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Serum copper levels in normal and pathologic pregnancies
Serum copper levels in normal and pathologic pregnancies J.
G.
E.
JUNGREIS,
W.
Z.
Jerusalem,
SCHENKER,
POLISHUK,
M.D. PH.D. M.D.
Israel
Serum copper levels were determined by a new method of atomic absorption spectroscopy in normal and pathologic pregnancies. The average serum copper in healthy men was 108 @g per cent compared to 129 tig per cent for normal nonpregnant women. The mean value of copper in umbilical cord blood was 55 pg per cent and amniotic fluid 30 pg per cent. In normal pregnancy, the serum copper shows a pogressive increase with the advancement of gestation. A decrease is noticed during labor and throughout the puerperium. Serum copper was elevated in cases of toxemia of pregnancy. Tkere is a correlation between the clinical severity and the serum copper level. Hypocupremia seems to us to be an index of placental insuficiency, since it was found in cases of postmaturity, premature rupture of the membranes, and in cases of spontaneous abortion.
THE RI s E 0 F serum copper level in pregnancy was first reported by Krebsl and later by others. 2-7 Correlation was observed between the elevation of copper levels followin,g oral or parenteral estrogen administration.*> g Correlation was also established between serum copper levels and urinary excretion of estriol.81 lo Since estriol excretion in urine during pregnancy is an accepted method for evaluating fetal and placenta’ function,ll it was considered of interest to study the serum copper in normal and pathoIogic pregnancies.
by atomic absorption spectroscopy. The advantages of this method are its high selectivity, sensitivity, and simple mode of performance. The ‘Perkin-Elmer atomic absorption spectrophotometer, Model 303, hollow copper cathode lamp was used. Procedure. One milliliter of bIood serum was diluted to 5 ml. with saline solution. The solution was then aspirated into airacetylene flame, and the atomic absorption of copper measured. Instrumental settings were: wavelength 3,255 A, slit 4, source 15 Ma., acetylene flow 9.0, air flow 9.0. The measurements were carried out with scale expansion of 10 times. The method of additions was used to prepare standard solutions. Known amounts of copper are added to a saline-diluted serum of known copper concentration, and a calibration curve for copper determination in blood serum is prepared. The latter is necessary, as the sample contains high and variable concentrations of matrix materials, whose effect on absorption is hard to duplicate with aqueous standards. All determina-
Method Five-milliliter venous blood specimens were obtained, with a tourniquet, from the antecubital vein. The blood was allowed to clot, :and the serum was separated. The copper content of blood serum was determined From the Department of ObstetricsGynecology, Hadassak University Hospital, and The DeQartment of Inorganrc Chemistry, The Hebrew University.
933
934
Schenker,
Jungreis,
and
A-ovcmber 15, 1969 .4m. J. Obst. B Gynec.
Polishuk
tions were made in duplicate and conventional statistical methods were employed. Material
A total of 966 blood serum copper determinations were performed. These included : 20 young men and 28 women in the fertile age. In another 9 patients the serum copper was studied during the menstrual cycle. Blood serum of umbilical cord was studied in 31 cases. In 14 cases copper levels of amniotic fluid obtained during elective cesarean section were determined. Serum copper was also determined in 282 clinically normal pregnancies: 81 cases in the first trimester, 77 cases in the second trimester, and 124 cases in the last trimester, 33 women were examined during labor, and 116 in the postpartum period, between 24 hours and 3 months after parturition. A further 18 patients attending the antenatal clinic were followed throughout pregnancy at approximately 4 to 6 week intervals. Serum copper was also studied in pathologic cases. These included 28 patients with toxemia of pregnancy, of whom 6 were cases of severe pre-eclampsia, 16 cases of rheumatic heart disease in functional Grade 23 in the third trimester, and 6 diabetic patients treated with insulin at 32 to 36 weeks of pregnancy. Twenty-one patients were admitted to the delivery room for premature rupture of membranes; of these 8 were at 32 to 36 weeks and 13 at 36 to 40 weeks. In 13 cases of clinically established postmaturity, 4 patients suffered from necrotic myomas during the second trimester of pregnancy. There was 38 cases of spontaneous
Table I. Mean serum copper levels No. Grouf~ Men Nonpregnant women Cord blood Amniotic fluid Normal -preg--
nancy
of
cases
Mean fi@
level %I
2
S.D.
