: 128 1: 128 1:256
1:256 1:256 1: 128 I : 256 1: 256 1: 256
1: 128 1:64 1:64 1: 128 1: 128 1: 128
I:64 1:64 1: 128 1: 128 1:256 1:256
1:64 1:64 1: 128 1:64 1:64 I:32
1:64 1: 128 1:256 1:64 1:64 1:64
1: 32
1: 1
I:2
I:5
1: 10
1:20
1: 50
1: 100
I:64 1: 128 1: 64 1: 128 1:256
One-step assay
Serum diluhons
1: 128 1:64
1: 16 1: 16
1: 32 I:16 I:8 I:4
1:64
1: 128
I:512
1: 128
1:64
I:64
1:32 1~32 I:32 1:32
1~64 1:64 1:64 1: 128 1: 128
1~64 1: 128 1:64 1: 128 1: 128
1: 128 1: 128 I:64 1:64 1:64
1:64 I:64 I:64 1:64 1: 32
1132 1:64 1: 32 1:32 1:32
1: 32 1:64 1: 32 1:32 1: 32
1:1
1:2
1:5
1: 10
1: 30
1:50
1:lOO
*Titers of one sperm-immobilizing serum in relation to the amount of guinea pig complement added. In the one-step assay, serum, sperm, and complement were mixed. In the two-step assay sperm and serum were mixed first and complement was added I bow later. matozoa at different temperature levels (4”, 20”. and 37” C) for I hour followed by an incubation for 18 hours at 4” C yielded the maximal amount of antibodies at 37” C. Applying lower temperatures for this first step revealed only an incomplete absorption. Tirnt, for nbsorfition. The incubation for the first absorption at 37” C was performed over periods of 10, 30, 60, and 120 minutes and 4, 18, and 24 hours. The heights of the precipitation peaks revealed that the maximal amount ctt. antibodies desorbed was reached aftt.r 1 hour. For the second absorption at 4” C absorption times ot 15. 30, and 60 minutes and 2. 4, 5, 8, and 18 hours were compared. After absorption during the first 2 hours the heights of precipitation peaks increased. At 2 hours of absorption at 4” C maximal precipitation heights were achieved. Therefore the first absorption at 37” C for 1 hour and the second absorption at 4” (1 for 2 hours were considered to be most satisfartor). pH s~aluc,c jar dissociation solution. The heights of the precipitation peaks for the desorbed antibodies were determined in five parallel tests in relation to the applied pH values of the dissociation solution (Fig. 6). Hypersalinc buffers, 100 ~1, (15 gm of NaCl in 100 ml of buffer) of different pH values were used. For pH values of 7.2. 6, and 5 a phosphate buffer (O.OOSM Sorensen buffer) and for pH values of 4.3 and 2 a citrate buffer (0.12M McIlvaine buffer) was applied. In the range between pH 6 and 4 more or less the same degree of dissociation could be found. At a pH lower than 2 further dissociation took place. Testing
the purified antibodies after neutralization revealed that only the proteins dissociated between pH 6 and 4 are sperm antibodies showing an agglutinating or immobilizing activity. Molarity ofthr dissociation solution. In order to establish a range of molarities different amounts of NaCl (total molarity 0.066M. O.l52M, 0.238M, 0.409M, 0.923M, 2.636M, and 4.346M) were added to Sorensen buffer, pH 5.0 (O.OSSM). The heights of the precipitation peaks of antibodies desorbed were determined in five parallel tests in relation to the tested molarities of the dissociation solution. With Sorensen buffer only, no dissociation resulted. An optimal dissociation took place at 0.238M. T~mprrutur~ for dissociation. Dissociation was performed for 1 hour at different temperatures (4”. 20”, and 37” C). In five parallel tests the resulting precipitation heights were related to the applied temperatures. Although only minor differences were found, 37” C seems to be the best temperature for dissociation. Time for dissociation. At 37” C dissociation was performed at different times (10, 15, and 30 minutes and 1, 2, 4, 10, and 24 hours). The heights of the precipitation peaks in five parallel tests were related to the time of dissociation. After 30 minutes dissociation had reached its maximum. One hour, however, was used for technical reasons in our routine tests. Rr~vcry rak. In order to determine the recovery rate of the extracted antibody complex after dissociation the supernate was neutralized to pH 7.2 with 0.1 NaHC03 in five parallel assays. Then 100 ~1 of serum
Volume
136
Number
1
Sperm
antibodies
loI 102
PBS
BAKER ‘;GffC
HANKS soiutlc?
apuc.dest
lo3
10'
in human
16
lo6
reproduction.
'07
10e
I
kq n'Jmber
113
CT
Fig. 5. Effect of total sperm count on heights of precipitation peaks in immunoelectrophoresis of agglutinating sperm-antibodies desorbed. Mean values and standard deviations from five parallel determinations.
Fig. 4. Effects of different washing solutions on the extent of immunoprecipitation. without sperm antibodies were added and the purification procedure was repeated. The second supernate was again determined immlinoelectrophoretitally. For agglutinating sera a recovery rate of 91.3 f 1.7% could be obtained. Immunoelectrophoresis of extracted sperm antibodies buffer system applied in immunoelectrophoresis. Electrophoresis was performed with different buffer systems (Fig. 7) (Tris-HCl, pH 9.0; Tris-EDTA, pH 8.9; Tris-citrate, pH 9.4; glycin-NaOH, pH 9.4) for 1 hour at 8 v/cm applying 1~1 of IgM and IgA standard immunoglobulins as reference immunoglobulins. The heights of the resulting precipitation peaks in each buffer system were determined and related to the antibody concentrations. For IgA and IgM Tris-citrate buffer (pH 9.4) revealed the highest values in the.dose-response curve. Therefore this buffer was used in the following experiments. Time for immunoelectrophoresi. In Tris-citrate buffer, pH 9.4, immunoelectrophoresis (Fig. 8) of standard IgM and IgA was performed in five parallel determinations. Three dilution steps were applied and three time intervals (1, 2, and 4 hours) tested. For each interval the heights of the precipitation peaks in relation to the antibody concentration were determined. At 4 v/cm for 4 hours the best linearity of the dose-response curve as well as the peaks resulted. Calibration curve for ZgA, ZgM, and ZgG. In seven or eight dilution steps the heights of the precipitation peaks for standard immunoglobulins A, M, and G were
Fig. 6. Effect of the pH of the dissociation solution in cleaving the sperm-antibody complex revealed in the heights of immunoprecipitation in immunoelectrophoresis of sperm-agglutinating antibodies desorbed. Dissociation for 60 minutes at 37” C. Mean value and standard deviations from five parallel determinations. determined in five parallel tests (IgA, 50 IU/ml = 0.84 mgiml; IgM, 400 IUiml = 3.5 mg/ml; IgG, 22 IUI ml = 19 1 mg/ml; Behring-Werke, Marburg, Germany) (Figs. 9 to 11). The calibration curves of IgA and IgM cross the zero point, enabling the migration of rabbit antibodies versus antihuman serum to be selected. The calibration curve for IgA is linear up to a concentration of 1 mg/ml only. Higher concentrations should be diluted beforehand. The accuracy is sufficient. The calibration curve for IgG can be used only for a semiquantitative determination (migration to cathode). Since it is known that IgG, due to its electrophoretic heterogenicity, reveals similar inappropriate results in
14
Mettler,
Shitwani,
and Grad1
January Am. J. Obstet.
