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Embryonic development in consecutive specimens from recurrent spontaneous abortions
Transactions of the Thirty-third Annual Meeting of the Society of Obstetricians and Gynaecologists of Canada
Embryonic development in consecutive specimens from recurrent spontaneous abortions BETTY BASIL Vancouver,
J. POLAND HO
YUEN
British
Columbia,
Canada
Although habitual abortion occurs in a relatively small number of patients, its successful management is often difficult., Etiologic factors may be either maternai or ernbryonk? or both; there is much information available on maternal factors but little is known of factors related to the embryo. Information obtained from examining and keryotyping the aborted Bmbryo or fetus may be of considerable importance in determining the possible cause of the abodion and subsequent investigation and counseling of the patient. At least two specimens from over 59 women who have had two or more consecutive spontaneous abortiins have been examined and the results show that: (1) Patients aborted co%eptuCs of the same developmental stage, i.e., embryo or fetus, in consecutive pregnancies. This suggests that investigation &ould be directed to factors which may be important in the stage of pregnancy indicated. (2) Late abortions were associated with normal fetal development and factors related to the uterine environment. (3) The conceptus in an ear& abortion was significantly more likely to have a cytogenetic abnormality, suggesting a problem at, or prior to, conception/fertilization. A case study is also included. (AM. J. OBSTET. GYNECOL. 130: 512, 1978.)
A LTHO u G H spontaneous abortion is a common event in a patient’s reproductive history, recurrence is relatively rare. Patients who have had two consecutive abortions are at greater risk of having a third and even a fourth,’ with the frustrations and unhappiness which accompany this problem. While considerable emphasis has been placed on the possible maternal factors which may be responsible for recurrent abortion, factors reFrom the Department University of British Supported by Medical MA 2740.
of Obstetrics
and Gynecology,
Columbia.
Research
Council
of Canada
Grant
Presented at the Thirty-third Annual Meeting of the Society of Obstetricians and Gynaecologists of Canada, Montreal, Quebec, Canada, June 14-18, 1977. Reprint requests: Dr. B. J. Poland, Vancouver General Hospital, Vancouver, Btitih Columbia, V5.Z IM9, Canada.
512
lated to embryonic and fetal development have received less attention until more recent years, when it has been shown that both morphologic* and cytogenet.? abnormalities of embryonic development are even more common than in sporadic spontaneous abortion. This study was undertaken to correlate the status of the conceptus in consecutive abortions with relevant maternal factors and to apply this information to the structure of a program of investigation and management. In the Embryology Study of the University of British Columbia, we have been carrying out a comprehensive examination of embryos and fetuses for 10 years. The only criteria for admission of the specimens to the study was that the embryo or fetus should be sufficiently intact for evaluation and that a maternal history should be available. During the course of the study embryos and fetuses from two or more consecu000%9378/78/05130-0512$00.40/O
0
1978
The C. V. Mosby
Co.
Volume Number
Embryonic development in recurrent abortions
130 5
tive abortions in 52 patients have been examined. Information from these patients and observations on the abortus form the basis for this study. The study was prospective in that at the time of admission of the first specimen it was not known that a second or even a third would be available.
Matetlals and methods The abortuses were examined in the Embryology Study from September, 1966, to January, 1977. These were examined by macro- or microdissection and categorized as embryo or fetus, normal or abnormal, and the abnormality was delineated. Tissue was submitted to the Cytogenetics Iaboratory, Department of Medical Genetics, and culture and subsequent chromosomal analysis were carried out under the direction of Dr. F. J. Dill. This investigative procedure was not available for specimens prior to 1971. The history was obtained directly from the patient by personal interview whenever possible; otherwise, it was obtained from the record of the attending physician.
