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Survey of 1120 Japanese women with a history of recurrent spontaneous abortions
123
Biology,44 (1992)123-130 0 1992ElsevierScience Publishers B.V. All rights reserved 002%2243/92/$05.00
European Journal of Obstetrics & Gynecology and Reproductive
EUROBS 01316
Survey of 1120 Japanese women with a history of recurrent spontaneous abortions Tsunehisa
Makino, Takabumi Hara, Chikahiro Oka, Kiwamu Toyoshima, Toshitaka Ken-ichi Iwasaki, Masakatsu Umeuchi and Rihachi Iizuka
Sugi,
Department of Obstetrics and Gynecology, School of Medicine, Keio Unioersiiy, Tokyo, Japan
Accepted for publication 15 October 1991
Summary
Our recent 7-year clinical survey showed that among the 1120 women with repeated spontaneous abortions registered in this clinic, 2898 out of a total of 3216 pregnancies (90.1%) had terminated in spontaneous abortion. Among these wastages, 84.2% occurred before 12 weeks of gestation, and 11.1 percent occurred between 12 and 15 weeks. Through routine examination of reproductive wastage, 82 (9.9%) of the 825 Japanese couples examined were shown to have either a chromosomal abnormality or normal variants in the wife and/or husband, thus demonstrating no racial difference in the incidence of chromosomal abnormalities in infertile patients in comparison with studies performed in other countries. One hundred and forty-seven congenital uterine anomalies (14.7%) were found in 1000 hysterosalpingographies, and 12 of 148 examined females were positive for anti-cardiolipin antibody. 393 other females with no major abnormalities likely to induce spontaneous abortions were indicated for immunotherapy. Ample time spent on genetic counseling prevented further reproductive wastage, and ideal metroplasty resulted in a successful post-operative pregnancy course in more than 85% of cases. Immunosuppressant and anticoagulant therapy decreased the serum titer of anti-cardiolipin antibody, enabling pregnancies to be maintained to term. Immunotherapy utilizing the husband’s lymphocytes also brought more than 80% of pregnancies to successful completion, with 200 deliveries achieved with this therapy. In contrast, 64.1% of pregnancies again terminated spontaneously in patients who were indicated for immunotherapy but did not receive treatment. The findings of the present study suggest that the causes of reproductive wastage, especially the etiology of early recurrent spontaneous abortion, are complex. However, well-organized routine examination of the causes of repetitive wastage has made it possible to identify appropriate treatments, resulting in marked reduction in human reproductive wastage. Abortion
Introduction Correspondence:
Dr. Tsunehisa Makino, Dept. of Obstetrics and Gynecology, School of Medicine, Keio University, Shin-
juku-ku,Tokyo 160,Japan.
The process of reproduction is extremely complex and inefficient [1,4]. It is estimated that
124
approximately 10 to 20% of known conceptions terminate in spontaneous abortion. Recent progress in hormone measurement has improved the sensitivity, specificity and assay time of conventional radio immunoassay. With this new approach, Wilcox et al. [23 demonstrated that 22% of conceptions terminated before pregnancy were detected clinically. Thus, spontaneous abortion is a common phenomenon and constitutes one of the major segments of what has been called reproductive or pregnancy wastage. In this study, 1120 Japanese women with a history of recurrent spontaneous abortions registered in our infertility clinic were surveyed for common background factors, including their age and the number and gestational weeks of their spontaneous abortions. The possible causes of these wastages were then surmised through comprehensive, routine examination of these patients. The effectiveness of the treatments for each etiology was also monitored through tracking their subsequent pregnancies over the past 7 years. As a control, the course of pregnancy was also monitored in patients who have had some abnormality but did not receive any appropriate therapy before pregnancy. These follow-up studies improved our methods of clinical examination and resulted in establishment of more effective therapies for common human reproductive wastages.
group was 3216; 2899 of these conceptions terminated in spontaneous abortion.
had
Congenital uterine anomaly
By the end of March 1990, hysterosalpingography had been performed on 1000 out of 1120 women, and 147 congenital uterine anomalies had been detected. A method in which the longitudinal iength of the uterine cavity (M) is compared with the dent of the uterine fundus (X) under hysterosalpingography was introduced to evaluate uterine deformities, and the incidence of abortion was analyzed as a function of the results of this evaluation (Fig. 1) [3]. A modified metroplasty was performed on 50 of these 147 patients. As a control, 47 other patients with anomalies were randomly selected, and their subsequent pregnancies were monitored without metroplasty. Chromosomal analysis
Chromosomal analysis of peripheral blood cells had been performed for both partners in 825 of the 1120 couples by the end of March 1990. The chromosomal preparations were analyzed after applying the trypsin G band method, and Qbanding or C-banding was also performed when necessary. For each individual, a minimum of 20 metaphase plates were counted, and at least 3 cells were karyotyped. Anti-phospholipid antibody syndrome
Subjects and Methods The possible causes of recurrent consecutive spontaneous abortions in 1120 couples (n = 1120) registered between 1984 and 1990 at Keio University Hospital in Tokyo were investigated. Genetic, immunologic, endocrine, infectious and uterine organic factors, including uterine myoma, were studied. The age distribution of the female partners was from 19 to 47 years old, with a mean age of 31.3 + 4.5 years (mean k SEMI. A spontaneous abortion was defined as a spontaneously terminated pregnancy in which urinary hCG was detected and D/C was performed. The mean number of spontaneous abortions was 2.9 + 0.7. The total number of past pregnancies in the patient
Using an enzyme-linked immunosorbent assay employing Harris’s standard serum, 148 women with a history of recurrent fetal loss were tested for serum anti-cardiolipin antibody. To differentiate early and late abortions, these women were divided into two groups. The spontaneous abortions in Group 1 were in the first trimester and those of Group 2 included wastages in the second trimester. The women diagnosed as having antiphospholipid antibody syndrome were then administered immunosuppressant or anticoagulant therapy. Immunotherapy for habitual spontaneous abortion Three hundred and ninety-three (n = 393)
women for whom the cause of abortion could not be surmised on the basis of our routine examina-
125
(1)
0 < f$ < -+
: arcuate
(2) + 5 6 < 1 : partial (3) $ + 1 Fig. 1. Evaluation
uterus
septate
: complete
uterus
septate
of uterine cavity deformity hysterosalpingogram.
