- Email: [email protected]
Effects of oral contraceptives on phospholipids of human cervical mucus
CURRENT INVESTIGATION
Effects of oral contraceptives on phospholipids of human cervical mucus ERIC
J.
SINGH,
JOSEPH
R.
SHEILA
BOSS,
Chicago,
M.S.,
PH.D.,
SWARTWOUT,
A.R.I.C. M.D.,
F.A.C.O.G.
B.A.
Illinois
Human cervical mucus phospholipids have been fractionated and quantitated in normal patients. Mucus phosphatides, as determined by thin-layer chromatography, include phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, sphingomyelin, lysophosphatidyl choline, cardiolipin, and glycerophosfihoryl choline. It is suggested that the phospholipid content and fatty acid compositions are related to the three phases of the menstrual cycle. The oral contracefitives change the amount and fatty acid composition of phospholipid in the cycle. The total fatty acids of phospholipids tend to become more saturated with the contraceptives.
THE AMOUNT, composition, and total fatty acid composition of phospholipids in human cervical mucus, during the normal menstrual cycle and under the influence of oral contraceptives, have not been reported previously. The procedures are also advanced for the separation of individual phosphatides with emphasis on their fatty acid composition. This paper is designed to serve a twofold purpose: first, to outline the present state of our knowledge of phospholipid com-
position in the cervical mucus; and second, to indicate how oral contraceptives can influence the secretion of cervical mucus phospholipids and its fatty acid composition. The data represent the first report of the amount of phospholipid, phospholipid components, fatty acid composition of total phospholipid, fatty acid composition of the phosphatides, and the effect of oral contraceptives on phospholipid. Materials
From the Department Gynecology, University
of Obstetrics of Chicago.
Ford
by Foundation.
Grant 690-0108 from
methods
All equipment employed in this study was thoroughly defatted with ethyl ether prior to use. Solvents were of AR grade and redistilled before use. A nitrogen atmosphere was maintained to minimize oxidation during various procedures. The samplesof cervical mucus were obtained from normal women and women ingesting oral contracep-
and
Presented in part at the symposium on lipids in “Reproductive Tissue” at the American Oil Chemists Society,’ Sixty-second Annual Meeting (May 2-6, 1971), Houston, Texas.
Supported
and
the
285
286 Singh, Swartwout,
400
300 r
January Am. J. Ohstrt.
and Boss
1
.
1.5. 192 Gynccol.
* CONTROL l OVULEN - 21 0 OVRAL . C - QUENS
. . A
.
Z-
. .
.
.
A
.
.
l : *
r A
6
10
8
12
14
16
CYCLE Fig. 1. The treated with
amount a daily
Table I. Composition components in human (entire normal cycle)
of mucus obtained from dose of contraceptives.
of phospholipid cervical mucus
Amount (%,I
Components Diphosphatidyl glycerol Glycerophosphoryl choline Phosphatidyl ethanolamine Phosphatidyl choline Phosphatidyl inositol Phosphatidyl serine Sphingomyelin Lysophosphatidyl choline
I
15.0 21.1 39.4 5.6
9.8 7.0 1.9
tives” by aspiration of the cervical canal. A group of 8 volunteers exhibiting normal menstrual cycles of 28 days were utilized. The volunteers first completed a control cycle with no medication and during the subsequent cycles received pills orally daily. Samples of mucus were collected daily by aspiration of the cervical canal and immediately weighed to the nearest milligram. The ages *C-Quens, Eli Lilly & Co., Indianapolis, Indiana; 21, G. D. Searle & Co., Chicago, Illinois; Ovral, Labs., Philadelphia, Pennsylvania.
OvulenWyeth
the
18
20
22
24
26
28
DAYS normal
patients
and
the patients
who
were
of the patients ranged from 20 to 22 years. None of the patients had clinical symptoms of genital infection. Lipids were extracted with the use of chloroform+methanol (2 : 1, v/v) with the use of the procedure of Folch and associates.’ For the variation study of phospholipids in the cycle, the lipids were extracted from the daily samples. For the analysis of fatty acid composition, the samples, pooled according to the cycle, were kept in chloroform:methano1 (2 : 1, v/v at 4’ C. until they were used. The phospholipids were isolated by Florisil column chromatography.2 The phospholipids were in a final fraction, and they were eluted with absolute methanol. Round spots of whole lipid components were placed on silica gel-impregnated glass fiber paper3 and uniformly sprayed with concentrated sulfuric acid and charred at 180’ C. for 20 minutes. Percentages were calculated with the use of a varicord densitometer48 5 equipped with a recorder. The phospholipid patterns were determined by thin-layer chromatography. The
Volume Number
112 2
Oral
contraceptive
effect
on
cervical
mucus
A CONTROL OVULEN 0 OVRAL n C-QUENS l
a2
30-
i P
9
11
13
15
17
CYCLE Fig. 2. The percentage amount treated cycles, respectively.