Results Fifty-two patients had 134 abortions of which the products of conception from 112 were examined. This was a total of 114 abortuses, including two sets of twins. Each specimen was categorized first as an embryo or fetus and then as normal or abnormal. The patients fell into one of three groups. Patients in group 1 aborted embryos consistently, in group 2 fetuses consistently, and in group 3 a mixture (Fig. 1). Forty patients, or 77 per cent, aborted conceptuses of the same category on consecutive occasions. In group 1, 60 out of 64 embryos (94 per cent) were abnormal, 52 of which had severe growth disorganization. Twenty-five of the 29 patients aborted only abnormal embryos. In group 2, only one of the 24 fetuses, a twin with spina bifida, was abnormal. Ten of the 11 patients aborted only normal fetuses. In group 3, 50 per cent of the patients aborted an embryo first and 50 per cent a fetus. There was an abnormality in seven first specimens and in eight second specimens. Twelve out of 13 embryos and three out of 13 fetuses were abnormal. Two of the fetuses had the phenotypes of chromosomal anomalies consistent with viability, X0 and trisomy 13. Karyotypes were obtained on 17 of the growthdisorganized embryos in this study. There were eight embryos with trisomy, five with triploidy, one with a translocation, and three with normal karyotypes. The translocation found in the embryo was unbalanced and was found in a balanced state in the mother’s karyotvpe. Growth-disorganized embryos have been
Group
1
29 Patients
AAAAAAAAAAAA
AAAAAAAAAAAAAAAA f AAAAAAAAAAAAAAAA A A
AAAAAAAAAAAA AAA Group
2
11 Pcttisnts
q uuooononou
A
F”~nuououo Group
3
513
A
q n
12 Patients
q AAAUA#O~#AU Auo~A13AAgAuA
Normal Abnormal
embryo. embryo
Normal
fetus
Abnormal
fetus
0
Fig. 1. Graphic representation of consecutive specimens in 52 patients. Double symbols indicate twins. I. Maternal of patients Table
Factors
Total abortions abortions per patient A&rage age (yr.) Irregular menstrual cycles Prior use of oral
factors in the three groups Group
1
Group 2
GTCW~ 3
78 2.7
26 2.3
30 2.5
31 12/25 = 48%
33 5/9 = 55%
31 5/l 1 = 45%
1l/29 = 33%
419 = 43%
4/l 1 = 36%
shown in other studies to have a chromosomal anomaly in over 80 per cent of cases.4 There were karyotypes obtained on four other abnormal embryos: three were trisomies and one was a triploid. It was possible to obtain a successful karyotype on two consecutive specimens in seven patients. In two patients, both specimens were trisomic; in two, trisomic and triploid; in three a trisomy followed an embryo with normal karyotype. Table I is an analysis of the maternal history in the three groups. The various factors do not differ significantly from group to group. The cycles designated irregular had a mean variation of 7 days or more. Oral contraception had been used prior to the first abortion but not subsequently.
Comment In group 1, the early failure of the pregnancy must be related to the lethal anomaly and the mechanisms which bring this about. The chromosomal anomalies were the same as those found in sporadic cases of spontaneous abortion, namely, trisomies and triploids. Although these anomalies have, as far as is known, a different mode of production, they existed in consecutive specimens of two patients. Trisomy has been associated with increased maternal chronologic age in both liveborn infants and abortuses.5* 6 Triploidy does not appear to be age related but has been considered to be
514
Poland and Ho hen
March
Am. J.
“0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Cycle Days
2. Plasma LH levels in control and Clomid cycles.
Fig. BOO7M) wo7i 2 P
500 400mo200 loo
-
0’
0
2
4
’
6
’
8
’
10
’
12
’
14
’
16
’
18
’
20
’
22
’
24
’
26
’
28
Cycle Days
3. Plasma estradiol levels in control and Clomid cycles.
Fig.
35 30 ’3
2520 I5 IO 50****** 0
0
‘( 2
4
6
8
10
12
I4
16
I8
20
22
24
26
28
Cycle Days
Fig.
