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Antibacterial activity of human amniotic fluid
Antibacterial activity of human amniotic fluid NIRA
BERGMAN,
BRUNO
BERCOVICI,
THEODORE
M.D.
SACKS,
M.MED.(
Jerusalem>
M.Sc
F.R.C.PATH.,
M.B.CH.B.,
PATH.)
Israel
Amniotic fluid samples from 2.5 of 28 women examined were found to possess antibacterial activity. This activity was directed against both gram-positive and gram-negative organisms and appeared to be related to the presence of varying combinations of different antibacterial factors. Lysozyme-like activity was present in only 9 of 24 fluid samples. Other factors differed in their heat stability and molecular weights.
T H A T A M N I o T I c fluid, like other body fluids, has an inherent ability to resist bacterial infections is suggested by the clinical observation that the fetus is protected even when ascending infections from the vagina and cervical canal are a decided possibilrarity with which abdominal ity.=, ’ The amniocentesis, a procedure frequently used today in the management of Rh incompatibility, is followed by infection also suggests the existence of some protective mechanism against bacterial infection. The reduction in the vaginal flora which is seen after membrane rupture and the escape of amniotic fluid33 ’ is further evidence of a possible antibacterial action. In addition to this indirect evidence, there have been several contradictory reports of attempts to demonstrate direct evidence of antibacterial activity of amniotic fluid. In 1949, Cattaneo5 reported
From the Departments of Clinical Microbiology and Obstetrics and Gynecology, Hadassah University and Hebrew University-Hadassah Medical School. y;;;ived
for
publication
March
Accepted
for
publication
May
antibacterial activity in amniotic fluid which suggested the presence of lysozyme, while Gusdon reported the presence of a substance bactericidal for B. subtilis, which did not appear to be lysozyme. Walsh, Hildebrandt, and Prystowsky7 and Sarkany and Gaylarde3 reported that amniotic fluid not only does not inhibit bacterial growth but actually supports it. Galask and Snyder,8 however, found that amniotic fluid was inhibitory for a number of bacterial genera (both grampositive and gram-negative), and later9 they attempted to identify the antibacterial factors in the fluid. In view of these contradictory findings, it was decided to investigate the presence of antibacterial factors in amniotic fluid in patients attending the Obstetrics and Gynecology Department of Hadassah University Hospital. Material
and
Amniotic
fluid. Fluid
methods
was obtained under aseptic conditions by abdominal paracentesis or at cesarean section from 28 women in the third trimester of pregnancy. None of the women had received antibiotics during the last month of their pregnancies. The amniotic fluid was used the same day or frozen at -20’ C. Each fluid was centrifuged at 11,000 g for 20 minutes at 4’ C. to remove particulate matter. The fluid was then
Hospital
31, IO, 1972.
Reprint requests: Prof. T. Sack:, Department of Clinical Microbiology, Hadassah University Hospital, Jerusalem, Israel.
520
Volume Number
114 4
Antibacterial
sterilized by filtration through a Seitz* filter or membrane filter.+ organisms. E. coli NCTC 104/18 and S~a~~yZ~coccus aureus (Oxford) NCTC 6571 (National Collection of Type Cultures, Colindale, London) were used, together with strains of Streptococcus uiridans, Streptococcus faecalis, Enterobacter, Candida, Pseudomonas aeruginosa, Klebsiella, Proteus mirabilis, Proteus morganii, and diphtheroid organisms isolated in our routine bacteriology laboratory. Stock cultures were maintained on nutrient agar (Difco Labs., Detroit, Michigan) slants, and fluid cultures were in trypticase soya broth (TSB, Difco) . Inhibitory activity. One-tenth milliliter of a lO-4 dilution of an 18 hour culture in TSB was added to 0.9 ml. of amniotic fluid to give a bacterial concentration, at time zero, of approximately 104 bacteria per milliliter. Bacterial counts were performed at 0, 7, and 24 hours by spreading 0.01 ml. of the inoculated fluid over the surface of an agar plate. Colonies were counted after 24 hours of incubation at 37O C. Fluids were regarded as inhibitory if the bacterial count at 24 hours was less than 106 per milliliter. Control cultures without amniotic fluid had counts of > lO* per milliliter. Where the count fell to 0, the fluids were labeled strongly inhibitory (possibly bactericidal). Sometimes the fluids inhibited growth after 7 hours but not after 24 hours-these were not regarded as inhibitory. An example of results obtained is shown in Table I. To exclude the possibility that the growth inhibition was due to lack of nutrients in the amniotic fluid samples, l/l0 volume of TSB was added to the fluid samples. This quantity of TSB added to normal saline supported full growth of the test organisms. The pH of the amniotic fluids was measured on Radiometer pH Meter 26 (London Co., Cleveland, Ohio). Heat resistance. Antibacterial activity of amniotic fluid was measured before and after heating to 50’ C. for 30 minutes, and to 100’ C. for 5 minutes and for 15 minutes. *Carlson Ford Grade tMillipore, 0.22~.
