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Uterine toxoplasma infections and repeated abortions
Uterine toxoplasma infections and repeated abortions BABILL STRAY-PEDERSEN, M.D. AI\"NE-MARIE LORENTZEN-STYR Oslo, Norway Serologic testing for Toxoplasma antibodies (dye test, indirect fluorescent antibody test on serum, and complement-fixation test) was done in 96 women with habitual abortions and 61 women with sporadic abortions. In 61 of these women, endometrial biopsies were examined for content of T. gondii (animal inoculation procedures, immunofluorescence microscopy). Control subjects were 59 women with no spontaneous abortions. In seven patients (one control patient and six with habitual abortion), tachyzoites of r. gondii were observed in repeated biopsies from the endometrium and in menstrual blood by the immunofluorescence method. In no case, however, could T. gondii be isolated from the endometrium by inoculations. Moreover, the serologic results obtained.amoog the patients with habitual abortion did not differ significantly from those obtained among the ones with sporadic abortion or among the control subjects. Five of the women with Toxoplasma-positive endometrium were serologically negative. Treatment of the Toxoplasma-positive women with antitoxoplasma drugs led to removal of parasites from the endometrium. T. gondii was not observed in the semen of the husbands. (AM. J. OasTET. GYNECOL. 1?8: 716, 1977.)
women with prior histories of spontaneous abortions were examined, and the results were compared to those obtained among 59 control patients. Since the majority of earlier investigations in this fie~d has been based on only one method for the diagnosis of Toxoplasma infections, it was found of interest to employ different serologic techniques as well as different types of tissue examinations.
IT Is GENERALLY accepted that during the acute stage of a primary infection with Toxoplasma gondii early in pregnancy, the parasite may invade the placenta and embryo and lead to spontaneous abortion. However. the possibility that transmission of parasites to the offspring may take place in a woman having a chronic or latent infection has been greatly discussed. 10 The incidence of spontaneous abortion has been found to be increased among women with chronic toxoplasmosis, as judged from serologic screenings3 • 5' 8 or from attempts to demonstrate the parasite in situ. 4 • M The results obtained in other investigations give no support to this hypothesis. 1 • 7• lt. 15 On the other hand, Kimball and associates6 have found chronic toxoplasmosis to be associated with sporadic abortions but not with habitual abortions. The aim of this retrospective study was to investigate the role of uterine infections with T. gondii in sporadic and habitual (recurrent) abortions. To this end, 157
Material and m....,_ Patients. Testing for Toxoplasma antibodies was done in 96 women with habitual abortions (three or more consecutive spontaneous abortions) and 61 women with sporadic abortions (two or more nonconsecutive spontaneous abortions). Control subjects were 59 women who had never experienced spontaneous abortions: 23 women with infertility problems, 16 women with irregular bleeding, and 20 women scheduled for elective abortion. The distribution of age among the abortion patients was almost identical to that among the control group (mean age 29.4 years). The women came from different districts of Norway. predominantly from the Oslo area. In 41 patients with habitual abortion and 20 patients with sporadic abortion who were admitted to the Department of Obstetrics and Gynaecology, Rikshospitalet, examination for general disorders. malformations of the genital tract, chromosomal aberrations.
From the Department of Obstetrics and Gynaecology, Rikshospitalet, University of Oslo, and the Norwegian Defense Microbiological Laboratory. Received for publication November 3, 1976. Accepted March 29. 1977. Reprint requests: Dr. Babill Stray-Pedersen, Department of Obstetrics and Gynaecology. Rikshospitalet, Oslo 1, Norway.
716
Toxoplasma infections and repeated abortions 717
Volume 128 Number 7
Table I. Serologic results
Group
GET-positive
IF~T-serum pcsitive
Patients (No.)
DT-pcsitive (%)
%
96 61
25 25 25
29 20 26
23
9
16 20 59
44
9 44 10 19
I
No.*
%
41 20 61
8
Abortion patients
Habitual abortion Sporadic abortion Total
Control patients InfertilitY Irregula~ bleeding Provoked abortion Total
157
10
19
9 9
I
No.*
84 45 129
9 13
0 7
*The number of abortion patients tested for IFAT-serum and CIT-antibodies.
endocrinologic dysfunctions, etc., was carried out. Semen and chromosomal analyses of the husbands were also performed. On the basis of these examinations, no pathologic condition was detected which could explain the reproductive failure. From these women and from the control patients, endometrial biopsies were taken and examined for content of parasites. Serologic methods. The dye test (DT) was performed with a modification of the classic SabinFeldman method. A titer of l: 4 or higher (final serum dilution) was considered as positive. The indirect fluorescent antibody test on serum (IFAT-serum) was done with the use of FITC-conjugated rabbit anti-human F(ab)2. A titer of l: 16 (initial serum dilution) was considered as positive. The complement-fiJ,~:ation test (CFT) was performed according to the recommended procedure with reagents provided by Behringwerke, A.G. A titer of 1 : 4 was considered as positive. Methods for demonstration of parasites in tissue. Tissue material. Endometrial tissue, menstrual blood, abortion material, or ejaculate ( 1 to 2 Gm.) was sent to the laboratory within four hours. When necessary, the tissue was homogenized in a mortar. Sterile isotonic saline solution (2 mi.) containing penicillin 500 I. U. per milliliter) and streptomycin (0.0 1 Gni. per milliliter) was added per gram of tissue or fluid. Preparations of 15 drops were made for the microscopic examinations. The remaining part of the suspension was kept at 4° C. overnight to be inoculated the next day. Indirect fluorescent antibody test an tissue. The test was performed according to the procedure previously described13 with the use of fluorescein-conjugated guinea pig anti-rabbit-F(ab)2. For each series of tissue preparations, one inhibition control was made. As references drop preparations of peritoneal ex:udate from mice infecteq with the RH-strain ofT. gondii were used.