Table II. Distribution menstrual
- 21
of fatty acids of human
12:o (Laurie acid) 13:o (Tridecanoic acid) 14:o (My&tic acid) 15:o (Pentadecanoic acid) 16:0 (Palmitic acid) 16:l (Palmitoleic acid) 17:o (Heptadecanoic acid) 17:l (Heptadecenoic acid) 18:O (Stearic acid) 18:l (Oleic acid) 18:2 (Linoleic acid) 30:o ( Arachidic acid) 2O:l (cis-5-Eicosenoic acid) 22:o (Behenic acid) 20~4 ( Arachidonic acid) 24:o (Lignoceric acid) 22:6 (Docosahexaenoic acid) Saturated Unsaturated Monounsaturated Polyunsaturated Total Total
of phospholipid
19
21
23
25
27 28
DAYS (in
cervical
micrograms
per
cent)
mucus phospholipids
in the
control
and
in
cycle
Fatty acid (Acid: No. of bond)
T =
287
25-
7
normal
phospholipids
odd even
Postmenstrual cycle (5-9 cycle days) (% +- S.D.) 3.1 0.5 3.9 1.3 23.2 1.6 1.1 0.5 21.0 17.6 7.8 1.3 1.6 3.1 5.9 6.5 T 65.0 35.0 21.3 13.7 3.4 96.6
+ + 2 +_ * 2 + 2 + 2 2 + + + t t +
0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.0
Mid-cycle (12-15 cycle days) (%’ + SD.) 3.6 2.0 9.0 2.0 26.5 7.2 2.0 2.0 12.5 14.2 5.4 0.3 2.0 2.0 5.4 3.7 T 63.6 36.2 25.4 10.8 8.0 91.8
+ k t + + -L t +_ 2 + k 2 c ” I t +
0.1 0.1 0.2 0.1 0.3 0.1 0.1 0.1 0.2 0.; 0.1 0.1 0.1 0.1 0.1 0.1 0.0
Premenstrual cycle (20-25 cycle days) ‘(70 z?z S.D.) 4.3 2.2 8.4 2.1 29.3 6.3 2.2 2.2 10.5 12.6 6.3 0.1 2.2 2.2 6.3 2.6 T 63.9 35.9 23.3 12.6
+ 2 + t + + 2 + t .k + t f ? t + 2
0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.t 0.2 0.2 0.1 0.0 0.1 0.1 0.2 0.1 0.0
8.7 91.1
trace.
extraction procedures, preparation of the plates and solvents, the procedure for phosphatide determination, and a typical thinlayer chromatography of phospholipids have been previously reported.6
A Barber Colman Model 5000 gas chromatograph with a dual hydrogen flame detector was used. The two U-shaped glass columns measured 6 feet by I/Z inch outside diameter and contained 3 per cent SE-30 on
288 Singh, Swartwout,
January Am. J. Obstet.