EKS.
activity
of
amniotic
fluid
521
Molecular weight. Weight of the inhibitory substances was estimated by centrifugal separation in a conical membrane* that retains substances with a molecular weight exceeding 50,000, or by passing the fluid under a pressure of 30 pounds per square inch through a membrane that retains particles with a molecular weight of > 100,OOO.t Activity. Measurement of lysozyme-like activity was recorded according to the method described by Galask and Snyder.9 Antibacterial spectrum. The spectrum was established by testing for inhibitory activity against 13 different species. Results Of the 28 fluid samples examined, 13 were tested against 4 bacteria only-Proteus mirabilis, E. coli, S. aureus, and S. faecalis. All but 3 inhibited at least one of the 4 test organisms. The other 15 fluids were tested against 12 organisms. Of these, each inhibited at least half of the test organisms and often many more. Thus, at least 25 of 28 fluid samples had antibacterial activity, and, of these, at least 23 were active against both gram-positive and gram-negative bacteria. Table II summarizes the results of the inbibitory activity against the various microorganisms tested. The pH of the fluids varied between 8.7 and 9.4; there was no correlation between pH and inhibitory activity. The effect of heat on the inhibitory activity is shown in Table III. In 11 of 18 fluids tested, inhibitory activity was retained after 100’ C. for 15 minutes, while in 7 it was lost. In 2 of these, the inhibitory activity was lost after 50’ C. for 30 minutes. Lysozyme-like activity was found in only 9 of the 24 fluids examinned for this characteristic. This activity was equivalent to from 0.3 7 per milliliter of lysozyme to 2.5 7 per milliliter in the different fluid samples. In 3 of the fluid samples examined, inhibitory activity was associated with a mo*Centriilo, xM 50, Amicon NV, ton, Massachusetts. tDiaflo xM 100, Amicon NV.
Amicon
Corp.,
Ix&g-
522
Bergman,
Bercovici,
Table I. Inhibitory assessed by viable bacteria
E. coli Stayphlococcus
Sacks
October Am. J. Obstet.
activity of amniotic counts of surviving
I Organism
and
I Medium
Amniotic TSB
fluid
Amniotic TSB
fluid
Time
fluid
Table II. Inhibitory fluid
samples
against
activity various
of amniotic test organisms
1 ;:ys
/jf!t$fh
(hr.)
organism &.)
&.)
4 x 104 3x 104
8
5 x 104 3x104
6 x 104 > 10s
x
>
15, 1972 Gymcd.
Z.) 10s 10s
0 >
10s
2 x 104 > 10s
lecular weight of less than 500,000; in 3, with a molecular weight of > 50,000; and, in 3, with both small and large fractions, Three of the fluids with inhibitory factors with a molecular weight of more than 50,000 were examined further and found to have fractions with molecular weights of > 100,000 and < 100,000. There was no obvious correlation between the various characteristics examined and the antibacterial spectrum of the different fluids. Table IV shows 6 examples of fluids with antibacterial activity, with a comparison of the various characteristics examined. From these results, it can be seen that a variety of inhibitory substances appear to be present. Comment Our results clearly support the findings of Galask and Snydera> g of the presence of antibacterial activity in amniotic fluid. Most of the fluid samples (25 of 28) that we examined were capable of at least inhibiting the growth of a wide range of bacteria and yeasts. Some of the fluids were even bactericidal for inocula of 104 bacteria per milliliter. Becaux of the small quantities of fluid obtained from each patient, we were unable to completely characterize the antibacterial factors present. It was obvious, however that the antibacterial activity was usually due to the presence of more than one factor. The concentration of these factors was low, and their activity was usually lost when the fluid was diluted one half. Lysozyme-like activity was found in 36
S. viridans S. albus Diphtheroids Candida albicans Proteus morganii Enterobacter Klebsiella E. coli Proteus mirabilis S. aweus Pseudomonas S. faecalis
14 16 IO 11 13 15 14 27 22 26 12 24
100 1OQ 100 100 84 75 71.4 66.6 54.5 42.3 33 16.6
Table III. activity
Effect of heating on inhibitory of amniotic fluid samples Znhibitory
No. of sambles
Before heating + +
11 5 Inhibitory
After for
+ +
+
2 *+ = absent.