The following demands had to be satisfied in order to designate a preparation as "Toxoplasma positive": The fluorescent structures had to be morphol~gically similar to the tachyzoites of T. gondii (being the same size and showing the same smooth contours and, the typical crescent shape), and at least three such structures had to be present in one drop of preparation. Mice inoculation. DT-negative female mice (MRIBOM), weighing 20 to 25 grams were used. An amount of 0.3 ml. of the tissue suspension was injected intraperitoneally and/or subcutaneously into six mice. Another two mice served as controls. Two weeks thereafter, three of the inoculated animals were bled and killed. The peritoneal exudates were examined for tachyzoites ofT. gondii and a suspension of the brains was injected into three other DT-negative mice. Between six and eight weeks after the primary inoculation, all animals were bled and killed. The blood samples were tested for DT-antibodies. May-Griinewald-Giemsa-stained microscopic preparations from the liver, spleen, and brain were examined for cysts. Statistical methods. The significant differences in our study were calculated by the chi-square test with the Yates correction for binomial distribution (p ::s 0.01 was selected as the fiducial point).
Results In Table I, the frequency of serum-positive women (DT, IFAT-serum, and CFT) in the different patient groups is listed. As seen from this table, 25 and 26 percent among the abortion patients were DT- and IFAT-serum positive, respectively, as compared to only 19 per cent among the control patients. However, this difference was not shown to be statistically significant. The frequency of CFT-antibodies for the abortion patients (9 per cent) was practically identical to that obtained for the control subjects (7 per cent). Moreover, the serologic results obtained among the patients with
718
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August I. 1977 .-\m . .J. Obstet. Cynt>col.
Tissue studies. Inoculation test1 . The attempts to isolate T. gondii from
Fig. I. Tachyzoites ofT. gondii in endometrial tissue (indirect Auorescent antibody test).
Table II. Demonstration of parasites in endometrial tissue by indirect fluorescent antibody test
Group Abortion patients
Habitual abortion Sporadic abortion Total
Patients examined (No.)
(No.)
41
6
20
0
61
6
23 16
I 0
20
0
Control patie'ilts
Infertility Irregular bleeding Provoked abortion Total
Patients with positive endlimetrium
59
habitual abortion did not seem to differ significantly from those obtained among the group with sporadic abortion. The positive titers were generally low. More than 90 per cent of the serum-positive women had DT titers below I :64. The titer levels among the serum-positive abortion patients were not different from those obtained for the serum-positive control subjects. No relationship appeared to exist between the titer levels of the serumpositive abortion patients and the actual number of their prior abortions (consecutive or nonconsecutive). The highest titers (DT = 1 : 1,024; I FATserum "" 1 : 1,024; CFT "" l : 80) were observed in an infertile woman 25 years of age who showed no other signs or symptoms of an acute infection with T. gondii.
any specimen of tissue m Huid (endometrium , menstrual blood , and abortion material) by inoculation into mice were unsuccessful in all cases. In no instance could tachyzoites or cysts ofT. gondii be demonstrated in tissue from the inoculated anima ls: also the a nimals did not show seroconversion. lndirt'cl .filwrt'.ill'n/ antibody le.\(\. In seven patiems, tachyzoites ofT. gondii were d e monstrated in the endometrial tissue by the immunofluorescence me thod . As shown in Table ll , one of these women represented a control (infertility) patient , whereas the remaining six belonged to the habitual abortion group. The endometrium samples from anotht:r three women (not listed in the table) were classified as ·Toxoplasma dubious,'' since they contained Jess than three tachyzoitcs per drop of prepara tion . These three patients represented one control (infertility) patieut and two patients with habitual abortion. The Toxoplasma-positive endometrium generally contained very few parasites. Most often, only I 0 1.0 15 tachyzoites per drop of preparation could be observed. However. in one patient (Case 7 in Table III) , the e ndometrium contained numerous parasites, about lOO tachyzoites per drop of preparation . Typically, the tachyzoites were scattered in the preparation. In some places , however. they appeared to be arranged in dusters (Fig. I). Real cysts were never observed. The greatest fraction of the parasites appeared to have an extracellular location, but parasites located intracellularly were also observed. Follow-up studies
Serologic studies. The serum reactions among 46 of the abortion patients, including the women with Toxoplasma-positive endometrium , were tested repeatedly during a period of two years. Except l(>r one patient , described below, th e serum reactions remained unchanged. In one patient with habitual abortion, increasing titers were observed during a pregnancy which started one year after the first examination. The endometrium from this woman had been classified as "Toxoplasma dubious." The original titers were DT = I : 8 and CFT = I :4. The highest titers obtained during the pregnancy were DT = I : 256 and CFT = I :8. Three months after the delivery, the titers were DT = 1.: 16 and CFT negative . The pregnancy appeared otherwise normal , and a normal delivery took place at term . The DT titer in the umbilical cord blood was I :256. The child was normal and healthy and DT negative at one year of age.