I, 1980 Gynecol.
10 t
I loo
w 2w
300
conceqlrcr
on CmglgAhl)
Fig. 7. Comparison of different buffer systems for the separation of IgM and IgA antibodies measured by the resulting heights of immunoprecipitates in immunoelectrophoresis of standard immunoglobulins (IgM and IgA) at different immunoglobulin levels. heqhts of PreClpltatlon (mm1
A 1
50-
hwhts
oi
t
I
L
100
200
>
300
: :--?
:‘c
.t--
:mg IgAiml)
Fig. 8. Effect of different separation times in the immunoelectrophoresis of IgM and IgA antibodies c)n the immunoprecipitation heights and concentration of standard immunoglobulins (IgM and IgA in Tris-citrate buffer). other buffer systems, a derivation, e.g.. by carbamylation,” should be performed. Metlmdologic and biologic errors. Three sera were extracted with spermatozoa from four different donot-s. Then the sperm samples of one donor were used on 4 different days, sperm samples of a second donor
on 3 different days, and sperm of the others for 1 day only. All determinations were performed in five parallel tests. The methodologic errors were negligible. The biologic error (same serum, same sperm donor, samples of different days) was -~7.1%. It was more or less equal to the error after using the same serum and
Volume Number
136 1
Sperm antibodies in human reproduction. I 115
15--
IO--
5--
Fig. 9. Calibration curve for the immunoelectrophoresis of IgA antibodies. Heights of immunoprecipitates in the immunoelectrophoresis of standard IgA in relation to the concentration.
Mean
values
and standard
deviations
from
five paral-
lel determinations. sperm samples same day.
of different
donors
(rt’7.9%)
Fig. 10. Calibration curve for the immunoelectrophoresis of IgM antibodies. Heights of immunoprecipitates in the immunoelectrophoresis of standard IgM in relation to the concentration. Mean values and standard deviations from five parallel determinations.
on the
Comment In sperm antibody work it is evident that there is an urgent need for standardization of techniques in order to compare results cited in the literature.5S ‘3 ii-~* i6, i*, I9 The sensitivity of the micro-sperm-agglutination method on tissue culture plates14 and its reproducibility seem to be better in this series than with the Franklin and Dukes test as found by the authors20 previously. Small amounts of sperm antibodies, however, can be determined only by immunoelectrophoresis and the determination following an antibody extraction allows the amount and character of the sperm specific antibodies to be determined but does not reveal their biologic effect. Traces of other serum proteins and immunoglobulins are also eluted with the extraction technique, thus preventing the desired quantitation of sperm antibodies with this method. The methodologic error for the micro-sperm-agglutination and immobilization tests as well as for the immunoelectrophoretic determination is reasonably small and only in the immunoelectrophoretic determination of IgG was the linearity of the calibration curves not sufficient. The fact that IgA antibodies were detected only in cervical mucus but never in serum agrees
Fig. 11. Calibration curve for the immunoelect;ophoresis of IgG antibodies. Heights of immunoprecipitates m the immunoelectrophoresis of standard IgG in relation to the concentration. Mean values and standard deviations from five determinations. Regression line (95% confidence level) from the same values.
116
t&tiller,
Shirwani,
and Grad1
January 1, 1980 Am. J. Obstet. Gynecol.
well with the results of Hulka and Omran,‘l who dettActc>d secretor)- IgA in the mucus membranes of the cervix The greatest error in the methods is the biologic error due to variation in sperm quality even from identical donora on different days. To use one or a few sperm donors ~tiith known good-quality sperm, as indicated by Friberg.” seemed to bring no improvement in the test results because the reactions of ejaculates from one donor on different days differ widely. To overcome this all tests were run in parallel. A known positivc and a kno\\n negative serum were always carried along and before assay the sperm material used was normed and characterized. Sperm motility, cell concentration. and morphology, as well as the absence of cell
detritus and foreign cells, were used as measures of this. Seminal plasma was not considered in the present study but investigation of its chemical characterization is urgently required, as its composition is closely linked to sperm motility. 22 The methodologic errors in this series were small and the biologic errors tolerable; the reproducibility and sensitivity of the sperm agglutination test were much better than using the sperm immobilization techniques. However, clinical correlation of these results needs to be explored further in a large screening study. We thank Ulla Bachmann-Diekmann, Renate Wittig, and E. Bertling for technical assistance and R. F. Harrison for help with the English text.
REFERENCES
Metchnikolf:
Etudes
sur la resorption
des cellules,
Ann.
Insi. Pasteur 13:737, 1899. Metalnikoff, S.: Etudes sur la spermatoxine, Pasteur 14~577, 1900. Shulman, S.: Tissue Specility and Autoimmunity,
Ann.
Inst. vol. 16,
New York. 1974, Springer Verlag. Jones, W. R.. Ing, R. M. Y., and Kaye, M. D.: A comparison ot‘ screening tests for antisperm activity in the serum 5.
6.
7.
8.
of infertile women, J. Reprod. Fertil. 32:357, 1973. Mettler, L.: Immunoiogie der weiblichen Sterilitit Fortschritte der Fertilitltsforschung, Band IV, Berlin, 1977, Grosse-Vedag. Kibrick, S.. Belding, D. L., and Merrill, B.: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatine agglutination test, Fertil. St&l. 3:430, 1952. Franklin, R. R.. and Dukes, C. D.: Antispermatozoa antibody and unexplained infertility. AM. J. OBSTET. GYstt.0~. 89~6. i964. Isojima. S . l-i. S. T., and Ashitaka, Y.: Immunologic analysis ol’ sperm-immobilizing factor found in sera of women Rith unexplained sterility, AM. J. OBSTET. GYNKOL.
