In vitro microagglutination of human spermatozoa by ethinyl estradiol - a brief communicationRama A Vaidya1, Sushama Manikeri2, Nargesh D Motashaw3, UK Sheth2
1 Division of Endocrinology, Institute for Research in Reproduction, I.C.M.R., India
2 Department of Pharmacology, Seth G. S. Medical College and K.E.M. Hospital, Parel, Bombay-400012, India
3 Department of Obstetrics and Gynaecology, Seth G. S. Medical College and K.E.M. Hospital, Parel, Bombay-400012, India
Microagglutination of human spermatozoa was induced in vitro in a dose dependent manner by the addition of the ethinyl estradiol to a serum with increased levels of β-lipoprotein. Surface changes in spermatozoa secondary to estrogens have been postulated; an analogues mechanism for the platelet changes in women on hormonal contraceptives is proposed.
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Antispermatozoal antibodies (ASA) have been incriminated as a cause of infertility. Franklin and Dukes in 1964 used sperm agglutination as a marker for the detection of ASA.  They reported that sera from 72.1% of women with unexplained infertility were positive for sperm microagglutination. Subsequently other investigators , have shown that the incidence of positive FD tests in sera of women with unexplained infertility is not as high as initially reported by Franklin and Dukes.  In one study spermagglutinating activity was found to be in 13% of women with. proven fertility.  It is now well established that sperm microagglutination is non specific for ASA.
In an attempt to understand the chemical nature of sperm agglutinins, Boettcher and Kay  examined spermmicroglutinating activity in different fractions of a positive serum. It was shown that spermaglutinating activity resided in β-lipoprotein fraction of the positive serum. They concluded from their data that the technique of microspermagglutination can detect spermagglutinins which were not immunoglohulins. Boettcher  has also shown that high proportion of individuals with elevated serum levels of steroid hormones (women on oral contraceptives, pregnant women and women on testosterone) were found to produce spermagglutinating activity in their sera.
In the present study, we have examined the possibility of in-vitro induction of microspermagglutination in previously negative sera by ethinyl estradiol. Sera from a child with elevated β-lipoproteins which were negative for microspermagglutination and from a healthy normally menstruating woman have been utilized.
It was planned -to use serum with elevated β-lipoprotein levels and which was relatively free of sex steroids. Blood samples from an 8 year old male child suffering from Fredrickson's syndrome Type II (familial hyper β-lipoproteinaemia) were obtained from Jerbai Wadia Children's Hospital. Sera were seperated and stored at -20°C till taken for incubation. β-lipoprotein concentration in the child's serum varied from 800 to 1000 mg%. Control sera were obtained from a healthy normally menstruating woman. She had not received any drugs for the past six months.
Pure ethinyl estradiol (EE) powder 500 mg. was dissolved in 0.1 ml of ethanol. Ethanol was allowed to evaporate till very small amount of ethanol was left so that EE could be obtained in a solution. Serial dilutions were then done to get 500 ng/ml of EE solution.
Spermatozoa were obtained from healthy fertile donors. Fresh semen was obtained from donors an hour before the incubation studies were done.
A freshly obtained semen specimen was evaluated for sperm count and motility. The count was adjusted to 50 million/ml using Tyrode's solution as diluent.
Child's serum with high β-lipoprotein content and serum from normally menstruating woman were incubated at 50'C for half an hour to destroy complement.
Six serological test tubes containing either Tyrode's solution or a serum with high β-lipoprotein content or from c normally menstruating woman, were set up for the experiment as shown in [Table 1]. EE in concentration of 100 no/ml of serum was added to serum with elevated β-lipoprotein (tube 2) and normal serum (tube 4). The same quantity of EE was added to Tyrode's solution which provided its control. The tubes were incubated at 37°C for 4 hours. At hourly interval, a drop was removed from each tube by micropipette and examined for microagglutination. Agglutination of any type was noted and read as-one plus for single agglutination in an average of every other high power field; two plus for single agglutination in every high power field; and three plus for more than a single agglutination in every high power field. Agglutination of only motile sperm was considered a positive reaction. Aggregates of dead sperm or live sperm attached to debris were considered a non-specific reaction.