uterus
by X/M
ratio in
tion of recurrent spontaneous abortion were treated with their husbands’ lymphocytes as immunotherapy [4]. The lymphocytes were separated aseptically from 30 ml of the husband’s venous blood by the Conray-Ficoll Method, and the lymphocyte concentration was adjusted to approximately 40 million/ml. Then this suspension was intradermally injected into the female partner up to 4 times at intervals of 2-4 weeks. As a randomized control, 64 other women indicated for immunotherapy who did not receive this treatment were monitored in their subsequent pregnancies. The outcome and course of pregnancies begun after this immunotherapy were investigated. The physical development of all children who were conceived and born after the immunotherapy was monitored up to the age of 3 years.
cases of normal variants detected in 825 couples. The 49 major chromosomal abnormalities consisted of 25 reciprocal translocations, 10 Robertsonian translocations, 3 inversions and 11 sex chromosome aneuploidies. The group with reciprocal translocations was the largest, consisting of 11 female and 14 male carriers. None of the reciprocal translocations involved the sex chromosomes. In one couple, both partners were carriers of a translocation. The incidence of chromosomal abnormalities was 5.9%, and the incidence when normal variants were included was 9.9%. Table III summarizes the frequency of abortions in the past pregnancies of the patients having each type of abnormality. In the 10 cases with Robertsonian translocations, the incidence of abortions seemed to be slightly lower, but there was no significant difference compared to the other types of abnormality. One hundred and forty seven congenital uterine anomalies were detected in 1000 hysterosalpingographies. By introducing the new X/M ratio to hysterosalpingography to evaluate uterine cavity deformities, Tables IV and V summarize the incidence of each anomaly and the early reproductive wastage frequency. As indicated, the largest group of anomalies is the 0
I N=994
Results The nine hundred and ninety-three spontaneous abortions confirmed from the patients’ histories are summarized in Fig. 2. As indicated, more than 84% of these wastages occurred before 12 weeks of gestation. Table I summarizes the possible causes and pathological findings for the 1120 couples, showing a wide variety in the etiology of recurrent reproductive wastage. Chromosomal abnormalities are generally classified as major or minor variants. Table II shows the 49 major chromosomal abnormalities and 33
0 4W
6W 5w
8W 7w
tow 9w
Fig. 2. Weeks of gestation
IZW IIW
13W
14w 16W . 15w 17w
at spontaneous patients.
1
2ow 22w 19W 21W 23W
abortion
in infertile
126 TABLE II Chromosomal abnormal in 825 couples with a history of recurrent spontaneous abortion 70-Z
n
*I 60.;:$‘;. 40- ;;-
iE 2. .; I 3
6MlY
2
3
%
Reciprocal translocation Robertsonian translocation Inversion (numbers 7, 11 and 12) Sex chromosome aneuploidy Normal variants
25 10 3 11 33
30.5 12.2 3.7 13.4 40.2
Total
82
100.0
Keio Univ. Hosp.
Fig. 3. Physical development of male children born to immunized mothers.
with anomalies. An ideal metroplasty was performed on 50 women with anomalies, and more than 85% of the postoperative pregnancies in 35 patients were successful, as indicated in Table VII. In contrast, 98.8% of the pregnancies in these 50 women had terminated in spontaneous abortion before the surgical treatment (Table VI), and 94.4% of pregnancies in the group of 47 randomized control women with anomalies also terminated spontaneously (Table VIII). Table IX shows that of 148 patients with a history of recurrent reproductive wastage tested
cm
6Mly
2
3
Keio Univ. Hosp.
Fig. 4. Physical development of female children born to immunized mothers.