20
108
10.3
28 31 14
129 55 30
12.7 8.5 7.3
139-297
11.2-39.3
282
abortion; 29 in the first trimester, 8 in the second trimester, and a case of hydatidiform mole. Results
The average serum copper level in healthy men (20 cases) was 108 Fg per cent (SD + 10.3) compared to 129 pg per cent (SD + 12.7) for normal nonpregnant women in the fertile age. The serum copper levels show certain fluctuations during the menstrual cycle which do not exceed the range of 15 to 20 ,ug per cent. The highest levels were between tenth and twentieth day of the cycle. The average serum copper level of umbilical cord was 55 pg per cent (t 8.5). No differences were found in male and female newborn levels. The mean level of copper in amniotic fluid was 30 pg per cent (5 7.3) (Table I). In normal pregnancy, the serum copper shows an increase, depending upon the time of gestation. A remarkable increase is noticed in the first trimester, and to a lesser extent in the second and third trimesters (Fig. 1) . Approximately the same results were obtained in the 18 patients that were followed throughout pregnancy and the postpartum period. A decrease in copper is seen at term and during delivery. In the postpartum period there is a slow decrease in copper, approaching the normal reported range between 8 to 12 weeks after parturition (Fig. 2). The decline is slower in women who breast-feed. The value obtained in the pre-eclamptic patients was higher than the value of copper serum in normal pregnancies at the same stage of gestation (Fig. 3). From the 6 cases we have followed of severe pre-eclampsia, there appears an evident correlation between the severity of the toxemia and the height of serum copper level. Higher serum levels were seen in patients with diabetes mellitus and rheumatic heart disease during pregnancy. In contrast, lower serum copper levels were observed in cases of premature rupture of the membranes and postmaturity (Fig. 3). Necrotic myoma during pregnancy increases serum level.
v01Ilme Number
105 Ii
Serum copper
during
pregnancy
Comment
The mean value of serum copper level in spontaneous abortion in the first trimester was 1,+2 pg per cent (30 patients), compared to 182 pg per cent (82 patients) in the sa:me weeks in normal pregnancies (Fig.
The advantages of the method of atomic absorption spectroscopy are its high selectivity, sensitivity, and simple mode of performance. In all the calorimetric methods for the determination of copper in serum, some preliminary operations are necessary: the proteins must be separated, the organicbound copper must be mineralized, and the
4). In a case of hydatidiform mole, at 12 weeks of gestation the serum copper was markedly elevated to 383 pg per cent.
26
22
TT ';;,
250
26
2.
T
17
Fig. 1.
Mean
serum
copper
(pg
weeks of pregnancy 76) at various stages of pregnancy.
5
10
100 0
15
935
20
25
30
Days postpartum Fig. 2. Mean sewn copper (~g %) during the puerperium.
35
40
33
936
Schenker,
Jungreis,
and
Polishuk
El
Normal
m
Toxemia
m
Heart
fggj
Premature Rupture Membranes
m
Postmaturity
350CI s
300-
9 Y G
250-
disease of
D!obetes
ii o E =I L
200-
rs
150-
100-
28
weeks
32
of
36
38-40
42- 43
pregnancy
Fig. 3. Mean serum copper (/lg %) in abnormal pregnancy.
n
NORMAL ABORTIONS
0
6
6-12
13-16
weeks
Fig. 4. Mean ous abortion.