Toxoplasma infections and repeated abortions
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719
Table III. Relevant data in patients with parasite-positive endometrium Husbands
Retrospective history Case No. 1 2 3 4 5 6
7
Diagnosis Habitual abortion Habitual abortion Habitual abortion Habitual abortion Habitual abortion Habitual abortion Infertility
Serolog_y
No. of prior spontaneous abortions
Duration of pregnancies (weeks)
DT
10 4 6
8-12 8-12 6-8 6-8 12-24 6-10
Neg.
4 4 4
0
Tissue studies. Four of the women with Toxoplasma-positive endometrium were examined repeatedly for parasites in the genital tract. In all of these patients, tachyzoites could be demonstrated by the immunofluorescence method in samples of endometrial tissue and/or menstrual blood. However, the inoculation tests in mice pretreated with cortisone acetate, were still negative. In one patient (Case 5 in Table III), tachyzoites were demonstrated by immunofluorescence microscopy in the placenta from an abortion which took place 18 months after the primary examination. Examination of the husbands. Attempts to demonstrate T. gondii in the semen from the husbands of the women with Toxoplasma-positive endometrium were performed. Parasites could not be demonstrated in any specimen by either the inoculation method or immunofluorescence microscopy. As shown in Table III, two of the husbands were DT positive. Treatment with pyrimethamine/sulfonamide. Four patients with Toxoplasma-positive endometrium were selected for treatment with antitoxoplasma drugs. The treatment was performed according to the following schedule; The first day 50 mg. of pyrimethamine and 6 Gm. of sulfonamide were given orally, then 25 mg. and 3 Gm. respectively, of these drugs per day were given during a period of four weeks. Folic acid and vitamin B were given as supplements. In one patient (Case 5), however, the treatment was stopped after one week because of the eruption of allergic skin reactions. Within few days after the treatment a curettage was performed, and the endometrial tissue was examined by immunofluorescence microscopy. The endometrium samples from three of the treated women were then designated as Toxoplasma negative, whereas the endometrium from one patient (Case 6), formerly classified as strongly positive, then contained very few parasites ( ''Toxoplasma dubious"). Three patients became pregnant immediately after
I
~eg.
Neg. Neg. I :64 1:32 Neg.
IFATserum
I
Neg. Neg. Neg. Neg. 1:64 I :64 Neg.
Serology CFT
DT
Neg. Neg. Neg.
Neg. Neg. 1:128 Neg. I :8 Neg. Not tested
~eg.
1:8 Neg. Neg.
I
CFT
Semen examination
Neg. Neg. Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg. Neg. Neg.
the treatment. The fourth patient changed her mind and continued to use contraception. One patient (Case 4) had a premature delivery in the twenty eighth gestational week, the child being normal and healthy and DT negative as was her mother. The second patient (Case 6) was delivered at term of a normal, healthy child, who was shown to be DT negative at three months of age. The third patient (Case 5), who had been treated for only one week, had a miscarriage in the twenty fourth gestational week. The placenta from this abortion was designated as "Toxoplasma dubious." She later had an abortion in the thirteenth gestational week. Unfortunately, the placenta was examined only by ordinary histologic techniques and showed "specific granulomatous infection."
Comment On the basis of the microscopic screening of endometrial tissue by the immunofluorescence method, an association between Toxoplasma infections in the uterus and the occurrence of habitual abortions might be suggested. As shown in Table II, tachyzoites ofT. gondii could be demonstrated in the endometrium samples from seven women, as many as six of whom belonged to the habitual abortion group. This could not represent accidental findings since the parasites could also be demonstrated in menstrual blood and in repeated endometrial biopsies as well as in abortion material from these patients. After treatment with antitoxoplasma drugs, the endometrium was shown to contain significantly smaller, if any, amounts of parasites. The fact that only tachyzoites ofT. gondii could be observed does not exclude the possibility that Toxoplasma cysts may exist in the uterine wall, as has been described by Remington and associates. 9 Such cysts could have ruptured for instance during the homogenization procedure or might be dispersed in tissue layers not accessible by curettage, i.e., in the myometrium.
720
Stray-Pedersen and Lorentzen-Styr Am.