TABLE I Abnormalities
detected in 1120 infertile women with a history of recurrent spontaneous abortion
Congenital uterine anomaly Uterine myoma Endometriosis Cervical incompetency Asherman’s syndrome Chromosomal abnormality Autoimmune diseases RA SLE MCTD Positive for anti-cardiolipin antibody Diabetes mellitus
147 23 7 12 9 82 5 2 1 24 5
Thyroid dysfunction Infections Syphilis RF Tuberculosis (lung) Hepatitis Miscellaneous Asthma Thrombosis Myasthenia gravis Muscular dystrophy Gong. heart disease
21 1 3 4 3 9 3 5 1 3
127 TABLE III Reproductive wastages in 82 carriers of abnormal chromosomes
Reciprocal translocation Robertsonian translocation Inversion Sex chromosome aneuploidy Normal variants
n
Total pregnancies
25 10 3 11 33
86 38 11 36 108 279
Total
Abortions or stillbirths
Live borns
Abortion rate (o/o)
80 30 10 32 95
6 8 1 4 13
93.0 78.9 90.9 88.9 88.0
247
32
88.5
TABLE IV
TABLE VII
Incidences of congenital uterine anomalies
Pregnancy course after metroplasty in 35 patients with congenital uterine anomalies
n
0 < X/M < l/3 (arcuate) l/3 < X/M < 1 (partial septate) X/M=1 (complete septate) Unicornuate Total
%
95 40 8 4
64.6 27.2 5.4 2.8
147
100.0
%
n
Abortion Premature labor Stillbirth Full-term
169 1 1 0
98.8 0.6 0.6 0.0
Total
171
100.0
TABLE V Number of spontaneous abortions in each group evaluated by X/M ratio in hysterosalpingogram No. of
abortions 0 < X/M < l/3 (Arcuate, n = 95) 292 l/3 < X/M < 1 (Partial septate, n = 40) 124 X/M = 1 (Complete septate, n = 8) 27 Unicornuate (n = 4) 13 Total
Per patient 3.1 3.1 3.4 3.3
TABLE VIII Pregnancy course in 47 randomized women with congenital uterine anomaly who did not receive any treatment n
%
Abortion Still birth Full-term delivery On-going pregnancy
152 1 7 1
94.4 0.6 4.4 0.6
Total
161
100.0
456
TABLE VI Reproductive wastage before metroplasty in 50 patients with congenital uterine anomalies n
%
Abortion On-going pregnancy Full-term delivery
5 4 26
14.3 11.4 74.3
Total
35
100.0
TABLE IX Distribution of anticardiolipin antibody positive patients with recurrent reproductive wastage (Group 1 and 2: see Subjects and Methods) Group
n
ACA positive
%
1 2
136 12
6 6
4.4 50.0
128 TABLE X Summary of the results after immunotherapy of 393 patients with a history of recurrent spontaneous abortion n
Total number of immunized patients
393
Total cases of pregnancy Number of patients giving birth Number of deliveries On-going pregnancy Abortions
239 171 200 23 16
for anti-cardiolipin antibody, 12 women were positive, when 6 IU was defined as the cut-off value for positive. Table X summarizes our recent results from performing immunotherapy for recurrent spontaneous abortions. As shown in this table, 239 women became pregnant after immunotherapy. Of these pregnant women, 171 patients delivered 200 full-term children and 23 other pregnancies are currently in the second trimester. These children are now up to three years of age, and Figs. 3 and 4 show that their physical development has been within the standards for Japan as a nation. In the randomized 64 control women, 64.1% of pregnancies were spontaneously terminated before 12 weeks of gestation. Discussion
In general, habitual recurrent miscarriage is defined as a sequence of three or more spontaneous abortions 112,131. In the present study, some women with a history of 2 recurrent spontaneous abortions were registered and then became
TABLE XI Prognosis of subsequent pregnancy in 64 randomized control women who did not receive immunotherapy n
%
Abortion On-going pregnancy Full-term delivery
41 5 18
64.1 7.8 28.1
Total
64
100.0
subjects for this survey because they strongly requested examination and treatment for their reproductive problems. The term ‘habitual’ means that after three consecutive abortions, the risk of subsequent loss is very high. Malpas was the first to describe a theoretical calculation of the probability of subsequent spontaneous abortion, and hypothesized that following three abortions, the likelihood of a subsequent abortion was 80-90% [5], although many other subsequent studies have reported lower percentages [12]. Our present study demonstrated that 2898 of 3216 conceptions in the 1120 registered patients with a history of recurrent miscarriage terminated in spontaneous abortion, for early reproductive loss rate of more than 90%. The mean number of abortions was 2.9 k 0.7, and the mean age was 31.1 f 3.6 years. Thus, repetitive spontaneous abortion is a problem that becomes increasingly serious for both the patient and the gynecologist, because the biological clock for female reproduction may soon expire unless the patients receive exact diagnosis and appropriate therapy for their reproductive problems. In this survey, it was found that the incidence of chromosomal abnormalities was 5.9% for major abnormalities and 4.0% for normal variants out of a total of 825 Japanese couples with a history of recurrent fetal loss. These results agree well with the findings of others [6,7], and indicate that there are no racial differences in this regard. There are several kinds of translocations, and some studies have reported that there is no excess of spontaneous abortions or malformations in families with translocations. However, our present study demonstrated that the incidence of reciprocal translocation is clearly higher in the group with a history of recurrent early reproductive loss, suggesting that this anomaly may play an important role in early reproductive wastage. Robertsonian translocations were detected in 10 cases, with more female than male carriers (8 : 2). Robertsonian translocations tend to cause sterility rather than miscarriage. Inversion 9 was found in 23 cases in the present study. The reported frequency of this inversion in the general population varies widely, from 0.01 to l.O%, with the differences probably due to the banding tech-
129