9. Riimke.
101:677, 1968. P.: immunity
of sperm,
in Textbook
13. Rose, N. Vyazov, antibodies 23: 175, 14. Friberg,
J. K., and and autoImmunol. on sperm-
agglut&ing antibodies in sera and seminal fluid; Acta Obstet. Gvnecol. Stand. b:Su~ol. 36. 1974. 15. Husted, i,, and Hjort, T.: M&otechnique for detection of sperm immobilization and cytotoxicity, in WHO Workshop on Techniques for Detection of Iso- and Autoantibodies to Human Spermatozoa, Aarhus, 15-19 July, 1974. 16. Boettcher, B., Hjort, T., Riimke, P., Shulman, S., and Vyazov, 0. E.: Auto- and iso-antibodies to antigens of the human reproductive system, Acta Pathol. Microbial. Stand. Sect. C, Suppl. No. 258, 1977. 17. Axelsen, N. H., Kr@ll, J., and Weeke, B.: Quantitative
immunoelectrophoresis, 1973.
Stand. J. Immunol.
4:Suppl.
I,
18. Kr@ll, J.: Rocket line immunoelectrophoresis, Stand. J. Immunol. 2:Suppl. 1, 1973. 19. Boettcher, B., Kay, D. J., Riimke, P., and Wright, L. E.: Human sera containing immunoglobulin and nonim-
munoglobulin 1971.
of Immun-
pathology, Vol. II, 1973, pp. 669-674. S., and Koyama, K.: Quantitative determination IO. Isojima, of sperm-immobilizing antibody in women, in WHO Workshop on Techniques for Detection of Iso- and Autoantibodies to Human Spermatozoa, Aarhus. 15- 19 July, 1974. 11. Shulman, S.: Reproduction and Antibody Response, 1975, CRC-Press. 12. Scott, J. S., and Jones, W. R.: Immunology of Human Reproduction, New York, 1976, Grune & Stratton, Inc.
R., Hjort, T., Riimke, P., Harper, M. 0. E.: Technique for detection of isoto human spermatozoa, Clin. Exp. 1976. 1.: Clinical and immunological studies
20.
Mettler,
sperm-agglutinins,
L., and Gradl,
T.:
Biol. Reprod.
Difficulty
of obtaining
5:235, repro-
ducibility in the Franklin and Dukes test for the detection of sperm-agglutinating antibodies in human sera, .J. Reprod. Fertil. 44~217, 1975. 21. Hulka,
J. F., and
potential NECOL.161:440, 99 --.
Omran,
K. F.: The
local antibody secretor,
AM.
uterine
cervix
J. OBSTET.
as GY-
1969.
Eliasson, R.: Standards for investigation Andrologia 3:49, 197 1.
of human
semen,
METTLER
D.
SHIRWANI
T.
GRADL
test methods
for sperm
Kid. Ckmun?; The four essential methods for sperm antibody detection are considered and evaluated. The micro-sperm-agglutination and micro-sperm-immobilization tests have been improved and optimized. Quantitative estimation of sperm antibodies was made by an immunoelectrophoresis technique after separation of antibodies from the sera and genital tract secretions by affinity chromatography. Antibodies were typed by iii-rocket immunoelectrophoresis. The pathologic of the modified tests were found to be smafl and the biologic errors were tolerable. Satisfactory results were achieved for sperm antibodii belonging to tgA and IgM, but not for other immunogfobulins. (AM. J. OBSTET. GYNECOL. 136906, 1960.)
THE EARLY experiments of Metchnikoff’ and Metalnikoff’.’ many authors have shown that human spermatozoa can be agglutinated or immobilized by certain human sera.“+,’ Various test methods have been developed to measure the actilitv of sera to agglutinate or immobilize human spermatozoa, such as the macro-agglutination test of Kibrick. Belding, and Merrill,” the microagglutmatlon test of Franklin and Dukes,’ and the micro-sperm-immobilization test developed by Isqjima, Li, and Ashiraka.’ It can be srrn from recent critical reviews”. a. ‘-12 that such methods, often modified by different workers, har~e a low rate of reproducibility and make reliable comparison of results impossible. In male sera, for example, the macro-agglutination test” appears to have some correl,trion of‘ results but has proved unsuitable for female se~a. SINCE
From the Department oj Obstetrrcs and Gynecology, l’niwr$~ of Kiel. (Cnder the direction of Professor K. Sfvnr?! I Re(e/:vd
jar- pubiiratlon
.4ccepttd
Februa~
Januq
I!,
197X.
14, 1979
Reprint requests: Dr.med. Dr.habil. L. Mettkr. Department of Obstetrics and G,vnecology, 2300 GPt7ZOTl~.
106
Kiel,
Dr.
errors
The first World Health Organization (WHO) workshop on iso- and autoantibodies to human spermatozoa in Aarhus in 1974 tried to establish some unification in the techniques used.‘” A definite improvement resulted from incorporating the tray agglutination modification of the microsperm-agglutination test developed by Friberg” and the tray micro-sperm-immobilization test suggested by Husted and Hjort.‘” Boettcher and associatesI edited the results of an international comparative study of antibodies to spermatozoa and other antigens detected in sera of infertile patients deposited in the WHO reference bank of reproductive immunology. These had been presented at the Second WHO Workshop on the techniques for the detection of iso- and autoantibodies in the sera of infertile patients held at Aarhus, Denmark, in 1976. Initially, a definite improvement resulted in incorporating the tests.“, I5 However, details of reproducibility of the methods were not sufficiently maintained unless the technical requirements were strictly adhered to. This paper reviews the technical details inherent in the methods, with special emphasis on their reproducibility. Methods are also described for quantitation and typing of any antibodies found present.
0002-Y37Ri80/010106+1I$Ol.lb/O~
IWOThe
C.V.M<)~h~
Co.
Volume Number
136 1
Sperm
Table I. Results of 15 sperm antibody containing sera and three cervical investigations with five different donors on the same day Serum No.
Tray agglutination
Tray immobilization
antibodies
in human
reproduction.
I
107
mucus samples tested in three parallel RIE*
LRIE*
1
2
64 32
1x
32 64 128
2x 2x 1x
-
64 16
3x 2x
-
256 128
4x 1x
1,064 512 256
3x lx 1x
1X
-
6 7
256 512 1,064
1x 2x 2x
8
32
64 128
4x lx
256 512
3x 2x
+
*gM
+ +
w I&TM IgG
+
w
I@ 512
5x
512 1,064 2,128
lx lx 3x
I!&
-
5x
9
-
32 64
2x 3x
10
-
64 128
2x 3x
11
-
32 64 128
2x 2x lx
*gG +
*gG
+
I@
12
64 128
3x 2x
13
8 16 32
1x 3x 2x
-
+
wf
14
32 64
3x 2x
-
+
IF+
+
*gG
-
15 Cervical
132 256
2x 3x
mucus:
A
256 512
3x 2x
B
32 64
4x 1x
c
64
5x
*RIE, rocket
immunoelectrophoresis;
-
IgA %A
-
W
LRIE, line-rocket immunoelectrophoresis.