4. Plasma progesterone
levels in control and Clomid cy-
cles. an accident of fertilization.’ The presence of the cytogenetic and morphologic anomalies suggests that the abortion was inevitable from conception. “Group 2 specimens consisted of normal fetuses, with one exception, which continued to develop in some cases to the beginning of the second trimester. It would appear that implantation, though initially successful, eventually failed and factors such as uterine abnormality, fibroids, incompetent cervix, or the quality of the endometrium would seem likely to be involved. Group 3 specimens were the result of both early and late abortion in the same patient and the result, presumably, of a combination of factors. These patients would require comprehensive investigation re-
1, 1978
Obstet. Gynecol.
lated to factors of both embryonic development and the uterine environment. In patients habitually aborting an early, abnormal embryo, it is necessary to search for factors persistently interfering with the production of a normal conceptus. The presence of a structural chromosomal anomaly in the parents leads to an increased frequency of an unbalanced karyotype in the conceptus, and has been found in two per cent of couples, affecting one or other parent, when there was a history of recurrent abortion.8 Parental karyotyping should therefore be done in such cases. Hertig,g from his work on early embryos in timed conceptions, states that, “If an oocyte lingers longer than 14 days in the follicle, it is likely to be a bad egg.” Considerable work has been done, using the rat as a model, on the relationship between delayed ovulation and subsequent conceptionsi A significant decrease in implantation and an increase in the number of abnormal and retarded conceptuses have been observed. Similar evidence is difficult to obtain in human subjects. Guerraro and Rojas I1 showed that in 1,000 cycles, which were monitored by a basal body temperature chart, the loss from spontaneous abortion was highest when conception occurred as a result of intercourse 3 or more days prior to, or 2 or more days after, ovulation. The activation of the process of meiosis and gamete maturation is triggered, according to Donahue’* and Edwards,i3 by the midcycle luteinizing hormone (LH) surge, though the mechanism is not yet completely understood. The follicle rupture coincides with the completion of the first meiotic division. The second meiotic division and extrusion of the second polar body occur at fertilization. It is, therefore, conceivable that inadequate pituitary gonadotropin secretion or delay in fertilization could result in intrafollicular aging and an abnormal conceptus. The uterine environment and particularly the quality of the endometrium is directly the result of estrogen stimulation during the follicular phase and of estrogen and progesterone produced by a healthy corpus luteum. An inadequate corpus luteum may lead to abortion without chromosomal or even morphologic anomalies in the fetus. In order to obtain some information on the enddcrine profile of these patients, and to explore a possible program of investigation, the hormonal levels of LH, estradiol, and progesterone of one patient, G. K., were monitored throughout two cycles. The first abortion was a trisomy, the second was not successfully cultured, and the third was a triploidy. Two consecutive menstrual cycles were studied. The
Volume Number
Embryonic development in recurrent abortions
1%) 5
first was a control cycle during which blood samples were obtained on alternate days, with an endometrial biopsy obtained on day 21. The second cycle was studied in the same manner except that clomiphene citrate (Clomid), 50 mg. daily for 5 days, was administered from day 5. Comparison between the two cycles indicates that the LH (Fig. 2), estradiol (Fig. 3), and progesterone (Fig. 4) levels in the Clomid cycle were higher than in the control cycle. The endometrial biopsy in the control cycle was out of phase by more than 2 days, whereas the biopsy in the treated cycle was restored to normal. In the cycle subsequent to the Clomid treatment the patient conceived her fourth pregnancy and carried it to term uneventfully. The plasma LH, estradiol, and progesterone levels, as well as the endome-
515
trial biopsies, obtained in these successive cycles suggest disordered follicular and luteal function which appear to be correctable by Clomid therapy. The possibility of abnormal follicular function and oocyte maturation resulting in the fertilization of abnormal oocytes should be considered as possible etioiogic factors in this patient’s abortions, although disorder of ovum transport and the uterine environment may also be important. Furthermore, improvement of follicular function by pharmacologic methods in which gonadotropin secretion is enhanced may have therapeutic potential in patients with a similar problem. Further research to elucidate the role of these mechanisms in these disorders appears indicated.