serum
copper
(pg % ) in spontane-
analytical procedure itself is generally tedious and time-consuming for routine analysis. In contrast, the proposed method requires no preliminaries, is very quick and reliable, and suits the clinical laboratory
perfectly. The disadvantage is the relatively high cost of the apparatus. A certain cyclic variation was found in the serum copper levels of the 9 women investigated throughout a menstrual cycle. The rise was at the late proliferative stage, contrary to the data of Sarrata,12 who found that the peak was at the premenstrual stage. We think that the rise in the late proliferative stage correlates with the increase in endogenie secretion of estrogen at that stage. It is not possible to determine ovulation by serum copper determination, as claimed by Dokumov.13 The copper content in the blood of the umbilical cord (55 pg per cent) was four to five times lower than that in the maternal blood. The same results were noticed by Taradaiko,14 who determined the copper in the umbilical artery, vein, retroplacental blood, and in several layers of the placenta. The conclusion from these findings is that copper cannot diffuse across the placenta, but accumulates in the Iayers of placenta and from there is tran&rred to the fetus by an active process accordii to need. No relation
was found
between
the
copper
level
Volume Number
105 fi
of the blood of the mother and that of the fetus; nor were there variations with respect to the age, parity, duration of pregnancy, and the weight of the newborn infant, as mentioned by others.141 l5 In normal pregnancy the copper level in the blood serum shows an increase, depending upon the time of gestation. The level of serum copper may be used as a diagnosis of pregnancy as early as the fourth week of gestation. A remarkable increase is seen in the first trimester and less in the second and the third trimesters. The peak is reached at 36 to 38 weeks of gestation and slowly declines at term and throughout puerperium, so the average values are back to the prepregnancy level 2 to 3 months post partum. Fro.m the results of copper determination in pal;hologic cases, it appears that besides changes in estrogenic activity, other factors also affect the serum copper levels. Of these, the liver plays a prominent role. The hypercupremia of pregnancy is probably due to a mobilization of copper from maternal tissues,2 especially from the liver,16 and is correlated with an increase in estrogens in the blood. 5, lo We found serum copper to be elevated in pre-eclamptic patients. This finding seems paradoxical, since it is known that toxemia of pregnancy is associated with low serum estrogen. I1 It could be explained by the work of Rasuli,16 who found that in women dying
Serum copper
during
pregnancy
937
from eclampsia, there was a depletion of copper content in the liver, from 800 to 40 pg per cent. Our findings in toxemia indicate that there is a correlation between the clinical severity of toxemia and the serum copper level. It is not the absolute figure per se that is an index of the severity of the disease but rather the abruptness of the change. The slight hypercupremia observed in patients with heart disease in pregnancy can be explained by finding of hypercupremia in congestive heart failure.17 The slightly increased level of serum copper in the few cases of diabetes mellitus may be related to the metabolic changes in the liver and due to mild or severe toxemia that usually complicates diabetes. In cases other than toxemia, diabetes, and congestive heart failure the finding of hypocupremia may be an indication of placental dysfunction. The lower levels of serum copper in cases of premature rupture of the membranes correlate with the findings of low estriol values in the urine in these cases, as reported by Taylor, Bruns, and Drose.l* Hypocupremia was also observed in cases of postmaturity. This may point to a correlation between low serum copper level and placental dysfunction.lg The marked hypoabortion once cupremia in spontaneous again indicates the relation between copper level and placental insufficiency.
REFERENCES
1. 2. 3.
4.
5. 6. 7. 8. 9.
Krebs, H. A.: Klin. Wchnschr. 7: 584, 1928. Fay, J., Cartwright, G. E., and Wintrobe, M. M.: J. Clin. Invest. 28: 488, 1945. Sachs, A., Levine, W. E., Griffith, W. O., and Hansen, C. H.: Am. J. Dis. Child. 56: 787, 1938. Lahey, M. E., Gubler, C. J., Cartwright, G. E.. and Wintrobe. M. M.: T. Clin. Invest. 32: 3i9, 1956. ’ ” Johnson, N.: Proc. Sot. Exper. Biol. & Med. 108: 518, 1961. De Jorge, F. B., Delascio, D., and Antunes, M. L.: Obst. & Gynec. 26: 225, 1965. O’Leary, J. A.. Novalis, G. S., and Vosburgh, G. J.: Obst. & Gynec. 28: 112, 1966. Russ, E., and Raymunt, L.: Proc. Sot. Exper. Biol. & Med. 92: 465, 1956. Turpin, R., Schmitt-Jubeau, H., and Jerome,
10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
H.: Compt. rend. Sot. biol. 144: 352, 1950. Studnitz, W., and Benin, D.: Acta endocrinol. 27: 245, 1958. Zondek. B.. and Pfiefer. V.: Acta obst. et gynec. dcan&nav. 38: 74i, 1959. Sarata, U.: Jap. J. M. SC. 3: 1, 1935. Dokumov, S.: Rev. Franc. . gynCc. et obst. 63: -. 37, 1968: Taradaiko, Y. V.: Akush. ginek. 39: 59, 1963. Lyko, J.: Acta med. polonia 8: 269, 1967. Rasuli, Z.: Akush. ainek. 39: 63. 1963. Tarlowska, A.: Polslki tygodnik lek. 20: 1309, 1965. Taylor, E. S., Bruns, P. D., and Drose, V. E.: Clin. Obst. & Gynec. 8: 550, 1965. Lundwall, F., and Stakemann, G.: Acta obst. et gynec. scandinav. 45: 301, 1966.