Material and methods Fifteen serum samples of female patients with sperm-agglutinating (n = 10) and sperm-immobilizing (n = 8) activity present, as ascertained by the microsperm-agglutinating? and micro-sperm-immobilizing* tests, were used to evaluate the reproducibility of the individual methods. Three sera had both agglutinating and immobilizing activity present. Three cervical mucus samples with sperm-agglutinating activity were examined as well.
The samples were selected from a group of 343 female patients with unknown causes of sterility. Samples from five different sperm donors had been tested in parallel on the same day and with the same donor on four different days to ensure quality control. Donor spermatozoa with motility under 75% and morphological abnormalities exceeding 5%, as well as samples containing leukocytes and cell debris, were discarded. Laboratory. The following methods were applied and optimal technical conditions established: (1) the
108
tvlettler,
Shitwani,
and Grad1
January 1, 1980 Am. J. Obstet. Gynecol.
Table II. Kesults of 15 sperm antibody containing sera and three cervical investigations with the same sperm donor on 4 different days Tray agglutination 64 128
lx 4x
32 128
1x IX
16 32
4x lx
256 128
3x 2x
256 1.004
3x 2x
256 512 1,064
3x lx lx
16 32
lx 4x
saline
(PBS):
. 2 Hz0
64
5x
256 512
4x lx -
RIE
LRIE
+
W
+
IgG
+
*@f IgG
+
IgG
+
IgM
256 512
2x 3x
+
I&
128 511 1,064
2x 2x lx
+
*gG W
+
w
32 64
lx 6x
+
W
128 256
4x lx
+
IgG
32 64 128
3x lx lx
+
I?5
+
I@
4x lx
8 16
3x 2x
+
W’
32 128
3x 2X
+
W’
+
IgG
+
*iit*
+
*Is*
+
*cc*
-
132
128 256
3x 2x
32 64
2x 3x
64
5x
micro-sperm-agglutination test as described by Friberg.’ ’ (2) the micro-sperm-immobilization test described as a rube-slide test by Isojima and associates’ and in tissue culture chambers by Husted and Hjort,‘” (3) affinity chromatography and rocket immunoelectrophoresis (RIE) (Axelsen, Kroll, and Weeke),” and (4) line-rocket immunoelectrophoresis (LRIE) (Kroll).” A number of buffers and solutions are required in the methodology. To avoid repetition they are described her<:: 4 X2 gm of’ Na,HPO,
immobilization
64 128
-
Phosphatr-hujjerrd
Tray
mucus samples tested in three parallel
5x
-
-
1.24 gm of KH,PO, 4.50 gm of NaCl in 1,000 ml of distilled Fructose
27.3 gm of 1.7 gm of 2.0 gm of 0.1 gm of Baker
water
bujjrr:
fructose Na,HPO, . 2 Hz0 NaCl KH*PO, in 1,000 ml of distilled
buffer:
30.0 gm of
D (+) glucose Hz0 3.06 gm of Na,HPO, . 2 Hz0 2.00 gm of NaCl 0.10 gm of KH2P04
water
Volume Number
136 1
Hank’s salt solution: commercial balanced salt solution, Serva, Heidelberg, Germany, and Gibco-Biocult, Glasgow, Great Britain TC 199 with Earle’s salts and L-glutamine: GibcoBiocult, Glasgow, Great Britain Fetal cay serum: Behring-Werke Marburg, Serva, Heidelberg, Germany Brinster medium: Gibco-Biocult, Glasgow, Great Britain, and Biological Company, New York Alsever solution, pH 6. I: 4.72 gm of N+HPO, 9 2 H,O 1.42 gm of citric acid . Hz0 20.5 gm of D (+) glucose . HZ0 4.2 gm of NaCl in 1,000 ml of distilled water I. Tray agglutination test. I4 Ejaculated spermatozoa from healthy donors (22 to 27 years of age) were incubated in glass tubes (inner diameter 1 cm) for 30 minutes at 37” C. Dead and immotile spermatozoa sedimented in the liquified ejaculate were discarded. In the supernatant fluid the sperm count was adjusted to 10 x lo6 spermatozoa/ml with glucose buffer. Then 2 ~1 of serum dilution (geometrical dilution series starting with 1: 4 dilution) were incubated with 1 ~1 of sperm dilution on tissue culture plates under paraffin oil in three parallel determinations (37” C, 90 minutes). The plates (M@ller coated, Moss, Norway) were incubated at 37” C for 90 minutes and examined under an inverted microscope at a magnification of x 100. Four steps were estimated: ali sperm agglutinated, +3; large sperm aggregates, few normal individually moving sperm, +2; small sperm aggregates, many individually moving sperm, + 1; no agglutinations, 0. The highest dilution revealing an agglutination of + 1 was considered to be the titer. At a magnification of x 600 the type of agglutination was determined (head-to-head, tail-to-tail, head-to-tail, tip-of-tail-to-tip-of-tail). In every experiment one known positive and one known negative serum were tested as reference points. The method was optimized for the time of sperm liquefication, sperm concentration, ratio of serum to sperm, incubation time, and dilution media for serum and sperm. The best conditions of each step were used to determine the methodologic and biologic variations. Ten positive agglutinating sera and three cervical mucus samples were tested with the sperm of five donors. All five donors showed good sperm motility (more than 75%) and a concentration of 50 to 80 . lo6 cells/ml. Sera were tested with the sperm of one donor on four different days and the sperm of five different donors on the same day. 2. Tray immobilization test. Is Sperm and serum dilu-
Sperm
antibodies
in human
reproduction.
I
109
1128" 1 64 '. 1 32
.