Admittedly, the attempts to isolate T. gondii by animal inoculation from the endometrial tissue of the parasite-positive patients were unsuccessful. However, whereas a positive inoculation test no doubt represents a great support to the diagnosis, the diagnostic significance of negative inoculation tests must he considered as questionable. Earlier investigations have shown that the greatest number of attempts to isolate T. gondii from the genital tract in women or abortion material have been unsuccessful. 6 · 11 · H. 1" The ex planation for the failure of the method could be, for instance, that the concentration of parasites in the endometrium is too low to initiate an infection in the inoculated animal or that we have to deal with special strains ofT. gondii with low virulence, viability, or antigenicity. 16 In view of the microscopic findings. the results obtained in the serologic screening of the patients would appear as rather contradictory. The frequency of positive serum reactions among the patients with habitual abortion was not found to be significantly different from that among the patients with sporadic abortion or the control patients (Table I). Moreover, the frequency of Toxoplasma antibodies is in fair agreement with the prevalence of 13 per cent observed among 20-year-old Norwegian recruits, 12 especially when the yearly incidence of seroconversion is equal to 3 to 5 per thousand as observed in a recent study of l 0,000 pregnant women from the Oslo area. 12 Therefore, the actual frequency of toxoplasmosis in our country is considerably lower than in southern Europe (France) but comparable with the incidence reported from London.11 Five of the seven women with Toxoplasma-positive
J.
August I. I ~77 Obster. Cvnecol.
endometrium were shown to he serum negative (Table I II). Since the sera from these tivc Komcn also have been tested at other laboratories.* it would seem unreasonable to suggest that the serologic techniques employed were insufficient, i.e .. with respect to the measurement of small concentrations of antibodies. It should also be emphasized that the detection ofT. gondii in tissue from serum-negative patients has also been reported by nther investigators. 2 • 4 · " Therefore. this observation necessarily raises questions not only about the specifi<.: properties of the parasite but also about the method of transmission and the exact nature of the immunologic relationship between the parasitt: and the host. The possibility of sexual transmission of parasites has been proposed after demonstration of tachyzoites in the seminal Huid. 2 This \\'aS not observed in the present study. In conclusion. further investigations seem to be necessary in order to definitely settle the question of whether tht: presence ofT. gondii in the uterine tissue actually is of pathogenetic signifkance in recurrent spontaneous abortions or if the parasites just behave as harmless inhabitants of the female genital tract. As indicated by the present study, such investigations ought to be based on a variety of diagnostic methods (immunofluores<.:ence techniques. improved serologic methods, tests for cellular immunity. etc.) since conventional serologic methods and inoculation procedures may be of limited value in this connection. We wish to thank Prof. K. Bj¢ro and Dr. T. Omland for advice and help during the performance of this work. *Statens Serum lnstitut, Copenhagen. Denmark. and Institute de Puericulture, Paris. France.
REFERENCES l. Berger, j., and Piekarski, G.:
2.
3.
4.
5.
6.
Die Bedeutung der Toxoplasma-Infektion fiir Schwangerschaftsverlauf und Kindesentwicklung, Geburtshilfe Frauenheilkd. 35: 89, 1975. Disko. R., Braveny, I., and Vogel, P.: Untersuchungen zum Vorkommen von Toxoplasma gondii im menschlichen Ejakulat, Z. Tropenmed. Parasitol. 22: 391, 1971. Hingorani, V., Prakash, 0., Chowdhry, P., and Kamalan, T. S.: Toxoplasmosis: Abortions and stillbirths, Indian J. Med. Res. 58: 967, 1970. Hoffbauer, H., Struck, E., Voss, H., and Werner, H.: Nachweis von Toxoplasmen in Menstrualblut mit Hilfe der Immunfluoreszenztechnik, Munch. Med. Wochenschr. 17: 969, 1969. Jones, M. H., Sever, J. L., Baker, T. H .. Hallatt, J. H., Goldenberg, E. D., Justus, K. M,, and Gilkeson, M. R.: Toxoplasmosis and abortion, A!>r. J. ORSTET. GYNECOL. 104:919, 1969. Kimball, A. C., Kean, B. H., and Fuchs, F.: The role of
toxoplasmosis in abortion, AM. J. 0BSTET. GvNECOL. lll: 219, 1971. 7. Koppe, J. G., Kloosterman, G. J., de Roever- Bonnet, H., Eckert-Struink, J. A., Lower-Sieger, D. H., and de Bruijne, J. I.: Toxoplasmosis and pregnancy, with a long-term follow-up of the children, Eur. J. Obstet. Gynecol. Reprod. Bioi. 4: 101, 1974. 8. Langer, H.: Intrauterine Toxoplasma-Infektion. Stuttgart, 1963, George Thieme Verlag, pp. 1-47. 9. Remington, J. S., Melton, M. L.. and Jacobs, L.: Chronic toxoplasma infection in the uterus, J. Lab. Clin. Med. 56:
879, 1960.
10. Remington,]. S.: Toxoplasmosis, in Charles, D., and Finland, M., editors: Obstetric and Perinatal Infections, Philadelphia, 1973, Lea & Febiger, Publishers, p.27. 11. Ruoss, C. F. and Bourne. G. L.: Toxoplasmosis in pregnancy, Br. J. Obstet. Gynaecol. 79: 1115, I 972. 12. Stray-Pedersen, B.: Toxoplasma infection and pregnancy. I-V. To be published.