niques. No treatment is available for patients diagnosed as carriers of an abnormal karyotype, and all such patients should be thoroughly counseled by specialists to avoid further unnecessary abortions. The uterus is an organ formed by the growth and fusion of paired primodia, and normal development depends totally on the regression of certain tissues as a result of this fusion. Thus, maldevelopment of the uterus is common, but nearly half of the cases of congenital anomalies of the uterus remain undiagnosed until difficulty in the maintenance of pregnancy is experienced. In the present study, 147 congenital uterine anomalies were detected in 1000 hysterosalpingographies. Since our previous report [3] demonstrated that the uterine septum is a tissue with an insufficient blood supply, we performed an ideal surgical resection of the ischemic area of the septum on these cases, constructing a new uterine cavity and establishing a normal cross-anastomosis of the uterine vessels. By this metroplasty, more than 85% of subsequent pregnancies were successfully maintained, whereas 169 out of 171 pre-operative gestations had terminated in reproductive wastage. This post-operative prognosis of pregnancy is also significant when compared to the prognosis for the randomized control group of women. Harris et al. were the first [8] to report a reliable method for measurement of anticardiolipin antibody, and it was demonstrated that this antibody was positive in patients with SLE and other autoimmune diseases. Recently, it was also reported that anti-cardiolipin antibody is associated with habitual abortion [9], suggesting a relatively high incidence of early productive wastage with anti-cardiolipin antibody. In the present study, 8.1% of 148 women examined were positive for anti-cardiolipin antibody, and in the group with a high antibody titer, the incidence of spontaneous abortion was higher in the second trimester than in the first trimester. Although some immunosuppressants and anticoagulants are recommended for reducing reproductive wastage, no concrete therapy has been recommended for anti-cardiolipin antibody syndrome. We previously reported successful results in the treatment
of this syndrome with adrenal steroids and aspirin [ 101. The immune response mechanism during human gestation is extremely complex. Part of this mechanism has been thought to be under genetic control. Many studies have reported the genetic significance of the major histocompatibility complex (MHC) in terms of its role in the maintenance of pregnancy, but the exact mechanism of its role in human reproduction remains to be elucidated. We have demonstrated that immunotherapy for recurrent spontaneous abortion by utilizing the husband’s lymphocytes induces anti-HLA blocking antibody in the immunized female partner, as shown in the one-way mixed lymphocyte culture (ML0 reaction [ll]. Anti-idiotype antibody has been demonstrated in the post-immunization serum of patients by two-color flowcytometry using FITC-labeled human immuno-y globulin and PE-labeled CD3 antibody [ll]. By this method, it was also demonstrated that the subset composition of T lymphocytes could be changed through immunotherapy, resulting in an increase in the suppressor T cells and a decrease in the cytotoxic T cells [l 11. All these findings may indicate that the maternal immune response can be altered by immunotherapy in a direction beneficial to the maintenance of pregnancy, and this could be one explanation for why clinical immunotherapy shows significant efficacy in the treatment of habitual abortion. The results of the present study indicate that there may be a selective mechanism in human reproduction which eliminates unfit conceptions, but that the number of pregnancy wastages can be minimized by comprehensive routine examination of each case and administration of appropriate therapy. Acknowledgements
The authors thank Ms. Hiroko Otsuka for her invaluable assistance throughout this study. They are also grateful to Dr. Takao Koike of Chiba University Hospital for generously supplying a standard serum of anti-cardiolipin antibody. This study, supported in part by a grant from the Ministry of Education of Japan (No. 01480400),
130
was presented by the first author at the VIIth World Congress on Human Reproduction in Helsinki, Finlarid, in 1990 as an invited lecture.
8
References 1 Reid DE, Ryan KJ, Benirschke K (Eds). Principles, Management of Human Reproduction. Philadelphia, PA: W.B. Saunders Company, 1972. 2 Wilcox AJ, Weinberg CR, O’Connor JF, Baird DD, Schlaterer JP, Canfield RE, Armstrong EG, Nisula BC. Incidence of early loss of pregnancy. N Engl J Med 1988;319:189-194. 3 Nakada K, Makino T, Tabuchi T, Iizuka R Analysis of congenital uterine anomalies in habitual abortions, evaluation of metroplasty. Jpn J Fertil Steril 1989;34:842-847. 4 Makino T Reproductive Wastage. The Proceedings of the XIIIth World Congress on Human Reproduction, Marrakesh, 1989. 5 Malpas P. A study of abortion sequences. J Obstet Gynecol Br Commonw 1938;45:931-949. 6 Simpson JL. Pregnancies in women with chromosomal abnormalities: genetic disease in pregnancy. New York: Academic Press, 1981;439. 7 Makino T, Tabuchi T, Nakada K, Iwasaki K, Tamura, S,
9
10
11
12 13
14
Iizuka R. Chromosomal analysis of 639 Japanese couples with repeated spontaneous abortions. Intern J, Fertil 1990;35:266-270. Harris EN, Gharavi AE, Boey ML, Pate1 BM, MacWorthYoung CG, Loizou S, Hughes GRV. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus Erythematosus. Lancet 1983;11:1211-1214. Lockshin MD, Qamar T, Druzin ML, Goei, S. Antibody to cardiolipin, lupus anticoagulant and fetal death. J Rheumatol 1987;14:259-262. Toyoshima K, Makino T, Sugi T, Nozawa S, Iizuka, R. Correlation between trimester of fetal wastage and anticardiolipin antibody titer. Intern J Fertil 1991;36:89-93. Sugi T, Makino TI, Maruyama T, Kim WK, Iizuka R. A possible mechanism of immunotherapy for patients with recurrent spontaneous abortions. Am J Reprod Immunol 1991;25:185-189. Stirrat GM. Recurrent miscarriage I: definition, epidemiology. Lancet 199O;i:673-675. WHO recommended definitions, terminology, format for statistical tables related to the perinatal period. Act Obstet Gynecol Stand 1977;56:247-253. Stirrat GM. Recurrent miscarriage II: clinical associations, causes, management. Lancet 1990$:728-733.