1 16 . 1 B .. I L .. 1 2 .' 1
2
3
Fig. 1. Effect of incubation
L
5
6
> t13ecrc
JiSi
time on sperm agglutination.
tions were prepared as described in above. Serum was inactivated at 57” C for 30 minutes. On tissue culture plates under paraffin oil, 1 ~1 of serum dilutions were mixed with 1 ~1 of sperm dilutions with a microsyringe (geometrical dilution steps in three parallels). The plates were incubated at 37” C for 1 hour. After this time 1~1 of guinea pig complement was added (guinea pig serum, Behring-Werke, Marburg, Germany). Following an additional hour of incubation at 37” C the degrees of immobilized spermatozoa were estimated under the inverted microscope at a magnification of x100. Four steps were estimated: 50% immotile sperm, 0; 50% immotile sperm, +; 75% immotile sperm, ++; 100% immotile sperm, + + + The highest dilution which revealed an immobilization of + in comparison to a negative control serum was considered to be the titer. Thus in every test one known positive and one known negative serum were tested in three parallel determinations. For optimization guinea pig complement was diluted in PBS and mixed in three parallel determinations with serum dilutions of a known sperm-immobilizing serum. In a one-step assay sperm, serum, and complement were mixed and incubated for 2 hours at 37” C. In a two-step assay serum and sperm were mixed and incubated for 1 hour at 37” C. Consecutively complement was added and the plates were incubated for an additional hour at 37” C. Finally the degree of immobilization was determined. Eight known sperm-immobilizing sera were tested with the sperm of five different donors. The sperm of one donor was used on 4 different days. 3. Affinity chromatography and RIE. AFFINITY
CHROMATOGRAPHY
(EXTRACTION
OF ANTI-
Spermatozoa were washed three times in PBS (centrifugation at 150 x g for 10 minutes at 15” C). At least 10’ spermatozoa were resuspended in 100 ~1 of serum and incubated at 37” C for 1 hour, followed by 2 BODIES).
110
Mettler,
Shitwani.
and Grad1 Am.
Titer
+
PBS
BAKER 1 -7; .>’
BRINSTER medium
TC 199 + /: -
._:
_’
Fig. 2. Effect i)f different media used to dilute serum in the \l~el-m-agglutrnarion test. hours at 4” (;. The sediment was washed twice with Baker buffer (150 x g, 10 minutes, 15” C) and resuspended in 100 ~1 of a dissociation solution for the elution of’ sperm-bound antibodies (10.0 gm of NaCl; 9.0 gm ol KH,PO,; 0.1 gm of Na,HPO., . 2Hz0 in 1,000 ml of distilled water) (pH 3.1). The mixture was incubated at 37” (; for I hour. After centrifugation the supernatant fluid containing the antibodies was quantified by RIF.. RIE (QUASTIFICATION OF ANTIBODIES). A 1% agarose gel Has prepared in Tris-citrate buffer, pH 9.4, at 95” C (!).2 gm of 7.ris-hydroxymethylaminomethan; 1.05 gm of’ citric. acicl-HZ0 in 1,000 ml of distilled water). After being cooled to 40 or 45” C, 8 ml of gel were mixed with 0.16 ml of rabbit antihuman serum (rabbit antiserum 1 s. human serum, Behring-Werke, Marburg, Germany). Glass plates, 10 by 10 cm, were covcred with this mixture. After solidification wells (2 mm in diameter) were cut 2 cm away from one edge. Then 3 ~1 of the &ted antibody solution were placed into thy wells with a microsyringe. Twenty minutes after filling the wells were sealed with one drop of agarose. Consecutively the plates were put into the electrophorests apparatus, where the buffer chambers contained T‘ris-citrate buffer. The connection between buffer and elec trode was maintained by a four-layered paper strip. The paper strips were sealed to the plates with liquid agarose. Electrophoresis was performed at 4 v/cm for S.5 hours. Then these plates were squeezed, washed. and stained with Coomassie blue.” The test results were multiplied by a iatio of the determined effective and theoretical values. A calibration curve reveals the antibody content. Purification of antibodies (affinity chromatography) was optimized with respect to the washing solutions for spermatozoa and to precipitate, the number of used spermatozoa for absorption, temperature for forming
January J. Oket.
1, 1980 Gvnecol.
the precipitate, and time and temperature for dissociation as well as composition of the dissociation solution. For immunoelectrophoresis the buffer system and the time were optimized. The recovery ratio for the purification step was determined. Optimal conditions of each step were used for the establishment of the calibration curves for IgA, IgM, and IgG. In seven or eight dilution steps the heights of the precipitation peaks for standard immunoglobulins, run in five parallel determinations, were determined (IgA, 50 IUiml = 0.84 mg/ml; IgM, 400 lU/ml = 3.5 mgiml; IgC, 22 IU/ml = 191 mg/ml; Behring-Werke, Marburg, Germany). In order to determine the methodologic and biologic standard deviations, 15 sera were examined with spermatozoa from four different donors for each. Spermatozoa of one donor were examined on 4 different days, of one donor on 3 different days, and of two donors only once. Every purification was performed in five parallel determinations. 4. Ident@&on ofantibody epe b LRIE. In eiectrophoresis IgG antibodies migrate toward the cathode, IgA and IgM antibodies to the anode. The differentiation between IgA and IgM antibodies was performed by LRIE.‘* From a 1% agarose gel in Tris-citrate buffer, pH 9.4, a strip 2 cm (wide) was cut along one side of the plate and filled with the eluted antibody solution (10%). In wells along the bottom line standard immunoglobulins were added (3 ~1). Electrophoresis was performed with 4 v/cm for 35 hours. Fusion of peak and line indicate the identity of the eluted antibody. The rest of the plate was covered with 6 ml of gel containing 2% antihuman rabbit serum.
Results The 15 selected sperm antibodies containing sera and cervical mucus samples revealed under the described optimal experimental conditions the results described in Tables I and II. In RIE all resulting precipitation peaks higher than 2 mm of migration were regarded as positive. lmmunoglobulins were not evaluated quantitatively. The three parallel investigations performed on the same day with one particular serum sample and one sperm donor always revealed identical results. Tray agglutination test. Timt, C$ liqu+ction. Aliquot sperm samples of one donor examined 5, 10, 20, 30, 45, 60, 120, and 180 minutes after ejaculation revealed no difference in sperm count of the supernate. In respect to sperm motility, an incubation period of 1 hour proved to be . optimal.