Volume 128 Number 7
13. Stray-Pedersen, S., Lorentzen-Styr, A.M., and Kaaresen, R.: The immunofluorescence method in the detection of Toxoplasma gondii. In press. 14. Struck, E., andJanitschke, K.: Anziichtungsversuche von Toxplasma gondii aus Plazenten und Abortmaterial und serologische Prufung der Frauen auf ToxoplasmaAntikorper, Z. Geburtshilfe Perinatal. 176: 324, 1972. 15. Thalhammer, 0.: Bemerkungen zur Frage des toxo-
Toxoplasma infections and repeated abortions
721
plasmabedingten Abortus, Arch. Gynaekol. 202: 96, 1964. 16. Werner, H.: Uber das Vorkommen von Toxoplasma gondii in den Generationsorganen bei Tier und Mensch, in Kirchhoff, H., and Langer, H., editors: Toxoplasmose, Praktische Fragen und Ergebnisse, Stuttgart, 1971, Georg Thieme Verlag, p. 20.
women with prior histories of spontaneous abortions were examined, and the results were compared to those obtained among 59 control patients. Since the majority of earlier investigations in this fie~d has been based on only one method for the diagnosis of Toxoplasma infections, it was found of interest to employ different serologic techniques as well as different types of tissue examinations.
IT Is GENERALLY accepted that during the acute stage of a primary infection with Toxoplasma gondii early in pregnancy, the parasite may invade the placenta and embryo and lead to spontaneous abortion. However. the possibility that transmission of parasites to the offspring may take place in a woman having a chronic or latent infection has been greatly discussed. 10 The incidence of spontaneous abortion has been found to be increased among women with chronic toxoplasmosis, as judged from serologic screenings3 • 5' 8 or from attempts to demonstrate the parasite in situ. 4 • M The results obtained in other investigations give no support to this hypothesis. 1 • 7• lt. 15 On the other hand, Kimball and associates6 have found chronic toxoplasmosis to be associated with sporadic abortions but not with habitual abortions. The aim of this retrospective study was to investigate the role of uterine infections with T. gondii in sporadic and habitual (recurrent) abortions. To this end, 157
Material and m....,_ Patients. Testing for Toxoplasma antibodies was done in 96 women with habitual abortions (three or more consecutive spontaneous abortions) and 61 women with sporadic abortions (two or more nonconsecutive spontaneous abortions). Control subjects were 59 women who had never experienced spontaneous abortions: 23 women with infertility problems, 16 women with irregular bleeding, and 20 women scheduled for elective abortion. The distribution of age among the abortion patients was almost identical to that among the control group (mean age 29.4 years). The women came from different districts of Norway. predominantly from the Oslo area. In 41 patients with habitual abortion and 20 patients with sporadic abortion who were admitted to the Department of Obstetrics and Gynaecology, Rikshospitalet, examination for general disorders. malformations of the genital tract, chromosomal aberrations.
From the Department of Obstetrics and Gynaecology, Rikshospitalet, University of Oslo, and the Norwegian Defense Microbiological Laboratory. Received for publication November 3, 1976. Accepted March 29. 1977. Reprint requests: Dr. Babill Stray-Pedersen, Department of Obstetrics and Gynaecology. Rikshospitalet, Oslo 1, Norway.
716
Toxoplasma infections and repeated abortions 717
Volume 128 Number 7
Table I. Serologic results
Group
GET-positive
IF~T-serum pcsitive
Patients (No.)
DT-pcsitive (%)
%
96 61
25 25 25
29 20 26
23
9
16 20 59
44
9 44 10 19
I
No.*
%
41 20 61
8
Abortion patients
Habitual abortion Sporadic abortion Total
Control patients InfertilitY Irregula~ bleeding Provoked abortion Total
157
10
19
9 9
I
No.*
84 45 129
9 13
0 7
*The number of abortion patients tested for IFAT-serum and CIT-antibodies.
endocrinologic dysfunctions, etc., was carried out. Semen and chromosomal analyses of the husbands were also performed. On the basis of these examinations, no pathologic condition was detected which could explain the reproductive failure. From these women and from the control patients, endometrial biopsies were taken and examined for content of parasites. Serologic methods. The dye test (DT) was performed with a modification of the classic SabinFeldman method. A titer of l: 4 or higher (final serum dilution) was considered as positive. The indirect fluorescent antibody test on serum (IFAT-serum) was done with the use of FITC-conjugated rabbit anti-human F(ab)2. A titer of l: 16 (initial serum dilution) was considered as positive. The complement-fiJ,~:ation test (CFT) was performed according to the recommended procedure with reagents provided by Behringwerke, A.G. A titer of 1 : 4 was considered as positive. Methods for demonstration of parasites in tissue. Tissue material. Endometrial tissue, menstrual blood, abortion material, or ejaculate ( 1 to 2 Gm.) was sent to the laboratory within four hours. When necessary, the tissue was homogenized in a mortar. Sterile isotonic saline solution (2 mi.) containing penicillin 500 I. U. per milliliter) and streptomycin (0.0 1 Gni. per milliliter) was added per gram of tissue or fluid. Preparations of 15 drops were made for the microscopic examinations. The remaining part of the suspension was kept at 4° C. overnight to be inoculated the next day. Indirect fluorescent antibody test an tissue. The test was performed according to the procedure previously described13 with the use of fluorescein-conjugated guinea pig anti-rabbit-F(ab)2. For each series of tissue preparations, one inhibition control was made. As references drop preparations of peritoneal ex:udate from mice infecteq with the RH-strain ofT. gondii were used.