G.
E.
JUNGREIS,
W.
Z.
Jerusalem,
SCHENKER,
POLISHUK,
M.D. PH.D. M.D.
Israel
Serum copper levels were determined by a new method of atomic absorption spectroscopy in normal and pathologic pregnancies. The average serum copper in healthy men was 108 @g per cent compared to 129 tig per cent for normal nonpregnant women. The mean value of copper in umbilical cord blood was 55 pg per cent and amniotic fluid 30 pg per cent. In normal pregnancy, the serum copper shows a pogressive increase with the advancement of gestation. A decrease is noticed during labor and throughout the puerperium. Serum copper was elevated in cases of toxemia of pregnancy. Tkere is a correlation between the clinical severity and the serum copper level. Hypocupremia seems to us to be an index of placental insuficiency, since it was found in cases of postmaturity, premature rupture of the membranes, and in cases of spontaneous abortion.
THE RI s E 0 F serum copper level in pregnancy was first reported by Krebsl and later by others. 2-7 Correlation was observed between the elevation of copper levels followin,g oral or parenteral estrogen administration.*> g Correlation was also established between serum copper levels and urinary excretion of estriol.81 lo Since estriol excretion in urine during pregnancy is an accepted method for evaluating fetal and placenta’ function,ll it was considered of interest to study the serum copper in normal and pathoIogic pregnancies.
by atomic absorption spectroscopy. The advantages of this method are its high selectivity, sensitivity, and simple mode of performance. The ‘Perkin-Elmer atomic absorption spectrophotometer, Model 303, hollow copper cathode lamp was used. Procedure. One milliliter of bIood serum was diluted to 5 ml. with saline solution. The solution was then aspirated into airacetylene flame, and the atomic absorption of copper measured. Instrumental settings were: wavelength 3,255 A, slit 4, source 15 Ma., acetylene flow 9.0, air flow 9.0. The measurements were carried out with scale expansion of 10 times. The method of additions was used to prepare standard solutions. Known amounts of copper are added to a saline-diluted serum of known copper concentration, and a calibration curve for copper determination in blood serum is prepared. The latter is necessary, as the sample contains high and variable concentrations of matrix materials, whose effect on absorption is hard to duplicate with aqueous standards. All determina-
Method Five-milliliter venous blood specimens were obtained, with a tourniquet, from the antecubital vein. The blood was allowed to clot, :and the serum was separated. The copper content of blood serum was determined From the Department of ObstetricsGynecology, Hadassak University Hospital, and The DeQartment of Inorganrc Chemistry, The Hebrew University.
933
934
Schenker,
Jungreis,
and
A-ovcmber 15, 1969 .4m. J. Obst. B Gynec.
Polishuk
tions were made in duplicate and conventional statistical methods were employed. Material
A total of 966 blood serum copper determinations were performed. These included : 20 young men and 28 women in the fertile age. In another 9 patients the serum copper was studied during the menstrual cycle. Blood serum of umbilical cord was studied in 31 cases. In 14 cases copper levels of amniotic fluid obtained during elective cesarean section were determined. Serum copper was also determined in 282 clinically normal pregnancies: 81 cases in the first trimester, 77 cases in the second trimester, and 124 cases in the last trimester, 33 women were examined during labor, and 116 in the postpartum period, between 24 hours and 3 months after parturition. A further 18 patients attending the antenatal clinic were followed throughout pregnancy at approximately 4 to 6 week intervals. Serum copper was also studied in pathologic cases. These included 28 patients with toxemia of pregnancy, of whom 6 were cases of severe pre-eclampsia, 16 cases of rheumatic heart disease in functional Grade 23 in the third trimester, and 6 diabetic patients treated with insulin at 32 to 36 weeks of pregnancy. Twenty-one patients were admitted to the delivery room for premature rupture of membranes; of these 8 were at 32 to 36 weeks and 13 at 36 to 40 weeks. In 13 cases of clinically established postmaturity, 4 patients suffered from necrotic myomas during the second trimester of pregnancy. There was 38 cases of spontaneous
Table I. Mean serum copper levels No. Grouf~ Men Nonpregnant women Cord blood Amniotic fluid Normal -preg--
nancy
of
cases
Mean fi@
level %I
2
S.D.