Volume 136 Number1
sperm concmtration. Comparing five different sperm concentrations ( 10S, 106. 10’. 2 X 107, and lo8 cells/ml) with respect to the titer of agglutination after incubation of 1 ~1 of sperm dilutions with 2 /*I of serum dilutions 10’ cells/ml revealed optimal results. Ratio of swum to spcrr~ The sperm agglutination titer was found to be substantially influenced by the ratio of serum to sperm. Beginning with ratios of 1: 100, 1 : 50, and 1: 10 (serum-sperm v/v) insignificant agglutination titers were registered. With ratios of 1: 5 and 1: 2 higher titers were achieved. The optimal titer resulted within a dilution range of 1: 1 and 5: 1 (serum-sperm v/v). Further reduction of sperm portions exerted a depressive effect on the agglutination reaction. Incubation time. The influence of incubation time on the sperm agglutination reaction was tested by studying agglutination titers of sperm antibody containing sera for time periods between 15 minutes and 6 hours (Fig. 1). The highest titers resulted after 4 hours. At this time, however, sperm motility is decreasing. Therefore an incubation time of 90 to 120 minutes was regarded as sufficient for proper evaluation. Dilution media for serum and sperm. With the use of different media for serum dilution (Fig. 2) Baker buffer revealed the highest agglutination titers. For sperm dilutions (Fig. 3) PBS, glucose buffer, fructose buffer, pyruvate buffer, Brinster medium, and TC 199 were compared. Optimal results were achieved by fructose buffer. With this buffer the motility of spermatozoa was, however, reduced. Therefore Baker buffer (glucose buffer) was selected for the test. The methodologic error (same serum, same sperm sample) amounted to * 1.6% (related to 2 log of titers). The biologic errors under different experimental conditions were: same serum, different days, different donors, 58.3%; same serum, different days, same donor, t 14.6%; same serum, same day, different donors, +- 12.5%; different sera, same day, same donor, -C2 1.2%. The differences in sperm quality (biologic error) from different donors and the differences in sperm quality from one donor on different days were similar. Due to these errors only sera with titers equal to or higher than 1: 8 were considered to be positive. Tray immobilization test. As indicated in Table III, in the one-step assay the highest titers were found at high concentrations of complement and serum. The two-step assay revealed a distribution pattern with two peaks, suggestive of a competitive reaction. The sperm immobilization reaction could be determined better in the two-step assay. The optimal ratio of serum to complement proved to be 1: 1 v/v. The methodologic error of this test examined in all eight positive sera with aliquot serum sam-
Sperm
PBS
antibodies
glucose Stiffer
in human
fructose buffer
Fig. 3. Effect of different agglutination reaction.
pyruva:e buiier
reproduction.
BRINSTER medium
I
111
TC 199 . ilet ;5 .,iFLP
media used to dilute sperm in the
ples and sperm samples of one donor on 1 day was less than 0.5% and thus is negligible. The biologic error, however, amounted to 28 to 35%: same serum, different days, different sperm donors, 229%; same serum, different days, same sperm donor, ?287&; same serum. same day, different sperm donors, ~35%. Titer differences between different sperm donors were more or less the same as between sperm samples from one donor on different days. Due to the rather high biologic error a serum, to be called positive, must show at least a positive reaction at a dilution of 1: 16. RIE.
Extraction EFFECT
of antibodies. OF DIFFERENT
WASHING
SOLUTIONS
ON
THE
The heights of the precipitation peaks (Fig. 4) were considered in relation to the different applied washing solutions. In all cases a total lo6 spermatozoa were used and washed twice with different media: PBS, Baker buffer, Hank’s solution, and distilled water. No immunoglobulins were absorbed with distilled water. Baker buffer, Hank’s solution, and PBS revealed no statistically significant heights of precipitation peaks. EXTENT
TOTAL
OF IMMUNOPRECIPITATION.
SPERM
COUNT
NECESSARY
FOR EXTRACTION
OF
In five parallel determinations 100 ~1 of serum samples with known sperm agglutinating activity were absorbed with a total of 103, 104, 105. 106, lo’, lOs, and 2 . 10s spermatozoa. The antibodies eluted were quantified by RIE. A total of lo4 spermatozoa was the lowest number giving a clear measurable precipitation peak (Fig. 5). The best reproducibility was found at a sperm count of lo6 spermatozoa. Temperature for absorption. The incubation of sperSPERM
ANTIBODIES.
112
Mettler,
Shirwani,
and Grad1
Table III. Effect of dilution immobilization test* Serum dilutions I: 128 I: 64 1 : 32 1 : 16 1. 8 I: 4 Compkm&
dilutions
January 1. 1980 Am. J. Obstet. Gynecol.
of serum or complement
on the one- and two-step
assay of the micro-sperm-
Two-step assay
I 1:64 1: 64 1:64 1: 128 1: 128 1:256
1:256 1:256 1: 128 I : 256 1: 256 1: 256
1: 128 1:64 1:64 1: 128 1: 128 1: 128
I:64 1:64 1: 128 1: 128 1:256 1:256
1:64 1:64 1: 128 1:64 1:64 I:32
1:64 1: 128 1:256 1:64 1:64 1:64
1: 32
1: 1
I:2
I:5
1: 10
1:20
1: 50
1: 100
I:64 1: 128 1: 64 1: 128 1:256
One-step assay
Serum diluhons
1: 128 1:64
1: 16 1: 16
1: 32 I:16 I:8 I:4
1:64
1: 128
I:512
1: 128
1:64
I:64
1:32 1~32 I:32 1:32
1~64 1:64 1:64 1: 128 1: 128
1~64 1: 128 1:64 1: 128 1: 128
1: 128 1: 128 I:64 1:64 1:64
1:64 I:64 I:64 1:64 1: 32
1132 1:64 1: 32 1:32 1:32
1: 32 1:64 1: 32 1:32 1: 32
1:1
1:2
1:5
1: 10
1: 30
1:50
1:lOO
*Titers of one sperm-immobilizing serum in relation to the amount of guinea pig complement added. In the one-step assay, serum, sperm, and complement were mixed. In the two-step assay sperm and serum were mixed first and complement was added I bow later. matozoa at different temperature levels (4”, 20”. and 37” C) for I hour followed by an incubation for 18 hours at 4” C yielded the maximal amount of antibodies at 37” C. Applying lower temperatures for this first step revealed only an incomplete absorption. Tirnt, for nbsorfition. The incubation for the first absorption at 37” C was performed over periods of 10, 30, 60, and 120 minutes and 4, 18, and 24 hours. The heights of the precipitation peaks revealed that the maximal amount ctt. antibodies desorbed was reached aftt.r 1 hour. For the second absorption at 4” C absorption times ot 15. 30, and 60 minutes and 2. 4, 5, 8, and 18 hours were compared. After absorption during the first 2 hours the heights of precipitation peaks increased. At 2 hours of absorption at 4” C maximal precipitation heights were achieved. Therefore the first absorption at 37” C for 1 hour and the second absorption at 4” (1 for 2 hours were considered to be most satisfartor). pH s~aluc,c jar dissociation solution. The heights of the precipitation peaks for the desorbed antibodies were determined in five parallel tests in relation to the applied pH values of the dissociation solution (Fig. 6). Hypersalinc buffers, 100 ~1, (15 gm of NaCl in 100 ml of buffer) of different pH values were used. For pH values of 7.2. 6, and 5 a phosphate buffer (O.OOSM Sorensen buffer) and for pH values of 4.3 and 2 a citrate buffer (0.12M McIlvaine buffer) was applied. In the range between pH 6 and 4 more or less the same degree of dissociation could be found. At a pH lower than 2 further dissociation took place. Testing
the purified antibodies after neutralization revealed that only the proteins dissociated between pH 6 and 4 are sperm antibodies showing an agglutinating or immobilizing activity. Molarity ofthr dissociation solution. In order to establish a range of molarities different amounts of NaCl (total molarity 0.066M. O.l52M, 0.238M, 0.409M, 0.923M, 2.636M, and 4.346M) were added to Sorensen buffer, pH 5.0 (O.OSSM). The heights of the precipitation peaks of antibodies desorbed were determined in five parallel tests in relation to the tested molarities of the dissociation solution. With Sorensen buffer only, no dissociation resulted. An optimal dissociation took place at 0.238M. T~mprrutur~ for dissociation. Dissociation was performed for 1 hour at different temperatures (4”. 20”, and 37” C). In five parallel tests the resulting precipitation heights were related to the applied temperatures. Although only minor differences were found, 37” C seems to be the best temperature for dissociation. Time for dissociation. At 37” C dissociation was performed at different times (10, 15, and 30 minutes and 1, 2, 4, 10, and 24 hours). The heights of the precipitation peaks in five parallel tests were related to the time of dissociation. After 30 minutes dissociation had reached its maximum. One hour, however, was used for technical reasons in our routine tests. Rr~vcry rak. In order to determine the recovery rate of the extracted antibody complex after dissociation the supernate was neutralized to pH 7.2 with 0.1 NaHC03 in five parallel assays. Then 100 ~1 of serum
Volume
136
Number
1
Sperm
antibodies
loI 102
PBS
BAKER ‘;GffC
HANKS soiutlc?