activtiy*
After 56’ C. for 30 min.
activity
100’ C. 15min. +
present;
-
=
inhibitov
activity
per cent of the fluids, but the results of heat resistance and molecular weight studies and the spectrum of activity which extended to gram-negative bacilli clearly indicated that other antibacterial factors were also present. The examples shown in Table IV show that antibacterial factors with the following characteristics were present in varying combinations in the different fluids: 1. Lysozyme-like activity (Nos. 13, 14, 17, 19, and 21). 2. A factor resistant to heating at 100’ C. with a molecular weight of less than 50,000 (No. 13). 3. A heat-labile factor ( 100° C.) with a molecular weight of > 100,000 (No. 14). 4. A factor resistant to heating at 100° C. with a molecular weight of > 100,000 (No. 17). 5. A heat-labile factor (lOO” Cl.) with a molecular weight of < 50,000 (No. 19).
Volume Number
Antibacterial
114 4
Table IV. Characteristics
of inhibitory
factors
in 6 fluid
activity
of amniotic
fluid
523
samples* Spectrum
Samfile No.
Lysozyme firesent
13 14 17 19 21 28 *t
Inbibitcuy = Not
activity
1
+
100’
=
inhibitory
N.D. t
t i t + t
Grampositive
1 >lOO,OOO
+
t t -
weight
1 >.50,000
< 50,000
-I.
+ + 4 4 present;
Molecular
resistance
56’
+ + + .I.t
=
N.D.
Heat
3/5 2/2 3/5 2/4 4/4 l/3
N:D. N.D. +
t t
Gramnegative 6/6 l/l 3/6 3/6 5/6 3/5
activity
done.
If we consider these various factors, we can see that they may be present singly or in combination. Fluid No. 21, for example, contains lysozyme-like activity and possibly a combination of Factors 2 and 4 or any of the heat-labile factors in combination with the heat-stable ones. The spectrum of antibacterial activity of these various factors singly or in combination is quite wide and extends to cover both gram-positive and gram-negative organisms. Those bacteria usually recognized as responsible for amnionitis were inhibited by fewer fluids than the species usually regarded
as nonpathogenic in this situation. Prevedourakis, Papadimitriou, and Ioannidoul” examined amniotic fluid samples from 117 women at term and found bacterial contamination in 9 (7.69 per cent). The bacteria present in these fluids (Proteus, E. coli, Enterococcus, Staphylococcus aweus, and Pseudomonas) were those that we found to be inhibited by a smaller proportion of the fluids than the other bacteria examined (see Table II). This supports the view that the antibacterial factors described in this paper play a role in combating amniotic fluid contamination in vivo.
REFERENCES
1. Blanc, W. A.: Gynaecologia 136: 101, 1953. 2. Benirschke, K.: Am. J. Dis. Child. 99: 714,
7. Walsh, H. R., Hildebrandt, sky, H.: AM. J. OBSTET.
1960. 3. 4.
1965.
Sarkany, I., and Gaylarde, Dermatol. 80: 241, 1968. Bercovici, B., and Diamant, Gynecol.
(In
C. C.: Br. J. Y.:
Obstet.
press.)
Cattaneo, P.: Clin. Ostet. Ginecol. 51: 60, 1949. 6. Gusdon, J. P.: J. Immunol. 88: 494, 1962.
5.
R., and Prystow93: 590,
GYNECOL.
8. 9.
Galask,
R. P., and Snyder,
OBSTET.
GYNECOL.
Galask,
R. P., and Snyder,
OBSTET.
GYNECOL.
102: 106:
I. S.: AM. J.
949, 59,
1968.
I. S.:
10. Prevedourakis, C., Papadimitriou, Ioannidou, A.: AM. J. OBSTET. 106:400,
1970.