Biology,44 (1992)123-130 0 1992ElsevierScience Publishers B.V. All rights reserved 002%2243/92/$05.00
European Journal of Obstetrics & Gynecology and Reproductive
EUROBS 01316
Survey of 1120 Japanese women with a history of recurrent spontaneous abortions Tsunehisa
Makino, Takabumi Hara, Chikahiro Oka, Kiwamu Toyoshima, Toshitaka Ken-ichi Iwasaki, Masakatsu Umeuchi and Rihachi Iizuka
Sugi,
Department of Obstetrics and Gynecology, School of Medicine, Keio Unioersiiy, Tokyo, Japan
Accepted for publication 15 October 1991
Summary
Our recent 7-year clinical survey showed that among the 1120 women with repeated spontaneous abortions registered in this clinic, 2898 out of a total of 3216 pregnancies (90.1%) had terminated in spontaneous abortion. Among these wastages, 84.2% occurred before 12 weeks of gestation, and 11.1 percent occurred between 12 and 15 weeks. Through routine examination of reproductive wastage, 82 (9.9%) of the 825 Japanese couples examined were shown to have either a chromosomal abnormality or normal variants in the wife and/or husband, thus demonstrating no racial difference in the incidence of chromosomal abnormalities in infertile patients in comparison with studies performed in other countries. One hundred and forty-seven congenital uterine anomalies (14.7%) were found in 1000 hysterosalpingographies, and 12 of 148 examined females were positive for anti-cardiolipin antibody. 393 other females with no major abnormalities likely to induce spontaneous abortions were indicated for immunotherapy. Ample time spent on genetic counseling prevented further reproductive wastage, and ideal metroplasty resulted in a successful post-operative pregnancy course in more than 85% of cases. Immunosuppressant and anticoagulant therapy decreased the serum titer of anti-cardiolipin antibody, enabling pregnancies to be maintained to term. Immunotherapy utilizing the husband’s lymphocytes also brought more than 80% of pregnancies to successful completion, with 200 deliveries achieved with this therapy. In contrast, 64.1% of pregnancies again terminated spontaneously in patients who were indicated for immunotherapy but did not receive treatment. The findings of the present study suggest that the causes of reproductive wastage, especially the etiology of early recurrent spontaneous abortion, are complex. However, well-organized routine examination of the causes of repetitive wastage has made it possible to identify appropriate treatments, resulting in marked reduction in human reproductive wastage. Abortion
Introduction Correspondence:
Dr. Tsunehisa Makino, Dept. of Obstetrics and Gynecology, School of Medicine, Keio University, Shin-
juku-ku,Tokyo 160,Japan.
The process of reproduction is extremely complex and inefficient [1,4]. It is estimated that
124
approximately 10 to 20% of known conceptions terminate in spontaneous abortion. Recent progress in hormone measurement has improved the sensitivity, specificity and assay time of conventional radio immunoassay. With this new approach, Wilcox et al. [23 demonstrated that 22% of conceptions terminated before pregnancy were detected clinically. Thus, spontaneous abortion is a common phenomenon and constitutes one of the major segments of what has been called reproductive or pregnancy wastage. In this study, 1120 Japanese women with a history of recurrent spontaneous abortions registered in our infertility clinic were surveyed for common background factors, including their age and the number and gestational weeks of their spontaneous abortions. The possible causes of these wastages were then surmised through comprehensive, routine examination of these patients. The effectiveness of the treatments for each etiology was also monitored through tracking their subsequent pregnancies over the past 7 years. As a control, the course of pregnancy was also monitored in patients who have had some abnormality but did not receive any appropriate therapy before pregnancy. These follow-up studies improved our methods of clinical examination and resulted in establishment of more effective therapies for common human reproductive wastages.
group was 3216; 2899 of these conceptions terminated in spontaneous abortion.
had
Congenital uterine anomaly
By the end of March 1990, hysterosalpingography had been performed on 1000 out of 1120 women, and 147 congenital uterine anomalies had been detected. A method in which the longitudinal iength of the uterine cavity (M) is compared with the dent of the uterine fundus (X) under hysterosalpingography was introduced to evaluate uterine deformities, and the incidence of abortion was analyzed as a function of the results of this evaluation (Fig. 1) [3]. A modified metroplasty was performed on 50 of these 147 patients. As a control, 47 other patients with anomalies were randomly selected, and their subsequent pregnancies were monitored without metroplasty. Chromosomal analysis
Chromosomal analysis of peripheral blood cells had been performed for both partners in 825 of the 1120 couples by the end of March 1990. The chromosomal preparations were analyzed after applying the trypsin G band method, and Qbanding or C-banding was also performed when necessary. For each individual, a minimum of 20 metaphase plates were counted, and at least 3 cells were karyotyped. Anti-phospholipid antibody syndrome
Subjects and Methods The possible causes of recurrent consecutive spontaneous abortions in 1120 couples (n = 1120) registered between 1984 and 1990 at Keio University Hospital in Tokyo were investigated. Genetic, immunologic, endocrine, infectious and uterine organic factors, including uterine myoma, were studied. The age distribution of the female partners was from 19 to 47 years old, with a mean age of 31.3 + 4.5 years (mean k SEMI. A spontaneous abortion was defined as a spontaneously terminated pregnancy in which urinary hCG was detected and D/C was performed. The mean number of spontaneous abortions was 2.9 + 0.7. The total number of past pregnancies in the patient
Using an enzyme-linked immunosorbent assay employing Harris’s standard serum, 148 women with a history of recurrent fetal loss were tested for serum anti-cardiolipin antibody. To differentiate early and late abortions, these women were divided into two groups. The spontaneous abortions in Group 1 were in the first trimester and those of Group 2 included wastages in the second trimester. The women diagnosed as having antiphospholipid antibody syndrome were then administered immunosuppressant or anticoagulant therapy. Immunotherapy for habitual spontaneous abortion Three hundred and ninety-three (n = 393)
women for whom the cause of abortion could not be surmised on the basis of our routine examina-
125
(1)
0 < f$ < -+
: arcuate
(2) + 5 6 < 1 : partial (3) $ + 1 Fig. 1. Evaluation
uterus
septate
: complete
uterus
septate
of uterine cavity deformity hysterosalpingogram.