20
108
10.3
28 31 14
129 55 30
12.7 8.5 7.3
139-297
11.2-39.3
282
abortion; 29 in the first trimester, 8 in the second trimester, and a case of hydatidiform mole. Results
The average serum copper level in healthy men (20 cases) was 108 Fg per cent (SD + 10.3) compared to 129 pg per cent (SD + 12.7) for normal nonpregnant women in the fertile age. The serum copper levels show certain fluctuations during the menstrual cycle which do not exceed the range of 15 to 20 ,ug per cent. The highest levels were between tenth and twentieth day of the cycle. The average serum copper level of umbilical cord was 55 pg per cent (t 8.5). No differences were found in male and female newborn levels. The mean level of copper in amniotic fluid was 30 pg per cent (5 7.3) (Table I). In normal pregnancy, the serum copper shows an increase, depending upon the time of gestation. A remarkable increase is noticed in the first trimester, and to a lesser extent in the second and third trimesters (Fig. 1) . Approximately the same results were obtained in the 18 patients that were followed throughout pregnancy and the postpartum period. A decrease in copper is seen at term and during delivery. In the postpartum period there is a slow decrease in copper, approaching the normal reported range between 8 to 12 weeks after parturition (Fig. 2). The decline is slower in women who breast-feed. The value obtained in the pre-eclamptic patients was higher than the value of copper serum in normal pregnancies at the same stage of gestation (Fig. 3). From the 6 cases we have followed of severe pre-eclampsia, there appears an evident correlation between the severity of the toxemia and the height of serum copper level. Higher serum levels were seen in patients with diabetes mellitus and rheumatic heart disease during pregnancy. In contrast, lower serum copper levels were observed in cases of premature rupture of the membranes and postmaturity (Fig. 3). Necrotic myoma during pregnancy increases serum level.
v01Ilme Number
105 Ii
Serum copper
during
pregnancy
Comment
The mean value of serum copper level in spontaneous abortion in the first trimester was 1,+2 pg per cent (30 patients), compared to 182 pg per cent (82 patients) in the sa:me weeks in normal pregnancies (Fig.
The advantages of the method of atomic absorption spectroscopy are its high selectivity, sensitivity, and simple mode of performance. In all the calorimetric methods for the determination of copper in serum, some preliminary operations are necessary: the proteins must be separated, the organicbound copper must be mineralized, and the
4). In a case of hydatidiform mole, at 12 weeks of gestation the serum copper was markedly elevated to 383 pg per cent.
26
22
TT ';;,
250
26
2.
T
17
Fig. 1.
Mean
serum
copper
(pg
weeks of pregnancy 76) at various stages of pregnancy.
5
10
100 0
15
935
20
25
30
Days postpartum Fig. 2. Mean sewn copper (~g %) during the puerperium.
35
40
33
936
Schenker,
Jungreis,
and
Polishuk
El
Normal
m
Toxemia
m
Heart
fggj
Premature Rupture Membranes
m
Postmaturity
350CI s
300-
9 Y G
250-
disease of
D!obetes
ii o E =I L
200-
rs
150-
100-
28
weeks
32
of
36
38-40
42- 43
pregnancy
Fig. 3. Mean serum copper (/lg %) in abnormal pregnancy.
n
NORMAL ABORTIONS
0
6
6-12
13-16
weeks
Fig. 4. Mean ous abortion.