REFERENCES 1. Poland,
J. R., Jones, D., and Trimble, B. K.: 127: 685, 1977. Poland, B. J.: Fertil. Steril. 22: 5, 1971. Boue, J. G., Boue, A., Lazar, P., and Gueguen, S.: AM. J. OBSTET. GYNECOL. 116: 6, 1973. Thiede, H. A., and Metcalfe, S.: AM. J. OBSTET. GYNECOL. 96: 1132, 1966. Penrose, L. S., and Smith, G. F. Down’s Anomaly, London, 1966, J. A. Churchill, Ltd. Boue, J. G., Boue, A., and Lazar, P.: In Aging Gametes, International Symposium, Seattle, 1973, Basel, 1974, S. Karger, AG. AM.
2. 3. 4. 5. 6.
B. J., Miller,
J. OBSTET.
GYNECOL
lnfomret~
7. Cam, D. H.: Adv. Hum. Genet. 2: 201, 1971. 8. Carr, D. H., and Gedeon, M. M.: Hum. Genet. 31: 93. 1976. 9. Hertig, A. T.: The overall problem in man, in Benirschke, K., Editor: Comparative Aspects of Reproductive Failure, Berlin, 1967, Springer, Verlag. 10. Freeman, M. E., Butcher, R. L., and Fugo, N. W.: Biol. Reprod. 4: 209, 1970. 11. Guerraro, R., and Rojas, 0.: N. Engf. J Med. 293: 12, 1975. 12. Donahue, R. P.: J. Exp. Zool. 16% 237, 1968. 13. Edwards, R. G.: Nature 208: 349, 1965.
for authors
Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: “The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published.” Authors will be consulted, when possible, regarding republication of their material.
Embryonic development in consecutive specimens from recurrent spontaneous abortions BETTY BASIL Vancouver,
J. POLAND HO
YUEN
British
Columbia,
Canada
Although habitual abortion occurs in a relatively small number of patients, its successful management is often difficult., Etiologic factors may be either maternai or ernbryonk? or both; there is much information available on maternal factors but little is known of factors related to the embryo. Information obtained from examining and keryotyping the aborted Bmbryo or fetus may be of considerable importance in determining the possible cause of the abodion and subsequent investigation and counseling of the patient. At least two specimens from over 59 women who have had two or more consecutive spontaneous abortiins have been examined and the results show that: (1) Patients aborted co%eptuCs of the same developmental stage, i.e., embryo or fetus, in consecutive pregnancies. This suggests that investigation &ould be directed to factors which may be important in the stage of pregnancy indicated. (2) Late abortions were associated with normal fetal development and factors related to the uterine environment. (3) The conceptus in an ear& abortion was significantly more likely to have a cytogenetic abnormality, suggesting a problem at, or prior to, conception/fertilization. A case study is also included. (AM. J. OBSTET. GYNECOL. 130: 512, 1978.)
A LTHO u G H spontaneous abortion is a common event in a patient’s reproductive history, recurrence is relatively rare. Patients who have had two consecutive abortions are at greater risk of having a third and even a fourth,’ with the frustrations and unhappiness which accompany this problem. While considerable emphasis has been placed on the possible maternal factors which may be responsible for recurrent abortion, factors reFrom the Department University of British Supported by Medical MA 2740.
of Obstetrics
and Gynecology,
Columbia.
Research
Council
of Canada
Grant
Presented at the Thirty-third Annual Meeting of the Society of Obstetricians and Gynaecologists of Canada, Montreal, Quebec, Canada, June 14-18, 1977. Reprint requests: Dr. B. J. Poland, Vancouver General Hospital, Vancouver, Btitih Columbia, V5.Z IM9, Canada.