and Boss
Table IV. Distribution of fatty acids of human cervical mucus phospholipids menstrual cycle with contraceptive ( C-Quens)
Table III. Distribution of fatty acids of human cervical mucus phospolipids in menstrual cycle with oral contraceptive (Ovral)
12:o 13:o
14:o 15:o 16:O
2.1 -+ 0.1
3.5 _+ 0.1
2.4+
1.0+0.1 6.3 2 0.1 2.1 + 0.1
1.7 + 0.1
1.2 + 0.1
8.92 0.2 3.5 20.1
5.9kO.l 2.45
17.9 f 0.2 5.42 0.1 1.0 c 0.1 1.720.1 l.O? 0.1 1.8 + 0.1
26.0 ? 0.3 7.3 s 0.1
16:l 17:o 17:l 18:O ia:1
14.6 +_ 0.2 11.4kO.l 4.2 _+0.1
ia:2 2O:l
12.5 t 0.2 8.9 + 0.1 5.4? 0.1
2.1 +-0.1
3.6tO.l 2 0.1 8.9 2 0.1 9.3 2 0.1 12.5 + 0.2 T +_0.0 T fO.O 3.1 2 0.1 8.3 2 0.2
22:o 20:4 24:0 22:6
Saturated Unsaturated Monounsaturated Polyunsaturated
0.1
0.1 29.4 ?I0.3 3.5-+ 0.1 1.2 + 0.1 1.220.1 12.92 0.1 8.2 to.1 4.7-+ 0.1 2.45 0.1 4.7 5 0.1 7.0 2 0.1 12.9-+ 0.1 T + 0.0
65.5 34.3
65.8 34.0
73.0 27.0
21.8
19.7
15.3
12.5
14.3
11.7
Total odd 5.1 Total even 94.7 T =
3.6
15, 1972 Gynecol.
12:o
4.0 + 0.1 1.9to.1 7.8f0.2 2.8f. 0.1 23.8f0.3 6.9 -+0.1 0.72 0.0 0.7 +_ 0.0
13:o
14:o 15:o
16:0 16:l
17:o 18:0 18:l 18:2 20:o PO:1 22:o 20:4 24~0 22:6
1.6kO.l 7.4 -i 0.2
1.9 to.1
3.1 c 0.1
30.5kO.3 5.6 + 0.1 0.4-f 0.0
21.920.3 6.02 0.1 0.22 0.0
68.5 31.3
4.8 ko.1 4.5 2 0.1 T 50.0 66.9 32.9
20.8
21.2
22.1
13.5
10.1
10.8
Total odd 6.1 Total even 94.1
5.1 94.4
93.3
T =
trace.
4.5 r 0.2
10.7 k 0.2 4.3 to.1 T to.0 65.7 34.3
Saturated Unsaturated Monounsaturated Polyunsaturated
91.1
0.1 0.1
1.OkO.l 1.6 + 0.1 15.3 t 0.2 15.2 50.1 19.0 t 0.2 11.9 -+0.2 13.5 + 0.2 13.3 i 0.1 2.8+ 0.1 5.5 + 0.1 6.0 F 0.1 0.3 50.1 0.42 0.0 0.2to.o 1.3 + 0.1 1.1 r 0.1 1.2 20.1 4.8? 0.1 3.6kO.l 4.5 to.1
17:l
8.7
4.5 r 1.85 7.4kO.l
in
4.620.1
2.8 + 0.1 T + 0.0
6.5
trace.
Table V. Distribution of total fatty acids of human cervical mucus phospholipids in entire menstrua Fatty acid (Acid:
No.
of
bond)
12:o
C-Quens
0.1 1.5+ 0.1 7.1+ 0.2
4.0 2 0.2 2.o‘L 0.1 8.02 0.3 4.0 + 0.2 24.0r 0.4 6.0 2 0.1 2.0 r0.2 2.0? 0.1
3.6+
13:o 14:o 15:o
16:0 16:l 17:o
17:l 18:0 18:l 18:2 20:o '2o:l 22:o 20:4 24:0
Saturated Unsaturated T =
Control (%)
trace.