uterus
by X/M
ratio in
tion of recurrent spontaneous abortion were treated with their husbands’ lymphocytes as immunotherapy [4]. The lymphocytes were separated aseptically from 30 ml of the husband’s venous blood by the Conray-Ficoll Method, and the lymphocyte concentration was adjusted to approximately 40 million/ml. Then this suspension was intradermally injected into the female partner up to 4 times at intervals of 2-4 weeks. As a randomized control, 64 other women indicated for immunotherapy who did not receive this treatment were monitored in their subsequent pregnancies. The outcome and course of pregnancies begun after this immunotherapy were investigated. The physical development of all children who were conceived and born after the immunotherapy was monitored up to the age of 3 years.
cases of normal variants detected in 825 couples. The 49 major chromosomal abnormalities consisted of 25 reciprocal translocations, 10 Robertsonian translocations, 3 inversions and 11 sex chromosome aneuploidies. The group with reciprocal translocations was the largest, consisting of 11 female and 14 male carriers. None of the reciprocal translocations involved the sex chromosomes. In one couple, both partners were carriers of a translocation. The incidence of chromosomal abnormalities was 5.9%, and the incidence when normal variants were included was 9.9%. Table III summarizes the frequency of abortions in the past pregnancies of the patients having each type of abnormality. In the 10 cases with Robertsonian translocations, the incidence of abortions seemed to be slightly lower, but there was no significant difference compared to the other types of abnormality. One hundred and forty seven congenital uterine anomalies were detected in 1000 hysterosalpingographies. By introducing the new X/M ratio to hysterosalpingography to evaluate uterine cavity deformities, Tables IV and V summarize the incidence of each anomaly and the early reproductive wastage frequency. As indicated, the largest group of anomalies is the 0
I N=994
Results The nine hundred and ninety-three spontaneous abortions confirmed from the patients’ histories are summarized in Fig. 2. As indicated, more than 84% of these wastages occurred before 12 weeks of gestation. Table I summarizes the possible causes and pathological findings for the 1120 couples, showing a wide variety in the etiology of recurrent reproductive wastage. Chromosomal abnormalities are generally classified as major or minor variants. Table II shows the 49 major chromosomal abnormalities and 33
0 4W
6W 5w
8W 7w
tow 9w
Fig. 2. Weeks of gestation
IZW IIW
13W
14w 16W . 15w 17w
at spontaneous patients.
1
2ow 22w 19W 21W 23W
abortion
in infertile
126 TABLE II Chromosomal abnormal in 825 couples with a history of recurrent spontaneous abortion 70-Z
n
*I 60.;:$‘;. 40- ;;-
iE 2. .; I 3
6MlY
2
3
%
Reciprocal translocation Robertsonian translocation Inversion (numbers 7, 11 and 12) Sex chromosome aneuploidy Normal variants
25 10 3 11 33
30.5 12.2 3.7 13.4 40.2
Total
82
100.0
Keio Univ. Hosp.
Fig. 3. Physical development of male children born to immunized mothers.
with anomalies. An ideal metroplasty was performed on 50 women with anomalies, and more than 85% of the postoperative pregnancies in 35 patients were successful, as indicated in Table VII. In contrast, 98.8% of the pregnancies in these 50 women had terminated in spontaneous abortion before the surgical treatment (Table VI), and 94.4% of pregnancies in the group of 47 randomized control women with anomalies also terminated spontaneously (Table VIII). Table IX shows that of 148 patients with a history of recurrent reproductive wastage tested
cm
6Mly
2
3
Keio Univ. Hosp.
Fig. 4. Physical development of female children born to immunized mothers.