512
lated to embryonic and fetal development have received less attention until more recent years, when it has been shown that both morphologic* and cytogenet.? abnormalities of embryonic development are even more common than in sporadic spontaneous abortion. This study was undertaken to correlate the status of the conceptus in consecutive abortions with relevant maternal factors and to apply this information to the structure of a program of investigation and management. In the Embryology Study of the University of British Columbia, we have been carrying out a comprehensive examination of embryos and fetuses for 10 years. The only criteria for admission of the specimens to the study was that the embryo or fetus should be sufficiently intact for evaluation and that a maternal history should be available. During the course of the study embryos and fetuses from two or more consecu000%9378/78/05130-0512$00.40/O
0
1978
The C. V. Mosby
Co.
Volume Number
Embryonic development in recurrent abortions
130 5
tive abortions in 52 patients have been examined. Information from these patients and observations on the abortus form the basis for this study. The study was prospective in that at the time of admission of the first specimen it was not known that a second or even a third would be available.
Matetlals and methods The abortuses were examined in the Embryology Study from September, 1966, to January, 1977. These were examined by macro- or microdissection and categorized as embryo or fetus, normal or abnormal, and the abnormality was delineated. Tissue was submitted to the Cytogenetics Iaboratory, Department of Medical Genetics, and culture and subsequent chromosomal analysis were carried out under the direction of Dr. F. J. Dill. This investigative procedure was not available for specimens prior to 1971. The history was obtained directly from the patient by personal interview whenever possible; otherwise, it was obtained from the record of the attending physician.
Results Fifty-two patients had 134 abortions of which the products of conception from 112 were examined. This was a total of 114 abortuses, including two sets of twins. Each specimen was categorized first as an embryo or fetus and then as normal or abnormal. The patients fell into one of three groups. Patients in group 1 aborted embryos consistently, in group 2 fetuses consistently, and in group 3 a mixture (Fig. 1). Forty patients, or 77 per cent, aborted conceptuses of the same category on consecutive occasions. In group 1, 60 out of 64 embryos (94 per cent) were abnormal, 52 of which had severe growth disorganization. Twenty-five of the 29 patients aborted only abnormal embryos. In group 2, only one of the 24 fetuses, a twin with spina bifida, was abnormal. Ten of the 11 patients aborted only normal fetuses. In group 3, 50 per cent of the patients aborted an embryo first and 50 per cent a fetus. There was an abnormality in seven first specimens and in eight second specimens. Twelve out of 13 embryos and three out of 13 fetuses were abnormal. Two of the fetuses had the phenotypes of chromosomal anomalies consistent with viability, X0 and trisomy 13. Karyotypes were obtained on 17 of the growthdisorganized embryos in this study. There were eight embryos with trisomy, five with triploidy, one with a translocation, and three with normal karyotypes. The translocation found in the embryo was unbalanced and was found in a balanced state in the mother’s karyotvpe. Growth-disorganized embryos have been
Group
1
29 Patients
AAAAAAAAAAAA
AAAAAAAAAAAAAAAA f AAAAAAAAAAAAAAAA A A
AAAAAAAAAAAA AAA Group
2
11 Pcttisnts
q uuooononou
A
F”~nuououo Group
3
513
A
q n
12 Patients
q AAAUA#O~#AU Auo~A13AAgAuA
Normal Abnormal
embryo. embryo
Normal
fetus
Abnormal
fetus
0
Fig. 1. Graphic representation of consecutive specimens in 52 patients. Double symbols indicate twins. I. Maternal of patients Table
Factors
Total abortions abortions per patient A&rage age (yr.) Irregular menstrual cycles Prior use of oral
factors in the three groups Group
1
Group 2
GTCW~ 3
78 2.7
26 2.3
30 2.5
31 12/25 = 48%
33 5/9 = 55%
31 5/l 1 = 45%
1l/29 = 33%
419 = 43%
4/l 1 = 36%
shown in other studies to have a chromosomal anomaly in over 80 per cent of cases.4 There were karyotypes obtained on four other abnormal embryos: three were trisomies and one was a triploid. It was possible to obtain a successful karyotype on two consecutive specimens in seven patients. In two patients, both specimens were trisomic; in two, trisomic and triploid; in three a trisomy followed an embryo with normal karyotype. Table I is an analysis of the maternal history in the three groups. The various factors do not differ significantly from group to group. The cycles designated irregular had a mean variation of 7 days or more. Oral contraception had been used prior to the first abortion but not subsequently.