1.8kO.l 26.3 kO.3 5.OkO.l 1.7kO.l
1.9-fO.l 14.6I! 0.3 14.8 -to.4 6.5 _+ 0.2 0.5 ? 0.1 1.9 10.1 2.4+ 0.1 5.850.2 4.3 2 0.3 63.8
35.9
(%)
Control (%)
0.1 1.520.1
3.or
6.3 2 0.2 1.520.1 26.0+ 0.3 4.5 + 0.2 1.5 t 0.1
C-Quens f%)
?I0.1 1.920.1 4.0
7.7 20.2 2.4 + 0.1 26.5 k0.4 5.7 + 0.1 0.5 t 0.1
10.0k 0.2
1.5+_0.1 15.0+ 0.3
8.1 50.2 6.0+ 0.2
16.5 + 0.2 6.02 0.1
10.3 to.2 4.0 + 0.2 0.8 2 0.1
2.8 2 0.2 6.0-c 0.2 5.OkO.l 63.5 36.5
5.0t 0.2 5.1 20.2 6.02 0.2 72.7 27.3
T kO.0 2.0 !: 0.1 6.0+ 0.2 a.o-+ 0.3 a.0 ko.3 68.0 32.0
0.9+ 0.1 2.0 kO.1
0.9kO.l 17.9t 0.3
1.3kO.l
Oral
contraceptive
60 to 80 mesh Gas Chrom P. The column, injector, and detector temperatures were 260, 260, and 270’ C., respectively. Helium was the carrier gas at 60 ml. per minute. With the system containing 15 per cent DEGS on 60 to 80 mesh Gas Chrom P, the column, injector, and detector temperatures were 210, 210, and 240° C. in the order stated. Helium flow was the same. Gas chromatographic analysis was performed directly on the methyl ester mixtures? of various components. The methyl ester fatty acid samples were dissolved in ether, and volumes of 2 ~1 were injected. The samples were hydrogenated as described elsewhere.8 Peaks were identified by use of references and the logarithmic plots of relative retention times vs. chain length and degree of unsaturation.” Results Figs. I and 2 show the variation of human cervical mucus and phospholipids during the itrenstrual cycle with and without oral contraceptives. It is noted from Fig. 1 that the amount of mucus decreased with oral contraceptives. It is observed from Fig. 2 that 1he percentage amount of phospholipids in mucus increased with contraceptives, and this increase remains almost constant in the cycle. The variation of phospholipid is most probably due to the higher and lower content of cycle with
and without
effect on cervical
mucus phospholipids
the other components in the total lipids. ‘Table I shows the distribution of various phosphatides in cervical mucus phospholipids. The main components of phospholipids are phosphatidyl ethanolamine, and lecithin, cardiolipin. Thin-layer chromatography indicates that lysolecithin is present in about 2.0 per cent concentration in mucus. Tables II to IV show the cyclic variations of fatty acid composition of phospholipids with and without contraceptives. In Table II. the noteworthy acid is palmitoleic; it varies from 1.6 to 7.2 per cent in the cycle. Stearic acid is 21.0 per cent in the postmenstrual cycle, while in the premenstrual cycle it is I03 per cent. The fatty acid composition also shows differences in the cycle. Table III presents the cyclic variations of fatty acid conq”‘sitions in the cycle with Ovral. The palmitic acid goes down in the mid-cycle while stearic acid almost remains constant. Table IV shows the cyclic variation of fatty acid compositions, with the USC of C-Quens as the oral contraceptive. Here, palmitic acid goes up in the mid-cycle, while linolcic acid varies from 2.8 to 6.0 per cent in the cycle. Table V presents the total fatty acid composition of phospholipids in the entire cycle with and without contraceptives. It is clear front the table that the fatty acid composition in phospholipids becomes more saturated with