serum
copper
(pg % ) in spontane-
analytical procedure itself is generally tedious and time-consuming for routine analysis. In contrast, the proposed method requires no preliminaries, is very quick and reliable, and suits the clinical laboratory
perfectly. The disadvantage is the relatively high cost of the apparatus. A certain cyclic variation was found in the serum copper levels of the 9 women investigated throughout a menstrual cycle. The rise was at the late proliferative stage, contrary to the data of Sarrata,12 who found that the peak was at the premenstrual stage. We think that the rise in the late proliferative stage correlates with the increase in endogenie secretion of estrogen at that stage. It is not possible to determine ovulation by serum copper determination, as claimed by Dokumov.13 The copper content in the blood of the umbilical cord (55 pg per cent) was four to five times lower than that in the maternal blood. The same results were noticed by Taradaiko,14 who determined the copper in the umbilical artery, vein, retroplacental blood, and in several layers of the placenta. The conclusion from these findings is that copper cannot diffuse across the placenta, but accumulates in the Iayers of placenta and from there is tran&rred to the fetus by an active process accordii to need. No relation
was found
between
the
copper
level
Volume Number
105 fi
of the blood of the mother and that of the fetus; nor were there variations with respect to the age, parity, duration of pregnancy, and the weight of the newborn infant, as mentioned by others.141 l5 In normal pregnancy the copper level in the blood serum shows an increase, depending upon the time of gestation. The level of serum copper may be used as a diagnosis of pregnancy as early as the fourth week of gestation. A remarkable increase is seen in the first trimester and less in the second and the third trimesters. The peak is reached at 36 to 38 weeks of gestation and slowly declines at term and throughout puerperium, so the average values are back to the prepregnancy level 2 to 3 months post partum. Fro.m the results of copper determination in pal;hologic cases, it appears that besides changes in estrogenic activity, other factors also affect the serum copper levels. Of these, the liver plays a prominent role. The hypercupremia of pregnancy is probably due to a mobilization of copper from maternal tissues,2 especially from the liver,16 and is correlated with an increase in estrogens in the blood. 5, lo We found serum copper to be elevated in pre-eclamptic patients. This finding seems paradoxical, since it is known that toxemia of pregnancy is associated with low serum estrogen. I1 It could be explained by the work of Rasuli,16 who found that in women dying
Serum copper
during
pregnancy
937
from eclampsia, there was a depletion of copper content in the liver, from 800 to 40 pg per cent. Our findings in toxemia indicate that there is a correlation between the clinical severity of toxemia and the serum copper level. It is not the absolute figure per se that is an index of the severity of the disease but rather the abruptness of the change. The slight hypercupremia observed in patients with heart disease in pregnancy can be explained by finding of hypercupremia in congestive heart failure.17 The slightly increased level of serum copper in the few cases of diabetes mellitus may be related to the metabolic changes in the liver and due to mild or severe toxemia that usually complicates diabetes. In cases other than toxemia, diabetes, and congestive heart failure the finding of hypocupremia may be an indication of placental dysfunction. The lower levels of serum copper in cases of premature rupture of the membranes correlate with the findings of low estriol values in the urine in these cases, as reported by Taylor, Bruns, and Drose.l* Hypocupremia was also observed in cases of postmaturity. This may point to a correlation between low serum copper level and placental dysfunction.lg The marked hypoabortion once cupremia in spontaneous again indicates the relation between copper level and placental insufficiency.
REFERENCES
1. 2. 3.
4.
5. 6. 7. 8. 9.
Krebs, H. A.: Klin. Wchnschr. 7: 584, 1928. Fay, J., Cartwright, G. E., and Wintrobe, M. M.: J. Clin. Invest. 28: 488, 1945. Sachs, A., Levine, W. E., Griffith, W. O., and Hansen, C. H.: Am. J. Dis. Child. 56: 787, 1938. Lahey, M. E., Gubler, C. J., Cartwright, G. E.. and Wintrobe. M. M.: T. Clin. Invest. 32: 3i9, 1956. ’ ” Johnson, N.: Proc. Sot. Exper. Biol. & Med. 108: 518, 1961. De Jorge, F. B., Delascio, D., and Antunes, M. L.: Obst. & Gynec. 26: 225, 1965. O’Leary, J. A.. Novalis, G. S., and Vosburgh, G. J.: Obst. & Gynec. 28: 112, 1966. Russ, E., and Raymunt, L.: Proc. Sot. Exper. Biol. & Med. 92: 465, 1956. Turpin, R., Schmitt-Jubeau, H., and Jerome,
10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
H.: Compt. rend. Sot. biol. 144: 352, 1950. Studnitz, W., and Benin, D.: Acta endocrinol. 27: 245, 1958. Zondek. B.. and Pfiefer. V.: Acta obst. et gynec. dcan&nav. 38: 74i, 1959. Sarata, U.: Jap. J. M. SC. 3: 1, 1935. Dokumov, S.: Rev. Franc. . gynCc. et obst. 63: -. 37, 1968: Taradaiko, Y. V.: Akush. ginek. 39: 59, 1963. Lyko, J.: Acta med. polonia 8: 269, 1967. Rasuli, Z.: Akush. ainek. 39: 63. 1963. Tarlowska, A.: Polslki tygodnik lek. 20: 1309, 1965. Taylor, E. S., Bruns, P. D., and Drose, V. E.: Clin. Obst. & Gynec. 8: 550, 1965. Lundwall, F., and Stakemann, G.: Acta obst. et gynec. scandinav. 45: 301, 1966.