Comment In group 1, the early failure of the pregnancy must be related to the lethal anomaly and the mechanisms which bring this about. The chromosomal anomalies were the same as those found in sporadic cases of spontaneous abortion, namely, trisomies and triploids. Although these anomalies have, as far as is known, a different mode of production, they existed in consecutive specimens of two patients. Trisomy has been associated with increased maternal chronologic age in both liveborn infants and abortuses.5* 6 Triploidy does not appear to be age related but has been considered to be
514
Poland and Ho hen
March
Am. J.
“0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
Cycle Days
2. Plasma LH levels in control and Clomid cycles.
Fig. BOO7M) wo7i 2 P
500 400mo200 loo
-
0’
0
2
4
’
6
’
8
’
10
’
12
’
14
’
16
’
18
’
20
’
22
’
24
’
26
’
28
Cycle Days
3. Plasma estradiol levels in control and Clomid cycles.
Fig.
35 30 ’3
2520 I5 IO 50****** 0
0
‘( 2
4
6
8
10
12
I4
16
I8
20
22
24
26
28
Cycle Days
Fig.
4. Plasma progesterone
levels in control and Clomid cy-
cles. an accident of fertilization.’ The presence of the cytogenetic and morphologic anomalies suggests that the abortion was inevitable from conception. “Group 2 specimens consisted of normal fetuses, with one exception, which continued to develop in some cases to the beginning of the second trimester. It would appear that implantation, though initially successful, eventually failed and factors such as uterine abnormality, fibroids, incompetent cervix, or the quality of the endometrium would seem likely to be involved. Group 3 specimens were the result of both early and late abortion in the same patient and the result, presumably, of a combination of factors. These patients would require comprehensive investigation re-
1, 1978
Obstet. Gynecol.
lated to factors of both embryonic development and the uterine environment. In patients habitually aborting an early, abnormal embryo, it is necessary to search for factors persistently interfering with the production of a normal conceptus. The presence of a structural chromosomal anomaly in the parents leads to an increased frequency of an unbalanced karyotype in the conceptus, and has been found in two per cent of couples, affecting one or other parent, when there was a history of recurrent abortion.8 Parental karyotyping should therefore be done in such cases. Hertig,g from his work on early embryos in timed conceptions, states that, “If an oocyte lingers longer than 14 days in the follicle, it is likely to be a bad egg.” Considerable work has been done, using the rat as a model, on the relationship between delayed ovulation and subsequent conceptionsi A significant decrease in implantation and an increase in the number of abnormal and retarded conceptuses have been observed. Similar evidence is difficult to obtain in human subjects. Guerraro and Rojas I1 showed that in 1,000 cycles, which were monitored by a basal body temperature chart, the loss from spontaneous abortion was highest when conception occurred as a result of intercourse 3 or more days prior to, or 2 or more days after, ovulation. The activation of the process of meiosis and gamete maturation is triggered, according to Donahue’* and Edwards,i3 by the midcycle luteinizing hormone (LH) surge, though the mechanism is not yet completely understood. The follicle rupture coincides with the completion of the first meiotic division. The second meiotic division and extrusion of the second polar body occur at fertilization. It is, therefore, conceivable that inadequate pituitary gonadotropin secretion or delay in fertilization could result in intrafollicular aging and an abnormal conceptus. The uterine environment and particularly the quality of the endometrium is directly the result of estrogen stimulation during the