AM.
J,
1970.
G., and GYNECOL.
BERGMAN,
BRUNO
BERCOVICI,
THEODORE
M.D.
SACKS,
M.MED.(
Jerusalem>
M.Sc
F.R.C.PATH.,
M.B.CH.B.,
PATH.)
Israel
Amniotic fluid samples from 2.5 of 28 women examined were found to possess antibacterial activity. This activity was directed against both gram-positive and gram-negative organisms and appeared to be related to the presence of varying combinations of different antibacterial factors. Lysozyme-like activity was present in only 9 of 24 fluid samples. Other factors differed in their heat stability and molecular weights.
T H A T A M N I o T I c fluid, like other body fluids, has an inherent ability to resist bacterial infections is suggested by the clinical observation that the fetus is protected even when ascending infections from the vagina and cervical canal are a decided possibilrarity with which abdominal ity.=, ’ The amniocentesis, a procedure frequently used today in the management of Rh incompatibility, is followed by infection also suggests the existence of some protective mechanism against bacterial infection. The reduction in the vaginal flora which is seen after membrane rupture and the escape of amniotic fluid33 ’ is further evidence of a possible antibacterial action. In addition to this indirect evidence, there have been several contradictory reports of attempts to demonstrate direct evidence of antibacterial activity of amniotic fluid. In 1949, Cattaneo5 reported
From the Departments of Clinical Microbiology and Obstetrics and Gynecology, Hadassah University and Hebrew University-Hadassah Medical School. y;;;ived
for
publication
March
Accepted
for
publication
May
antibacterial activity in amniotic fluid which suggested the presence of lysozyme, while Gusdon reported the presence of a substance bactericidal for B. subtilis, which did not appear to be lysozyme. Walsh, Hildebrandt, and Prystowsky7 and Sarkany and Gaylarde3 reported that amniotic fluid not only does not inhibit bacterial growth but actually supports it. Galask and Snyder,8 however, found that amniotic fluid was inhibitory for a number of bacterial genera (both grampositive and gram-negative), and later9 they attempted to identify the antibacterial factors in the fluid. In view of these contradictory findings, it was decided to investigate the presence of antibacterial factors in amniotic fluid in patients attending the Obstetrics and Gynecology Department of Hadassah University Hospital. Material
and
Amniotic
fluid. Fluid
methods
was obtained under aseptic conditions by abdominal paracentesis or at cesarean section from 28 women in the third trimester of pregnancy. None of the women had received antibiotics during the last month of their pregnancies. The amniotic fluid was used the same day or frozen at -20’ C. Each fluid was centrifuged at 11,000 g for 20 minutes at 4’ C. to remove particulate matter. The fluid was then
Hospital
31, IO, 1972.
Reprint requests: Prof. T. Sack:, Department of Clinical Microbiology, Hadassah University Hospital, Jerusalem, Israel.
520
Volume Number
114 4
Antibacterial
sterilized by filtration through a Seitz* filter or membrane filter.+ organisms. E. coli NCTC 104/18 and S~a~~yZ~coccus aureus (Oxford) NCTC 6571 (National Collection of Type Cultures, Colindale, London) were used, together with strains of Streptococcus uiridans, Streptococcus faecalis, Enterobacter, Candida, Pseudomonas aeruginosa, Klebsiella, Proteus mirabilis, Proteus morganii, and diphtheroid organisms isolated in our routine bacteriology laboratory. Stock cultures were maintained on nutrient agar (Difco Labs., Detroit, Michigan) slants, and fluid cultures were in trypticase soya broth (TSB, Difco) . Inhibitory activity. One-tenth milliliter of a lO-4 dilution of an 18 hour culture in TSB was added to 0.9 ml. of amniotic fluid to give a bacterial concentration, at time zero, of approximately 104 bacteria per milliliter. Bacterial counts were performed at 0, 7, and 24 hours by spreading 0.01 ml. of the inoculated fluid over the surface of an agar plate. Colonies were counted after 24 hours of incubation at 37O C. Fluids were regarded as inhibitory if the bacterial count at 24 hours was less than 106 per milliliter. Control cultures without amniotic fluid had counts of > lO* per milliliter. Where the count fell to 0, the fluids were labeled strongly inhibitory (possibly bactericidal). Sometimes the fluids inhibited growth after 7 hours but not after 24 hours-these were not regarded as inhibitory. An example of results obtained is shown in Table I. To exclude the possibility that the growth inhibition was due to lack of nutrients in the amniotic fluid samples, l/l0 volume of TSB was added to the fluid samples. This quantity of TSB added to normal saline supported full growth of the test organisms. The pH of the amniotic fluids was measured on Radiometer pH Meter 26 (London Co., Cleveland, Ohio). Heat resistance. Antibacterial activity of amniotic fluid was measured before and after heating to 50’ C. for 30 minutes, and to 100’ C. for 5 minutes and for 15 minutes. *Carlson Ford Grade tMillipore, 0.22~.