contraceptives __-
Control (%) 3.0 r 0.1 1.7kO.l 6.5 +_ 0.2
--
1.7 to.1 L’i.5 2 0.3 4.5 2 0.2 1.7iO.l 1.5 + 0.1 1.3.6 + 0.3 17.0 -+ 0.3 5..5 5 0.1 0.4 + 0.1 ‘) ““0.1 -._23 + 0.2 6.” ? 0.1 4 5 2 0.2 63:; 36.9
Ovral mi 3.0 + 0.1 1.5 -e 0.1 6.5 +_ 0.2 2.5 k 0.2 26.0 _+ 0.4 i.5 t 0.2 1.:!+01 1.8-fO:l 14.0 -c 0.2 9.0 f 0.2 4.5 t 0.1 o..i + 0.1 2.5 + 0.1 3.0 -c 0.2 7.0 i- 0.3 9.5 + 0.” 69.7 30.3
Control (%) 3.5 + 0.2 1.5 + 0.1 7.0 t 0.2 1.5 + 0.1 26.3 k 0.5 5.0 f 0.2 1.5 + 0.1 1.8 im 0.1 14.5 +_ 0.2 16.9 + 0.3 6.5 + 0.1 0.5 r 0.1 2.040.1 2.5 5 0.1 6.0 5 0.2 3.0+ 0.1 61.8 38.2
289
Omal (%J 4.0 f 0.2 1.6 + 0.1 6.8 + 0.1 2.5 rt 0.1 30.0 t 0 L’ 5.0 * 0:1 1.5 i 0.1 1.7 f 0.1 14.0 + 0.3 11.2’O.L’ 5.5 t 0.2 0.2 + 0.1 1.5 5 0.1 2.5 5 0.1 6.0 + 0.1 6.0 _C0.1 69.1 30.9
Control (%J 4.0 t I.52 7.0 + 1.32 25.0 2 5.5 5 1.5 2 1.4 t 1”.9+0:1 16.8 t 7.0 5 0.3 It 1 ..i k 3.0 2 3.0 t 6.0 + 63.7 37.2
0.1 1 0.2 0.1 0.3 0.2 0.1 0.1 0:3 0.3 0.1 0.1 0.1 0.1 0.1
-_Ui? 01 (%.I
4.0 f 0.1 1.8 -4. 0.1 6.0 -+ 0.2 3.0 ?: 0.2 29.0 ? 0.2 4.5 -! 0.1 1 .n + 0.1 1.O.kO.l Ii.0 i- 0.2 12.0 + 0.2 5.0 IO.1 0.3 kO.1 1 i -t I). 1 3.0 + 0.1 7.nto.2 5.7 t 0.2 69.0 31.0
290
Sigh,
Swartwout,
and
Boss
Table VI. Fatty acid compositions Fatty acid (Acid: No. of bond)
SPH
4.0 t 0.2 0.6 2 0.1 7.4 i: 0.2 3.02 0.1 18.8 +_0.2 6.8 + 0.1 1.820.1 1.7 + 0.1 9.4 + 0.2 11.8 + 0.2 5.0 _+0.2 1.7 2 0.1
12:o
13:o l4:O 15:o 16:0 16:l 17:o 17:l la:o la: i 18:2 20:o 2O:l 21:ot 22:o 20:4 23:0$ ‘24:O Saturated Odd Even Unsaturated Monoenes Polyenes Even No. of double bond/ molecule
2.3 + 0.1 5.8 i 0.2 3.OkO.l 16.9 + 0.3 74.7 10.7 64.0 25.3 20.3 5.0 23.6 0.3
CL, Diphosphatidyl phosphatidyl inositol; *Entire
pool
tHeneicosan& $Tricosanoic
sample
glycerol; GPC, PS, phosphatidyl of 3 normal
of phosphntides CL + GPC
LPC
2.8 + 0.2 1.2CO.l 5.6 -+ 0.2 1.7kO.l 24.6 + 0.3 5.3 2 0.2 1.2ro.1 2.9 + 0.1 9.2 + 0.2 13.9 + 0.3 4.2 + 0.1 2.3 +_0.1 5.8 2 0.2
4.1 2 0.1 0.5 2 0.1 7.5 2 0.2 1.6” 0.1 24.8 It 0.3 6.8 2 0.2 1.450.1 1.6 + 0.1 12.9 + 0.2 22.5 _+0.3 7.2 + 0.2 1.4 + 0.1 79+0.1 -.- 0.8 +- 0.1 1.6kO.l 2.9 +_0.1 57.9 3.5 54.4 41.9 33.1 8.8 40.3
a.7 +_0.3 3.5 + 0.1 7.0 +_0.2 64.3 4.1 60.2 35.6 27.9 7.7 32.7 0.5
0.5
glycerophosphoryl serine; SPH, cycles
(cycles
were
of human
PS
cervical
muws+ ------. PE
PI
3.7 2 0.1 0.6 -+0.1 6.5 + 0.2 1.2 2 0.1 26.4 5 0.2 6.2 + 0.1 1.2 + 0.1 1.4kO.l 17.9 _+0.2 20.3 + 0.3 6.2fO.l 1.420.1 1.5 * 0.1
4.4 !: 0.2 0.5 + 0.1 8.2 _+0.1 1.5 k 0.1 26.9 + 0.3 7.2 _+0.2 1.5 + 0.1 2.0 + 0.1 11.7fO.Z 13.8 + 0.2 5.9 2 0.1 1.0%0.1 3.2 _+0.1
2:8+0.1 1.2 _+0.1
3.2 +_0.1 0.5 _+0.1 6.6 + 0.2 1.6 + 0.1 25.8 + 0.3 8.1 _+0.2 2.2 5 0.1 3.2 + 0.1 9”?0 1 9:s -+ 0:1 2.7 f 0.1 1.020.1 2.7 k 0.1 1.6f-0.1 8.6 _+0.2
0.6 2 0.1 3.1 2 0.1
3.3 2 0.1 2.0-+0.1
5.2 * 0.1 69.3 6.0 63.3 30.6 25.2 5.4 28.2
13.4 + 0.2 65.1 4.3 60.8 34.9 23.6 11.3 31.7
1.7 +_0.1 61.3 3.0 58.2 38.7 29.4 9.3 37.3
6.9 2 0.2 65.9 3.5 62.4 34.1 36.2 7.9 32.1
3.5 2 0.1 1.2 -+ 0.1 12.5 2 0.2 2.4 + 0.1 29.4 5 0.4 5.4 2 0.2 2.4 2 0.1 2.4 i: 0.1 9.9 f: 0.2 13.2 _+0.2 4”tol i- k 0:o 4.2 _+0.1
0.4
PC
~~.