follicular phase and of estrogen and progesterone produced by a healthy corpus luteum. An inadequate corpus luteum may lead to abortion without chromosomal or even morphologic anomalies in the fetus. In order to obtain some information on the enddcrine profile of these patients, and to explore a possible program of investigation, the hormonal levels of LH, estradiol, and progesterone of one patient, G. K., were monitored throughout two cycles. The first abortion was a trisomy, the second was not successfully cultured, and the third was a triploidy. Two consecutive menstrual cycles were studied. The
Volume Number
Embryonic development in recurrent abortions
1%) 5
first was a control cycle during which blood samples were obtained on alternate days, with an endometrial biopsy obtained on day 21. The second cycle was studied in the same manner except that clomiphene citrate (Clomid), 50 mg. daily for 5 days, was administered from day 5. Comparison between the two cycles indicates that the LH (Fig. 2), estradiol (Fig. 3), and progesterone (Fig. 4) levels in the Clomid cycle were higher than in the control cycle. The endometrial biopsy in the control cycle was out of phase by more than 2 days, whereas the biopsy in the treated cycle was restored to normal. In the cycle subsequent to the Clomid treatment the patient conceived her fourth pregnancy and carried it to term uneventfully. The plasma LH, estradiol, and progesterone levels, as well as the endome-
515
trial biopsies, obtained in these successive cycles suggest disordered follicular and luteal function which appear to be correctable by Clomid therapy. The possibility of abnormal follicular function and oocyte maturation resulting in the fertilization of abnormal oocytes should be considered as possible etioiogic factors in this patient’s abortions, although disorder of ovum transport and the uterine environment may also be important. Furthermore, improvement of follicular function by pharmacologic methods in which gonadotropin secretion is enhanced may have therapeutic potential in patients with a similar problem. Further research to elucidate the role of these mechanisms in these disorders appears indicated.
REFERENCES 1. Poland,
J. R., Jones, D., and Trimble, B. K.: 127: 685, 1977. Poland, B. J.: Fertil. Steril. 22: 5, 1971. Boue, J. G., Boue, A., Lazar, P., and Gueguen, S.: AM. J. OBSTET. GYNECOL. 116: 6, 1973. Thiede, H. A., and Metcalfe, S.: AM. J. OBSTET. GYNECOL. 96: 1132, 1966. Penrose, L. S., and Smith, G. F. Down’s Anomaly, London, 1966, J. A. Churchill, Ltd. Boue, J. G., Boue, A., and Lazar, P.: In Aging Gametes, International Symposium, Seattle, 1973, Basel, 1974, S. Karger, AG. AM.
2. 3. 4. 5. 6.
B. J., Miller,
J. OBSTET.
GYNECOL
lnfomret~
7. Cam, D. H.: Adv. Hum. Genet. 2: 201, 1971. 8. Carr, D. H., and Gedeon, M. M.: Hum. Genet. 31: 93. 1976. 9. Hertig, A. T.: The overall problem in man, in Benirschke, K., Editor: Comparative Aspects of Reproductive Failure, Berlin, 1967, Springer, Verlag. 10. Freeman, M. E., Butcher, R. L., and Fugo, N. W.: Biol. Reprod. 4: 209, 1970. 11. Guerraro, R., and Rojas, 0.: N. Engf. J Med. 293: 12, 1975. 12. Donahue, R. P.: J. Exp. Zool. 16% 237, 1968. 13. Edwards, R. G.: Nature 208: 349, 1965.
for authors
Most of the provisions of the Copyright Act of 1976 became effective on January 1, 1978. Therefore, all manuscripts must be accompanied by the following statement, signed by each author: “The undersigned author(s) transfers all copyright ownership of the manuscript entitled (title of article) to The C. V. Mosby Company in the event the work is published. The author(s) warrants that the article is original, is not under consideration by another journal, and has not been previously published.” Authors will be consulted, when possible, regarding republication of their material.