apuc.dest
lo3
10'
in human
16
lo6
reproduction.
'07
10e
I
kq n'Jmber
113
CT
Fig. 5. Effect of total sperm count on heights of precipitation peaks in immunoelectrophoresis of agglutinating sperm-antibodies desorbed. Mean values and standard deviations from five parallel determinations.
Fig. 4. Effects of different washing solutions on the extent of immunoprecipitation. without sperm antibodies were added and the purification procedure was repeated. The second supernate was again determined immlinoelectrophoretitally. For agglutinating sera a recovery rate of 91.3 f 1.7% could be obtained. Immunoelectrophoresis of extracted sperm antibodies buffer system applied in immunoelectrophoresis. Electrophoresis was performed with different buffer systems (Fig. 7) (Tris-HCl, pH 9.0; Tris-EDTA, pH 8.9; Tris-citrate, pH 9.4; glycin-NaOH, pH 9.4) for 1 hour at 8 v/cm applying 1~1 of IgM and IgA standard immunoglobulins as reference immunoglobulins. The heights of the resulting precipitation peaks in each buffer system were determined and related to the antibody concentrations. For IgA and IgM Tris-citrate buffer (pH 9.4) revealed the highest values in the.dose-response curve. Therefore this buffer was used in the following experiments. Time for immunoelectrophoresi. In Tris-citrate buffer, pH 9.4, immunoelectrophoresis (Fig. 8) of standard IgM and IgA was performed in five parallel determinations. Three dilution steps were applied and three time intervals (1, 2, and 4 hours) tested. For each interval the heights of the precipitation peaks in relation to the antibody concentration were determined. At 4 v/cm for 4 hours the best linearity of the dose-response curve as well as the peaks resulted. Calibration curve for ZgA, ZgM, and ZgG. In seven or eight dilution steps the heights of the precipitation peaks for standard immunoglobulins A, M, and G were
Fig. 6. Effect of the pH of the dissociation solution in cleaving the sperm-antibody complex revealed in the heights of immunoprecipitation in immunoelectrophoresis of sperm-agglutinating antibodies desorbed. Dissociation for 60 minutes at 37” C. Mean value and standard deviations from five parallel determinations. determined in five parallel tests (IgA, 50 IU/ml = 0.84 mgiml; IgM, 400 IUiml = 3.5 mg/ml; IgG, 22 IUI ml = 19 1 mg/ml; Behring-Werke, Marburg, Germany) (Figs. 9 to 11). The calibration curves of IgA and IgM cross the zero point, enabling the migration of rabbit antibodies versus antihuman serum to be selected. The calibration curve for IgA is linear up to a concentration of 1 mg/ml only. Higher concentrations should be diluted beforehand. The accuracy is sufficient. The calibration curve for IgG can be used only for a semiquantitative determination (migration to cathode). Since it is known that IgG, due to its electrophoretic heterogenicity, reveals similar inappropriate results in
14
Mettler,
Shitwani,
and Grad1
January Am. J. Obstet.
I, 1980 Gynecol.
10 t
I loo
w 2w
300
conceqlrcr
on CmglgAhl)
Fig. 7. Comparison of different buffer systems for the separation of IgM and IgA antibodies measured by the resulting heights of immunoprecipitates in immunoelectrophoresis of standard immunoglobulins (IgM and IgA) at different immunoglobulin levels. heqhts of PreClpltatlon (mm1
A 1
50-
hwhts
oi
t
I
L
100
200
>
300
: :--?
:‘c
.t--
:mg IgAiml)
Fig. 8. Effect of different separation times in the immunoelectrophoresis of IgM and IgA antibodies c)n the immunoprecipitation heights and concentration of standard immunoglobulins (IgM and IgA in Tris-citrate buffer). other buffer systems, a derivation, e.g.. by carbamylation,” should be performed. Metlmdologic and biologic errors. Three sera were extracted with spermatozoa from four different donot-s. Then the sperm samples of one donor were used on 4 different days, sperm samples of a second donor
on 3 different days, and sperm of the others for 1 day only. All determinations were performed in five parallel tests. The methodologic errors were negligible. The biologic error (same serum, same sperm donor, samples of different days) was -~7.1%. It was more or less equal to the error after using the same serum and
Volume Number
136 1
Sperm antibodies in human reproduction. I 115
15--
IO--
5--
Fig. 9. Calibration curve for the immunoelectrophoresis of IgA antibodies. Heights of immunoprecipitates in the immunoelectrophoresis of standard IgA in relation to the concentration.