Since in a pilot experiment positive results were obtained when EE was added to hyper B-lipoprotein serum, an experiment for a dose response was done at various concentration of EE ranging from 10 ng/ml of serum to 500 ng/ml of serum. The same protocol was observed for Tyrode's solution as control and for normal serum.
From the set of six tubes only tube No. 2 which contained serum with elevated β-lipoprotein plus 100 ng/ml of EE, showed a positive response at 1 hr. Marked sperm agglutination was seen where 5 to 10 spermatozoa were clumped together. Such clumps were two to three per high power field. The agglutinated spermatozoa were actively motile. None of the other tubes displayed even an occasional sperm agglutination.
[Table 2] indicates spermagglutination response at various concentrations of EE. Ethinyl estradiol at the final concentration of 500 ng/ml could not be judged for inducing agglutination because sperm motility at this concentration was seriously affected. Ethinyl estradiol at 10 ng/ml did not produce any agglutination. The first indication of sperm agglutination was seen at 50 ng/ml of EE. At concentrations of 100 ng/ml and 200 ng/ml marked agglutination was noticed. However, at 200 ng/ml concentration, sperm motility was observed to be decreased. Once again the control tubes with Tyrode's solution and tubes containing normal serum showed no spermagglutination. This confirms the earlier results where EE failed to induce any sperm agglutination in spermatozoa incubated with either normal serum or Tyrode's solution.
Boettcher fractionated sperm agglutinating sera and has shown that spermagglutinating activity in the human sera resided in the β-lipoprotein fraction and not in the gamma-globulin fraction. The presence of spermagglutinins in sera from women  taking oral contraceptives and pregnant women' suggested that increased levels of steroids might cause sera to become spermagglutinating.  Spermagglutinins in the sera were suggested to be steroids bound to the β-lipoprotiens. The study suggested that microspermagglutination is not specific for detecting ASA. Estrogen binding globulins from the uteri of the rabbit" and cows are found in the first peak on gel filteration of Sephadex G-200 as has been spermagglutinating fraction (13-lipoprotein) in the above experiments. It has been demonstrated that sex steroids bind strongly to rabbit, boar and bull spermatozoa.  One of us (R.AV) working on sperm capacitation of rabbit spermatozoa had observed that actively motile spermatozoa flushed from uteri of estrogen dominated female rabbits were agglutinated head to head within two hours after mating.  In the present study it was possible to induce spermagglutination in-vitro by synthetic estrogens. A dose response effect was seen in child's serum which had high β-lipoprotein content. Sperm agglutination could not be induced in spermatozoa incubated either in normal serum or in Tyrode's solution.
Enhancement of platelet aggregation and adhesiveness by B-lipoprotein has been reported.  A synthetic estrogenethinyl estradiol had the highest binding platelets - followed by estradiol, estrone and progesterone.  Pollar et al  found the significant increase in platelet aggregation in woman taking estrogen-progestin oral contraceptives.  Elkeles et al  described an increased response to ADP in man taking estrogens. Synthetic estrogen is a common dominator for the induction of spermagglutination either in vivo or in vitro as in the present study and for the altered responses in platelet. ,
The important correlates of the capacitation phenomenon like the removal of tetracycline fluorescence  from rabbit spermatozoa, change in net surface negative charge  of rabbit spermatozoa and their head to head agglutination  when incubated in rabbit uterus are estrogen dependent. It has been shown that progesterone or contraceptive steroids with synthetic progestine interfere with some membrane processes like the removal of tetracycline fluorescence.  In the review of coagulation studies in women using hormonal contraceptives, Dugdale and Masi  had suggested that estrogen increases platelet function and accelerates clotting. From the cited literature ,, and the present experiments we postulate that estradiol and particularly synthetic estrogen may cause surface changes in cells like spermatozoa and platelets that enhance their agglutination.
Increase in β-lipoprotein in women during hormonal contraceptives may further enhance agglutination in the mentioned cells. 
[Table 1], [Table 2]