0.7
choline; PE, phosphatidyl ethanolamine; sphingomyelin; LPC, Iysophosphatidyl choline.
0.6 I%,
phosphatidyl
0.5 choline;
PI.
28 days).
acid acid.
the use of oral contraceptives. In general, it changes from 63.0 to 69.0 per cent in saturation. The total composition of phospholipid fatty acids, however, did not differ significantly between the control or treated groups. Table VI illustrates the composition of phosphatide fatty acids. Sphingomyelin contains the highest percentage amount of odd acids as compared to other components. It is noted that lecithin-, phosphatidyl ethanolamineand cardiolipid-type fractions are most unsaturated, while the sphingomyelin is most saturated.
3. The lecithin and phosphatidyl ethanolamine are the main components of phospholipid. 4. Cyclic variations in fatty acid composition were observed in the cycle. 5. With contraceptives the fatty acid becomes more saturated. 6. Among phosphatides, the numbers of double bonds per molecule in diphosphatidyl glycerol + glycerophosphoryl choline, phosphatidyl ethanolamine, lecithin, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, and lysolecithin are 0.5, 0.6, 0.7, 0.5, 0.5: 0.3, and 0.5, respectively.
Results
1. The amount of total mucus decreased with the use of oral contraceptives. 2. The percentage amount of phospholipids increased with contraceptives.
Comment
Cervical mucus is a complex mixture of inorganic and organic substances and enzymes, bearing a similarity to a dual phase gel, with
Oral
contraceptive
hydration during the proliferative phase and dehydration during the secretory phase.‘O Spem migration was accelerated in hydrolyzed cervical mucus.l’ Contraceptives affect the cervical mucus viscosity, making it difficult for the spermatozoa to penetrate it and ascend.‘” Recently, it has been suggestedthat phospholipids take part in viscosity13 in the mucus and hence are responsible for sperm migration as one of the components. Viscosity is very important in the mid-
REFERENCES
1. Folch, J., Lees, M., and Stanley, G. H. S. : J, Biol. Chem. 226: 497, 1957. 2. Carroll, K. K.: J. Lipid Res. 2: 135, 1961. 3. Hamilton, J. G., Swartwout, J, R., and Miller, OTT.: Biochem. Biophys. Res. Commun. 5: 226, 1961. 4. Blank, M. L., Schmit, J. A., and Privett, 0. S.: J. Am. Oil Chem. Sot. 41: 371, 1964. .5. Privett, 0. S., and Blank, M. L.: J. Am. Oil Chem. Sot. 40: 70, 1963. 6. Singh, E. J., and Gerishbein, L. L.: J. Chromatogr. 31: 20, 1967.
effect on cervical
mucus phospholipids
291
cycle. It has been demonstrated that pills have complex effects on the secretion of the cervical mucus. It is noted that with the contraceptives the phospholipids increased in the cycle. These drugs presumably act lo inhibit ovulation either directly or indirectly, and they also change the amount and cornposition of phospholipid fatty acids. It may be possible that phospholipid takes part in the elasticity of mucus.
7.
8. 9. 10. 11. 12. 13.
Luddy, F. E., Barford, R. A., and RiemenSchneider, R. W.: J. Am. Oil Chem. Sot. 37: 447, 1960. Singh, E. J., Gerishbein, L. L., and O’Neill. H. J.: Lipids 1: 274, 1966. Woodford, F. P., and Van Gent, C. M.: J. Lipid Res. 1: 188, 1960. Platt, H. A.: J. Reprod. Med. 3: 131, 1969. Moghissi, K. S., and Syner, F. N.: Frrtil. Steril. 21: 234, 1970. Sten. J., and Sting. K.: Fertil. Steril. 21: 307. 1970. Lewis, R. W.: Lipids 5: 917. 1970.