Mean
values
and standard
deviations
from
five paral-
lel determinations. sperm samples same day.
of different
donors
(rt’7.9%)
Fig. 10. Calibration curve for the immunoelectrophoresis of IgM antibodies. Heights of immunoprecipitates in the immunoelectrophoresis of standard IgM in relation to the concentration. Mean values and standard deviations from five parallel determinations.
on the
Comment In sperm antibody work it is evident that there is an urgent need for standardization of techniques in order to compare results cited in the literature.5S ‘3 ii-~* i6, i*, I9 The sensitivity of the micro-sperm-agglutination method on tissue culture plates14 and its reproducibility seem to be better in this series than with the Franklin and Dukes test as found by the authors20 previously. Small amounts of sperm antibodies, however, can be determined only by immunoelectrophoresis and the determination following an antibody extraction allows the amount and character of the sperm specific antibodies to be determined but does not reveal their biologic effect. Traces of other serum proteins and immunoglobulins are also eluted with the extraction technique, thus preventing the desired quantitation of sperm antibodies with this method. The methodologic error for the micro-sperm-agglutination and immobilization tests as well as for the immunoelectrophoretic determination is reasonably small and only in the immunoelectrophoretic determination of IgG was the linearity of the calibration curves not sufficient. The fact that IgA antibodies were detected only in cervical mucus but never in serum agrees
Fig. 11. Calibration curve for the immunoelect;ophoresis of IgG antibodies. Heights of immunoprecipitates m the immunoelectrophoresis of standard IgG in relation to the concentration. Mean values and standard deviations from five determinations. Regression line (95% confidence level) from the same values.
116
t&tiller,
Shirwani,
and Grad1
January 1, 1980 Am. J. Obstet. Gynecol.
well with the results of Hulka and Omran,‘l who dettActc>d secretor)- IgA in the mucus membranes of the cervix The greatest error in the methods is the biologic error due to variation in sperm quality even from identical donora on different days. To use one or a few sperm donors ~tiith known good-quality sperm, as indicated by Friberg.” seemed to bring no improvement in the test results because the reactions of ejaculates from one donor on different days differ widely. To overcome this all tests were run in parallel. A known positivc and a kno\\n negative serum were always carried along and before assay the sperm material used was normed and characterized. Sperm motility, cell concentration. and morphology, as well as the absence of cell
detritus and foreign cells, were used as measures of this. Seminal plasma was not considered in the present study but investigation of its chemical characterization is urgently required, as its composition is closely linked to sperm motility. 22 The methodologic errors in this series were small and the biologic errors tolerable; the reproducibility and sensitivity of the sperm agglutination test were much better than using the sperm immobilization techniques. However, clinical correlation of these results needs to be explored further in a large screening study. We thank Ulla Bachmann-Diekmann, Renate Wittig, and E. Bertling for technical assistance and R. F. Harrison for help with the English text.
REFERENCES
Metchnikolf:
Etudes
sur la resorption
des cellules,
Ann.
Insi. Pasteur 13:737, 1899. Metalnikoff, S.: Etudes sur la spermatoxine, Pasteur 14~577, 1900. Shulman, S.: Tissue Specility and Autoimmunity,
Ann.
Inst. vol. 16,
New York. 1974, Springer Verlag. Jones, W. R.. Ing, R. M. Y., and Kaye, M. D.: A comparison ot‘ screening tests for antisperm activity in the serum 5.
6.
7.
8.
of infertile women, J. Reprod. Fertil. 32:357, 1973. Mettler, L.: Immunoiogie der weiblichen Sterilitit Fortschritte der Fertilitltsforschung, Band IV, Berlin, 1977, Grosse-Vedag. Kibrick, S.. Belding, D. L., and Merrill, B.: Methods for the detection of antibodies against mammalian spermatozoa. II. A gelatine agglutination test, Fertil. St&l. 3:430, 1952. Franklin, R. R.. and Dukes, C. D.: Antispermatozoa antibody and unexplained infertility. AM. J. OBSTET. GYstt.0~. 89~6. i964. Isojima. S . l-i. S. T., and Ashitaka, Y.: Immunologic analysis ol’ sperm-immobilizing factor found in sera of women Rith unexplained sterility, AM. J. OBSTET. GYNKOL.
9. Riimke.
101:677, 1968. P.: immunity
of sperm,
in Textbook
13. Rose, N. Vyazov, antibodies 23: 175, 14. Friberg,
J. K., and and autoImmunol. on sperm-
agglut&ing antibodies in sera and seminal fluid; Acta Obstet. Gvnecol. Stand. b:Su~ol. 36. 1974. 15. Husted, i,, and Hjort, T.: M&otechnique for detection of sperm immobilization and cytotoxicity, in WHO Workshop on Techniques for Detection of Iso- and Autoantibodies to Human Spermatozoa, Aarhus, 15-19 July, 1974. 16. Boettcher, B., Hjort, T., Riimke, P., Shulman, S., and Vyazov, 0. E.: Auto- and iso-antibodies to antigens of the human reproductive system, Acta Pathol. Microbial. Stand. Sect. C, Suppl. No. 258, 1977. 17. Axelsen, N. H., Kr@ll, J., and Weeke, B.: Quantitative
immunoelectrophoresis, 1973.
Stand. J. Immunol.
4:Suppl.
I,
18. Kr@ll, J.: Rocket line immunoelectrophoresis, Stand. J. Immunol. 2:Suppl. 1, 1973. 19. Boettcher, B., Kay, D. J., Riimke, P., and Wright, L. E.: Human sera containing immunoglobulin and nonim-
munoglobulin 1971.
of Immun-
pathology, Vol. II, 1973, pp. 669-674. S., and Koyama, K.: Quantitative determination IO. Isojima, of sperm-immobilizing antibody in women, in WHO Workshop on Techniques for Detection of Iso- and Autoantibodies to Human Spermatozoa, Aarhus. 15- 19 July, 1974. 11. Shulman, S.: Reproduction and Antibody Response, 1975, CRC-Press. 12. Scott, J. S., and Jones, W. R.: Immunology of Human Reproduction, New York, 1976, Grune & Stratton, Inc.
R., Hjort, T., Riimke, P., Harper, M. 0. E.: Technique for detection of isoto human spermatozoa, Clin. Exp. 1976. 1.: Clinical and immunological studies
20.
Mettler,
sperm-agglutinins,
L., and Gradl,
T.:
Biol. Reprod.
Difficulty
of obtaining
5:235, repro-
ducibility in the Franklin and Dukes test for the detection of sperm-agglutinating antibodies in human sera, .J. Reprod. Fertil. 44~217, 1975. 21. Hulka,
J. F., and
potential NECOL.161:440, 99 --.
Omran,
K. F.: The
local antibody secretor,
AM.
uterine
cervix
J. OBSTET.
as GY-
1969.
Eliasson, R.: Standards for investigation Andrologia 3:49, 197 1.
of human
semen,