EKS.
activity
of
amniotic
fluid
521
Molecular weight. Weight of the inhibitory substances was estimated by centrifugal separation in a conical membrane* that retains substances with a molecular weight exceeding 50,000, or by passing the fluid under a pressure of 30 pounds per square inch through a membrane that retains particles with a molecular weight of > 100,OOO.t Activity. Measurement of lysozyme-like activity was recorded according to the method described by Galask and Snyder.9 Antibacterial spectrum. The spectrum was established by testing for inhibitory activity against 13 different species. Results Of the 28 fluid samples examined, 13 were tested against 4 bacteria only-Proteus mirabilis, E. coli, S. aureus, and S. faecalis. All but 3 inhibited at least one of the 4 test organisms. The other 15 fluids were tested against 12 organisms. Of these, each inhibited at least half of the test organisms and often many more. Thus, at least 25 of 28 fluid samples had antibacterial activity, and, of these, at least 23 were active against both gram-positive and gram-negative bacteria. Table II summarizes the results of the inbibitory activity against the various microorganisms tested. The pH of the fluids varied between 8.7 and 9.4; there was no correlation between pH and inhibitory activity. The effect of heat on the inhibitory activity is shown in Table III. In 11 of 18 fluids tested, inhibitory activity was retained after 100’ C. for 15 minutes, while in 7 it was lost. In 2 of these, the inhibitory activity was lost after 50’ C. for 30 minutes. Lysozyme-like activity was found in only 9 of the 24 fluids examinned for this characteristic. This activity was equivalent to from 0.3 7 per milliliter of lysozyme to 2.5 7 per milliliter in the different fluid samples. In 3 of the fluid samples examined, inhibitory activity was associated with a mo*Centriilo, xM 50, Amicon NV, ton, Massachusetts. tDiaflo xM 100, Amicon NV.
Amicon
Corp.,
Ix&g-
522
Bergman,
Bercovici,
Table I. Inhibitory assessed by viable bacteria
E. coli Stayphlococcus
Sacks
October Am. J. Obstet.
activity of amniotic counts of surviving
I Organism
and
I Medium
Amniotic TSB
fluid
Amniotic TSB
fluid
Time
fluid
Table II. Inhibitory fluid
samples
against
activity various
of amniotic test organisms
1 ;:ys
/jf!t$fh
(hr.)
organism &.)
&.)
4 x 104 3x 104
8
5 x 104 3x104
6 x 104 > 10s
x
>
15, 1972 Gymcd.
Z.) 10s 10s
0 >
10s
2 x 104 > 10s
lecular weight of less than 500,000; in 3, with a molecular weight of > 50,000; and, in 3, with both small and large fractions, Three of the fluids with inhibitory factors with a molecular weight of more than 50,000 were examined further and found to have fractions with molecular weights of > 100,000 and < 100,000. There was no obvious correlation between the various characteristics examined and the antibacterial spectrum of the different fluids. Table IV shows 6 examples of fluids with antibacterial activity, with a comparison of the various characteristics examined. From these results, it can be seen that a variety of inhibitory substances appear to be present. Comment Our results clearly support the findings of Galask and Snydera> g of the presence of antibacterial activity in amniotic fluid. Most of the fluid samples (25 of 28) that we examined were capable of at least inhibiting the growth of a wide range of bacteria and yeasts. Some of the fluids were even bactericidal for inocula of 104 bacteria per milliliter. Becaux of the small quantities of fluid obtained from each patient, we were unable to completely characterize the antibacterial factors present. It was obvious, however that the antibacterial activity was usually due to the presence of more than one factor. The concentration of these factors was low, and their activity was usually lost when the fluid was diluted one half. Lysozyme-like activity was found in 36
S. viridans S. albus Diphtheroids Candida albicans Proteus morganii Enterobacter Klebsiella E. coli Proteus mirabilis S. aweus Pseudomonas S. faecalis
14 16 IO 11 13 15 14 27 22 26 12 24
100 1OQ 100 100 84 75 71.4 66.6 54.5 42.3 33 16.6
Table III. activity
Effect of heating on inhibitory of amniotic fluid samples Znhibitory
No. of sambles
Before heating + +
11 5 Inhibitory
After for
+ +
+
2 *+ = absent.