The following demands had to be satisfied in order to designate a preparation as "Toxoplasma positive": The fluorescent structures had to be morphol~gically similar to the tachyzoites of T. gondii (being the same size and showing the same smooth contours and, the typical crescent shape), and at least three such structures had to be present in one drop of preparation. Mice inoculation. DT-negative female mice (MRIBOM), weighing 20 to 25 grams were used. An amount of 0.3 ml. of the tissue suspension was injected intraperitoneally and/or subcutaneously into six mice. Another two mice served as controls. Two weeks thereafter, three of the inoculated animals were bled and killed. The peritoneal exudates were examined for tachyzoites ofT. gondii and a suspension of the brains was injected into three other DT-negative mice. Between six and eight weeks after the primary inoculation, all animals were bled and killed. The blood samples were tested for DT-antibodies. May-Griinewald-Giemsa-stained microscopic preparations from the liver, spleen, and brain were examined for cysts. Statistical methods. The significant differences in our study were calculated by the chi-square test with the Yates correction for binomial distribution (p ::s 0.01 was selected as the fiducial point).
Results In Table I, the frequency of serum-positive women (DT, IFAT-serum, and CFT) in the different patient groups is listed. As seen from this table, 25 and 26 percent among the abortion patients were DT- and IFAT-serum positive, respectively, as compared to only 19 per cent among the control patients. However, this difference was not shown to be statistically significant. The frequency of CFT-antibodies for the abortion patients (9 per cent) was practically identical to that obtained for the control subjects (7 per cent). Moreover, the serologic results obtained among the patients with
718
Stray-PederSen and Lorentzen-Styr
August I. 1977 .-\m . .J. Obstet. Cynt>col.
Tissue studies. Inoculation test1 . The attempts to isolate T. gondii from
Fig. I. Tachyzoites ofT. gondii in endometrial tissue (indirect Auorescent antibody test).
Table II. Demonstration of parasites in endometrial tissue by indirect fluorescent antibody test
Group Abortion patients
Habitual abortion Sporadic abortion Total
Patients examined (No.)
(No.)
41
6
20
0
61
6
23 16
I 0
20
0
Control patie'ilts
Infertility Irregular bleeding Provoked abortion Total
Patients with positive endlimetrium
59
habitual abortion did not seem to differ significantly from those obtained among the group with sporadic abortion. The positive titers were generally low. More than 90 per cent of the serum-positive women had DT titers below I :64. The titer levels among the serum-positive abortion patients were not different from those obtained for the serum-positive control subjects. No relationship appeared to exist between the titer levels of the serumpositive abortion patients and the actual number of their prior abortions (consecutive or nonconsecutive). The highest titers (DT = 1 : 1,024; I FATserum "" 1 : 1,024; CFT "" l : 80) were observed in an infertile woman 25 years of age who showed no other signs or symptoms of an acute infection with T. gondii.
any specimen of tissue m Huid (endometrium , menstrual blood , and abortion material) by inoculation into mice were unsuccessful in all cases. In no instance could tachyzoites or cysts ofT. gondii be demonstrated in tissue from the inoculated anima ls: also the a nimals did not show seroconversion. lndirt'cl .filwrt'.ill'n/ antibody le.\(\. In seven patiems, tachyzoites ofT. gondii were d e monstrated in the endometrial tissue by the immunofluorescence me thod . As shown in Table ll , one of these women represented a control (infertility) patient , whereas the remaining six belonged to the habitual abortion group. The endometrium samples from anotht:r three women (not listed in the table) were classified as ·Toxoplasma dubious,'' since they contained Jess than three tachyzoitcs per drop of prepara tion . These three patients represented one control (infertility) patieut and two patients with habitual abortion. The Toxoplasma-positive endometrium generally contained very few parasites. Most often, only I 0 1.0 15 tachyzoites per drop of preparation could be observed. However. in one patient (Case 7 in Table III) , the e ndometrium contained numerous parasites, about lOO tachyzoites per drop of preparation . Typically, the tachyzoites were scattered in the preparation. In some places , however. they appeared to be arranged in dusters (Fig. I). Real cysts were never observed. The greatest fraction of the parasites appeared to have an extracellular location, but parasites located intracellularly were also observed. Follow-up studies
Serologic studies. The serum reactions among 46 of the abortion patients, including the women with Toxoplasma-positive endometrium , were tested repeatedly during a period of two years. Except l(>r one patient , described below, th e serum reactions remained unchanged. In one patient with habitual abortion, increasing titers were observed during a pregnancy which started one year after the first examination. The endometrium from this woman had been classified as "Toxoplasma dubious." The original titers were DT = I : 8 and CFT = I :4. The highest titers obtained during the pregnancy were DT = I : 256 and CFT = I :8. Three months after the delivery, the titers were DT = 1.: 16 and CFT negative . The pregnancy appeared otherwise normal , and a normal delivery took place at term . The DT titer in the umbilical cord blood was I :256. The child was normal and healthy and DT negative at one year of age.