TABLE I Abnormalities
detected in 1120 infertile women with a history of recurrent spontaneous abortion
Congenital uterine anomaly Uterine myoma Endometriosis Cervical incompetency Asherman’s syndrome Chromosomal abnormality Autoimmune diseases RA SLE MCTD Positive for anti-cardiolipin antibody Diabetes mellitus
147 23 7 12 9 82 5 2 1 24 5
Thyroid dysfunction Infections Syphilis RF Tuberculosis (lung) Hepatitis Miscellaneous Asthma Thrombosis Myasthenia gravis Muscular dystrophy Gong. heart disease
21 1 3 4 3 9 3 5 1 3
127 TABLE III Reproductive wastages in 82 carriers of abnormal chromosomes
Reciprocal translocation Robertsonian translocation Inversion Sex chromosome aneuploidy Normal variants
n
Total pregnancies
25 10 3 11 33
86 38 11 36 108 279
Total
Abortions or stillbirths
Live borns
Abortion rate (o/o)
80 30 10 32 95
6 8 1 4 13
93.0 78.9 90.9 88.9 88.0
247
32
88.5
TABLE IV
TABLE VII
Incidences of congenital uterine anomalies
Pregnancy course after metroplasty in 35 patients with congenital uterine anomalies
n
0 < X/M < l/3 (arcuate) l/3 < X/M < 1 (partial septate) X/M=1 (complete septate) Unicornuate Total
%
95 40 8 4
64.6 27.2 5.4 2.8
147
100.0
%
n
Abortion Premature labor Stillbirth Full-term
169 1 1 0
98.8 0.6 0.6 0.0
Total
171
100.0
TABLE V Number of spontaneous abortions in each group evaluated by X/M ratio in hysterosalpingogram No. of
abortions 0 < X/M < l/3 (Arcuate, n = 95) 292 l/3 < X/M < 1 (Partial septate, n = 40) 124 X/M = 1 (Complete septate, n = 8) 27 Unicornuate (n = 4) 13 Total
Per patient 3.1 3.1 3.4 3.3
TABLE VIII Pregnancy course in 47 randomized women with congenital uterine anomaly who did not receive any treatment n
%
Abortion Still birth Full-term delivery On-going pregnancy
152 1 7 1
94.4 0.6 4.4 0.6
Total
161
100.0
456
TABLE VI Reproductive wastage before metroplasty in 50 patients with congenital uterine anomalies n
%
Abortion On-going pregnancy Full-term delivery
5 4 26
14.3 11.4 74.3
Total
35
100.0
TABLE IX Distribution of anticardiolipin antibody positive patients with recurrent reproductive wastage (Group 1 and 2: see Subjects and Methods) Group
n
ACA positive
%
1 2
136 12
6 6
4.4 50.0
128 TABLE X Summary of the results after immunotherapy of 393 patients with a history of recurrent spontaneous abortion n
Total number of immunized patients
393
Total cases of pregnancy Number of patients giving birth Number of deliveries On-going pregnancy Abortions
239 171 200 23 16
for anti-cardiolipin antibody, 12 women were positive, when 6 IU was defined as the cut-off value for positive. Table X summarizes our recent results from performing immunotherapy for recurrent spontaneous abortions. As shown in this table, 239 women became pregnant after immunotherapy. Of these pregnant women, 171 patients delivered 200 full-term children and 23 other pregnancies are currently in the second trimester. These children are now up to three years of age, and Figs. 3 and 4 show that their physical development has been within the standards for Japan as a nation. In the randomized 64 control women, 64.1% of pregnancies were spontaneously terminated before 12 weeks of gestation. Discussion
In general, habitual recurrent miscarriage is defined as a sequence of three or more spontaneous abortions 112,131. In the present study, some women with a history of 2 recurrent spontaneous abortions were registered and then became
TABLE XI Prognosis of subsequent pregnancy in 64 randomized control women who did not receive immunotherapy n
%
Abortion On-going pregnancy Full-term delivery
41 5 18
64.1 7.8 28.1
Total
64
100.0
subjects for this survey because they strongly requested examination and treatment for their reproductive problems. The term ‘habitual’ means that after three consecutive abortions, the risk of subsequent loss is very high. Malpas was the first to describe a theoretical calculation of the probability of subsequent spontaneous abortion, and hypothesized that following three abortions, the likelihood of a subsequent abortion was 80-90% [5], although many other subsequent studies have reported lower percentages [12]. Our present study demonstrated that 2898 of 3216 conceptions in the 1120 registered patients with a history of recurrent miscarriage terminated in spontaneous abortion, for early reproductive loss rate of more than 90%. The mean number of abortions was 2.9 k 0.7, and the mean age was 31.1 f 3.6 years. Thus, repetitive spontaneous abortion is a problem that becomes increasingly serious for both the patient and the gynecologist, because the biological clock for female reproduction may soon expire unless the patients receive exact diagnosis and appropriate therapy for their reproductive problems. In this survey, it was found that the incidence of chromosomal abnormalities was 5.9% for major abnormalities and 4.0% for normal variants out of a total of 825 Japanese couples with a history of recurrent fetal loss. These results agree well with the findings of others [6,7], and indicate that there are no racial differences in this regard. There are several kinds of translocations, and some studies have reported that there is no excess of spontaneous abortions or malformations in families with translocations. However, our present study demonstrated that the incidence of reciprocal translocation is clearly higher in the group with a history of recurrent early reproductive loss, suggesting that this anomaly may play an important role in early reproductive wastage. Robertsonian translocations were detected in 10 cases, with more female than male carriers (8 : 2). Robertsonian translocations tend to cause sterility rather than miscarriage. Inversion 9 was found in 23 cases in the present study. The reported frequency of this inversion in the general population varies widely, from 0.01 to l.O%, with the differences probably due to the banding tech-
129