activtiy*
After 56’ C. for 30 min.
activity
100’ C. 15min. +
present;
-
=
inhibitov
activity
per cent of the fluids, but the results of heat resistance and molecular weight studies and the spectrum of activity which extended to gram-negative bacilli clearly indicated that other antibacterial factors were also present. The examples shown in Table IV show that antibacterial factors with the following characteristics were present in varying combinations in the different fluids: 1. Lysozyme-like activity (Nos. 13, 14, 17, 19, and 21). 2. A factor resistant to heating at 100’ C. with a molecular weight of less than 50,000 (No. 13). 3. A heat-labile factor ( 100° C.) with a molecular weight of > 100,000 (No. 14). 4. A factor resistant to heating at 100° C. with a molecular weight of > 100,000 (No. 17). 5. A heat-labile factor (lOO” Cl.) with a molecular weight of < 50,000 (No. 19).
Volume Number
Antibacterial
114 4
Table IV. Characteristics
of inhibitory
factors
in 6 fluid
activity
of amniotic
fluid
523
samples* Spectrum
Samfile No.
Lysozyme firesent
13 14 17 19 21 28 *t
Inbibitcuy = Not
activity
1
+
100’
=
inhibitory
N.D. t
t i t + t
Grampositive
1 >lOO,OOO
+
t t -
weight
1 >.50,000
< 50,000
-I.
+ + 4 4 present;
Molecular
resistance
56’
+ + + .I.t
=
N.D.
Heat
3/5 2/2 3/5 2/4 4/4 l/3
N:D. N.D. +
t t
Gramnegative 6/6 l/l 3/6 3/6 5/6 3/5
activity
done.
If we consider these various factors, we can see that they may be present singly or in combination. Fluid No. 21, for example, contains lysozyme-like activity and possibly a combination of Factors 2 and 4 or any of the heat-labile factors in combination with the heat-stable ones. The spectrum of antibacterial activity of these various factors singly or in combination is quite wide and extends to cover both gram-positive and gram-negative organisms. Those bacteria usually recognized as responsible for amnionitis were inhibited by fewer fluids than the species usually regarded
as nonpathogenic in this situation. Prevedourakis, Papadimitriou, and Ioannidoul” examined amniotic fluid samples from 117 women at term and found bacterial contamination in 9 (7.69 per cent). The bacteria present in these fluids (Proteus, E. coli, Enterococcus, Staphylococcus aweus, and Pseudomonas) were those that we found to be inhibited by a smaller proportion of the fluids than the other bacteria examined (see Table II). This supports the view that the antibacterial factors described in this paper play a role in combating amniotic fluid contamination in vivo.
REFERENCES
1. Blanc, W. A.: Gynaecologia 136: 101, 1953. 2. Benirschke, K.: Am. J. Dis. Child. 99: 714,
7. Walsh, H. R., Hildebrandt, sky, H.: AM. J. OBSTET.
1960. 3. 4.
1965.
Sarkany, I., and Gaylarde, Dermatol. 80: 241, 1968. Bercovici, B., and Diamant, Gynecol.
(In
C. C.: Br. J. Y.:
Obstet.
press.)
Cattaneo, P.: Clin. Ostet. Ginecol. 51: 60, 1949. 6. Gusdon, J. P.: J. Immunol. 88: 494, 1962.
5.
R., and Prystow93: 590,
GYNECOL.
8. 9.
Galask,
R. P., and Snyder,
OBSTET.
GYNECOL.
Galask,
R. P., and Snyder,
OBSTET.
GYNECOL.
102: 106:
I. S.: AM. J.
949, 59,
1968.
I. S.:
10. Prevedourakis, C., Papadimitriou, Ioannidou, A.: AM. J. OBSTET. 106:400,
1970.
AM.
J,
1970.
G., and GYNECOL.