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Table III. Relevant data in patients with parasite-positive endometrium Husbands
Retrospective history Case No. 1 2 3 4 5 6
7
Diagnosis Habitual abortion Habitual abortion Habitual abortion Habitual abortion Habitual abortion Habitual abortion Infertility
Serolog_y
No. of prior spontaneous abortions
Duration of pregnancies (weeks)
DT
10 4 6
8-12 8-12 6-8 6-8 12-24 6-10
Neg.
4 4 4
0
Tissue studies. Four of the women with Toxoplasma-positive endometrium were examined repeatedly for parasites in the genital tract. In all of these patients, tachyzoites could be demonstrated by the immunofluorescence method in samples of endometrial tissue and/or menstrual blood. However, the inoculation tests in mice pretreated with cortisone acetate, were still negative. In one patient (Case 5 in Table III), tachyzoites were demonstrated by immunofluorescence microscopy in the placenta from an abortion which took place 18 months after the primary examination. Examination of the husbands. Attempts to demonstrate T. gondii in the semen from the husbands of the women with Toxoplasma-positive endometrium were performed. Parasites could not be demonstrated in any specimen by either the inoculation method or immunofluorescence microscopy. As shown in Table III, two of the husbands were DT positive. Treatment with pyrimethamine/sulfonamide. Four patients with Toxoplasma-positive endometrium were selected for treatment with antitoxoplasma drugs. The treatment was performed according to the following schedule; The first day 50 mg. of pyrimethamine and 6 Gm. of sulfonamide were given orally, then 25 mg. and 3 Gm. respectively, of these drugs per day were given during a period of four weeks. Folic acid and vitamin B were given as supplements. In one patient (Case 5), however, the treatment was stopped after one week because of the eruption of allergic skin reactions. Within few days after the treatment a curettage was performed, and the endometrial tissue was examined by immunofluorescence microscopy. The endometrium samples from three of the treated women were then designated as Toxoplasma negative, whereas the endometrium from one patient (Case 6), formerly classified as strongly positive, then contained very few parasites ( ''Toxoplasma dubious"). Three patients became pregnant immediately after
I
~eg.
Neg. Neg. I :64 1:32 Neg.
IFATserum
I
Neg. Neg. Neg. Neg. 1:64 I :64 Neg.
Serology CFT
DT
Neg. Neg. Neg.
Neg. Neg. 1:128 Neg. I :8 Neg. Not tested
~eg.
1:8 Neg. Neg.
I
CFT
Semen examination
Neg. Neg. Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg. Neg. Neg.
the treatment. The fourth patient changed her mind and continued to use contraception. One patient (Case 4) had a premature delivery in the twenty eighth gestational week, the child being normal and healthy and DT negative as was her mother. The second patient (Case 6) was delivered at term of a normal, healthy child, who was shown to be DT negative at three months of age. The third patient (Case 5), who had been treated for only one week, had a miscarriage in the twenty fourth gestational week. The placenta from this abortion was designated as "Toxoplasma dubious." She later had an abortion in the thirteenth gestational week. Unfortunately, the placenta was examined only by ordinary histologic techniques and showed "specific granulomatous infection."
Comment On the basis of the microscopic screening of endometrial tissue by the immunofluorescence method, an association between Toxoplasma infections in the uterus and the occurrence of habitual abortions might be suggested. As shown in Table II, tachyzoites ofT. gondii could be demonstrated in the endometrium samples from seven women, as many as six of whom belonged to the habitual abortion group. This could not represent accidental findings since the parasites could also be demonstrated in menstrual blood and in repeated endometrial biopsies as well as in abortion material from these patients. After treatment with antitoxoplasma drugs, the endometrium was shown to contain significantly smaller, if any, amounts of parasites. The fact that only tachyzoites ofT. gondii could be observed does not exclude the possibility that Toxoplasma cysts may exist in the uterine wall, as has been described by Remington and associates. 9 Such cysts could have ruptured for instance during the homogenization procedure or might be dispersed in tissue layers not accessible by curettage, i.e., in the myometrium.
720
Stray-Pedersen and Lorentzen-Styr Am.
Admittedly, the attempts to isolate T. gondii by animal inoculation from the endometrial tissue of the parasite-positive patients were unsuccessful. However, whereas a positive inoculation test no doubt represents a great support to the diagnosis, the diagnostic significance of negative inoculation tests must he considered as questionable. Earlier investigations have shown that the greatest number of attempts to isolate T. gondii from the genital tract in women or abortion material have been unsuccessful. 6 · 11 · H. 1" The ex planation for the failure of the method could be, for instance, that the concentration of parasites in the endometrium is too low to initiate an infection in the inoculated animal or that we have to deal with special strains ofT. gondii with low virulence, viability, or antigenicity. 16 In view of the microscopic findings. the results obtained in the serologic screening of the patients would appear as rather contradictory. The frequency of positive serum reactions among the patients with habitual abortion was not found to be significantly different from that among the patients with sporadic abortion or the control patients (Table I). Moreover, the frequency of Toxoplasma antibodies is in fair agreement with the prevalence of 13 per cent observed among 20-year-old Norwegian recruits, 12 especially when the yearly incidence of seroconversion is equal to 3 to 5 per thousand as observed in a recent study of l 0,000 pregnant women from the Oslo area. 12 Therefore, the actual frequency of toxoplasmosis in our country is considerably lower than in southern Europe (France) but comparable with the incidence reported from London.11 Five of the seven women with Toxoplasma-positive
J.