niques. No treatment is available for patients diagnosed as carriers of an abnormal karyotype, and all such patients should be thoroughly counseled by specialists to avoid further unnecessary abortions. The uterus is an organ formed by the growth and fusion of paired primodia, and normal development depends totally on the regression of certain tissues as a result of this fusion. Thus, maldevelopment of the uterus is common, but nearly half of the cases of congenital anomalies of the uterus remain undiagnosed until difficulty in the maintenance of pregnancy is experienced. In the present study, 147 congenital uterine anomalies were detected in 1000 hysterosalpingographies. Since our previous report [3] demonstrated that the uterine septum is a tissue with an insufficient blood supply, we performed an ideal surgical resection of the ischemic area of the septum on these cases, constructing a new uterine cavity and establishing a normal cross-anastomosis of the uterine vessels. By this metroplasty, more than 85% of subsequent pregnancies were successfully maintained, whereas 169 out of 171 pre-operative gestations had terminated in reproductive wastage. This post-operative prognosis of pregnancy is also significant when compared to the prognosis for the randomized control group of women. Harris et al. were the first [8] to report a reliable method for measurement of anticardiolipin antibody, and it was demonstrated that this antibody was positive in patients with SLE and other autoimmune diseases. Recently, it was also reported that anti-cardiolipin antibody is associated with habitual abortion [9], suggesting a relatively high incidence of early productive wastage with anti-cardiolipin antibody. In the present study, 8.1% of 148 women examined were positive for anti-cardiolipin antibody, and in the group with a high antibody titer, the incidence of spontaneous abortion was higher in the second trimester than in the first trimester. Although some immunosuppressants and anticoagulants are recommended for reducing reproductive wastage, no concrete therapy has been recommended for anti-cardiolipin antibody syndrome. We previously reported successful results in the treatment
of this syndrome with adrenal steroids and aspirin [ 101. The immune response mechanism during human gestation is extremely complex. Part of this mechanism has been thought to be under genetic control. Many studies have reported the genetic significance of the major histocompatibility complex (MHC) in terms of its role in the maintenance of pregnancy, but the exact mechanism of its role in human reproduction remains to be elucidated. We have demonstrated that immunotherapy for recurrent spontaneous abortion by utilizing the husband’s lymphocytes induces anti-HLA blocking antibody in the immunized female partner, as shown in the one-way mixed lymphocyte culture (ML0 reaction [ll]. Anti-idiotype antibody has been demonstrated in the post-immunization serum of patients by two-color flowcytometry using FITC-labeled human immuno-y globulin and PE-labeled CD3 antibody [ll]. By this method, it was also demonstrated that the subset composition of T lymphocytes could be changed through immunotherapy, resulting in an increase in the suppressor T cells and a decrease in the cytotoxic T cells [l 11. All these findings may indicate that the maternal immune response can be altered by immunotherapy in a direction beneficial to the maintenance of pregnancy, and this could be one explanation for why clinical immunotherapy shows significant efficacy in the treatment of habitual abortion. The results of the present study indicate that there may be a selective mechanism in human reproduction which eliminates unfit conceptions, but that the number of pregnancy wastages can be minimized by comprehensive routine examination of each case and administration of appropriate therapy. Acknowledgements
The authors thank Ms. Hiroko Otsuka for her invaluable assistance throughout this study. They are also grateful to Dr. Takao Koike of Chiba University Hospital for generously supplying a standard serum of anti-cardiolipin antibody. This study, supported in part by a grant from the Ministry of Education of Japan (No. 01480400),
130
was presented by the first author at the VIIth World Congress on Human Reproduction in Helsinki, Finlarid, in 1990 as an invited lecture.
8
References 1 Reid DE, Ryan KJ, Benirschke K (Eds). Principles, Management of Human Reproduction. Philadelphia, PA: W.B. Saunders Company, 1972. 2 Wilcox AJ, Weinberg CR, O’Connor JF, Baird DD, Schlaterer JP, Canfield RE, Armstrong EG, Nisula BC. Incidence of early loss of pregnancy. N Engl J Med 1988;319:189-194. 3 Nakada K, Makino T, Tabuchi T, Iizuka R Analysis of congenital uterine anomalies in habitual abortions, evaluation of metroplasty. Jpn J Fertil Steril 1989;34:842-847. 4 Makino T Reproductive Wastage. The Proceedings of the XIIIth World Congress on Human Reproduction, Marrakesh, 1989. 5 Malpas P. A study of abortion sequences. J Obstet Gynecol Br Commonw 1938;45:931-949. 6 Simpson JL. Pregnancies in women with chromosomal abnormalities: genetic disease in pregnancy. New York: Academic Press, 1981;439. 7 Makino T, Tabuchi T, Nakada K, Iwasaki K, Tamura, S,
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Iizuka R. Chromosomal analysis of 639 Japanese couples with repeated spontaneous abortions. Intern J, Fertil 1990;35:266-270. Harris EN, Gharavi AE, Boey ML, Pate1 BM, MacWorthYoung CG, Loizou S, Hughes GRV. Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in systemic lupus Erythematosus. Lancet 1983;11:1211-1214. Lockshin MD, Qamar T, Druzin ML, Goei, S. Antibody to cardiolipin, lupus anticoagulant and fetal death. J Rheumatol 1987;14:259-262. Toyoshima K, Makino T, Sugi T, Nozawa S, Iizuka, R. Correlation between trimester of fetal wastage and anticardiolipin antibody titer. Intern J Fertil 1991;36:89-93. Sugi T, Makino TI, Maruyama T, Kim WK, Iizuka R. A possible mechanism of immunotherapy for patients with recurrent spontaneous abortions. Am J Reprod Immunol 1991;25:185-189. Stirrat GM. Recurrent miscarriage I: definition, epidemiology. Lancet 199O;i:673-675. WHO recommended definitions, terminology, format for statistical tables related to the perinatal period. Act Obstet Gynecol Stand 1977;56:247-253. Stirrat GM. Recurrent miscarriage II: clinical associations, causes, management. Lancet 1990$:728-733.