August I. I ~77 Obster. Cvnecol.
endometrium were shown to he serum negative (Table I II). Since the sera from these tivc Komcn also have been tested at other laboratories.* it would seem unreasonable to suggest that the serologic techniques employed were insufficient, i.e .. with respect to the measurement of small concentrations of antibodies. It should also be emphasized that the detection ofT. gondii in tissue from serum-negative patients has also been reported by nther investigators. 2 • 4 · " Therefore. this observation necessarily raises questions not only about the specifi<.: properties of the parasite but also about the method of transmission and the exact nature of the immunologic relationship between the parasitt: and the host. The possibility of sexual transmission of parasites has been proposed after demonstration of tachyzoites in the seminal Huid. 2 This \\'aS not observed in the present study. In conclusion. further investigations seem to be necessary in order to definitely settle the question of whether tht: presence ofT. gondii in the uterine tissue actually is of pathogenetic signifkance in recurrent spontaneous abortions or if the parasites just behave as harmless inhabitants of the female genital tract. As indicated by the present study, such investigations ought to be based on a variety of diagnostic methods (immunofluores<.:ence techniques. improved serologic methods, tests for cellular immunity. etc.) since conventional serologic methods and inoculation procedures may be of limited value in this connection. We wish to thank Prof. K. Bj¢ro and Dr. T. Omland for advice and help during the performance of this work. *Statens Serum lnstitut, Copenhagen. Denmark. and Institute de Puericulture, Paris. France.
REFERENCES l. Berger, j., and Piekarski, G.:
2.
3.
4.
5.
6.
Die Bedeutung der Toxoplasma-Infektion fiir Schwangerschaftsverlauf und Kindesentwicklung, Geburtshilfe Frauenheilkd. 35: 89, 1975. Disko. R., Braveny, I., and Vogel, P.: Untersuchungen zum Vorkommen von Toxoplasma gondii im menschlichen Ejakulat, Z. Tropenmed. Parasitol. 22: 391, 1971. Hingorani, V., Prakash, 0., Chowdhry, P., and Kamalan, T. S.: Toxoplasmosis: Abortions and stillbirths, Indian J. Med. Res. 58: 967, 1970. Hoffbauer, H., Struck, E., Voss, H., and Werner, H.: Nachweis von Toxoplasmen in Menstrualblut mit Hilfe der Immunfluoreszenztechnik, Munch. Med. Wochenschr. 17: 969, 1969. Jones, M. H., Sever, J. L., Baker, T. H .. Hallatt, J. H., Goldenberg, E. D., Justus, K. M,, and Gilkeson, M. R.: Toxoplasmosis and abortion, A!>r. J. ORSTET. GYNECOL. 104:919, 1969. Kimball, A. C., Kean, B. H., and Fuchs, F.: The role of
toxoplasmosis in abortion, AM. J. 0BSTET. GvNECOL. lll: 219, 1971. 7. Koppe, J. G., Kloosterman, G. J., de Roever- Bonnet, H., Eckert-Struink, J. A., Lower-Sieger, D. H., and de Bruijne, J. I.: Toxoplasmosis and pregnancy, with a long-term follow-up of the children, Eur. J. Obstet. Gynecol. Reprod. Bioi. 4: 101, 1974. 8. Langer, H.: Intrauterine Toxoplasma-Infektion. Stuttgart, 1963, George Thieme Verlag, pp. 1-47. 9. Remington, J. S., Melton, M. L.. and Jacobs, L.: Chronic toxoplasma infection in the uterus, J. Lab. Clin. Med. 56:
879, 1960.
10. Remington,]. S.: Toxoplasmosis, in Charles, D., and Finland, M., editors: Obstetric and Perinatal Infections, Philadelphia, 1973, Lea & Febiger, Publishers, p.27. 11. Ruoss, C. F. and Bourne. G. L.: Toxoplasmosis in pregnancy, Br. J. Obstet. Gynaecol. 79: 1115, I 972. 12. Stray-Pedersen, B.: Toxoplasma infection and pregnancy. I-V. To be published.
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13. Stray-Pedersen, S., Lorentzen-Styr, A.M., and Kaaresen, R.: The immunofluorescence method in the detection of Toxoplasma gondii. In press. 14. Struck, E., andJanitschke, K.: Anziichtungsversuche von Toxplasma gondii aus Plazenten und Abortmaterial und serologische Prufung der Frauen auf ToxoplasmaAntikorper, Z. Geburtshilfe Perinatal. 176: 324, 1972. 15. Thalhammer, 0.: Bemerkungen zur Frage des toxo-
Toxoplasma infections and repeated abortions
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plasmabedingten Abortus, Arch. Gynaekol. 202: 96, 1964. 16. Werner, H.: Uber das Vorkommen von Toxoplasma gondii in den Generationsorganen bei Tier und Mensch, in Kirchhoff, H., and Langer, H., editors: Toxoplasmose, Praktische Fragen und Ergebnisse, Stuttgart, 1971, Georg Thieme Verlag, p. 20.