Effects of oral contraceptives on phospholipids of human cervical mucus ERIC
J.
SINGH,
JOSEPH
R.
SHEILA
BOSS,
Chicago,
M.S.,
PH.D.,
SWARTWOUT,
A.R.I.C. M.D.,
F.A.C.O.G.
B.A.
Illinois
Human cervical mucus phospholipids have been fractionated and quantitated in normal patients. Mucus phosphatides, as determined by thin-layer chromatography, include phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl inositol, sphingomyelin, lysophosphatidyl choline, cardiolipin, and glycerophosfihoryl choline. It is suggested that the phospholipid content and fatty acid compositions are related to the three phases of the menstrual cycle. The oral contracefitives change the amount and fatty acid composition of phospholipid in the cycle. The total fatty acids of phospholipids tend to become more saturated with the contraceptives.
THE AMOUNT, composition, and total fatty acid composition of phospholipids in human cervical mucus, during the normal menstrual cycle and under the influence of oral contraceptives, have not been reported previously. The procedures are also advanced for the separation of individual phosphatides with emphasis on their fatty acid composition. This paper is designed to serve a twofold purpose: first, to outline the present state of our knowledge of phospholipid com-
position in the cervical mucus; and second, to indicate how oral contraceptives can influence the secretion of cervical mucus phospholipids and its fatty acid composition. The data represent the first report of the amount of phospholipid, phospholipid components, fatty acid composition of total phospholipid, fatty acid composition of the phosphatides, and the effect of oral contraceptives on phospholipid. Materials
From the Department Gynecology, University
of Obstetrics of Chicago.
Ford
by Foundation.
Grant 690-0108 from
methods
All equipment employed in this study was thoroughly defatted with ethyl ether prior to use. Solvents were of AR grade and redistilled before use. A nitrogen atmosphere was maintained to minimize oxidation during various procedures. The samplesof cervical mucus were obtained from normal women and women ingesting oral contracep-
and
Presented in part at the symposium on lipids in “Reproductive Tissue” at the American Oil Chemists Society,’ Sixty-second Annual Meeting (May 2-6, 1971), Houston, Texas.
Supported
and
the
285
286 Singh, Swartwout,
400
300 r
January Am. J. Ohstrt.
and Boss
1
.
1.5. 192 Gynccol.
* CONTROL l OVULEN - 21 0 OVRAL . C - QUENS
. . A
.
Z-
. .
.
.
A
.
.
l : *
r A
6
10
8
12
14
16
CYCLE Fig. 1. The treated with
amount a daily
Table I. Composition components in human (entire normal cycle)
of mucus obtained from dose of contraceptives.
of phospholipid cervical mucus
Amount (%,I
Components Diphosphatidyl glycerol Glycerophosphoryl choline Phosphatidyl ethanolamine Phosphatidyl choline Phosphatidyl inositol Phosphatidyl serine Sphingomyelin Lysophosphatidyl choline
I
15.0 21.1 39.4 5.6
9.8 7.0 1.9
tives” by aspiration of the cervical canal. A group of 8 volunteers exhibiting normal menstrual cycles of 28 days were utilized. The volunteers first completed a control cycle with no medication and during the subsequent cycles received pills orally daily. Samples of mucus were collected daily by aspiration of the cervical canal and immediately weighed to the nearest milligram. The ages *C-Quens, Eli Lilly & Co., Indianapolis, Indiana; 21, G. D. Searle & Co., Chicago, Illinois; Ovral, Labs., Philadelphia, Pennsylvania.
OvulenWyeth
the
18
20
22
24
26
28
DAYS normal
patients
and
the patients
who
were
of the patients ranged from 20 to 22 years. None of the patients had clinical symptoms of genital infection. Lipids were extracted with the use of chloroform+methanol (2 : 1, v/v) with the use of the procedure of Folch and associates.’ For the variation study of phospholipids in the cycle, the lipids were extracted from the daily samples. For the analysis of fatty acid composition, the samples, pooled according to the cycle, were kept in chloroform:methano1 (2 : 1, v/v at 4’ C. until they were used. The phospholipids were isolated by Florisil column chromatography.2 The phospholipids were in a final fraction, and they were eluted with absolute methanol. Round spots of whole lipid components were placed on silica gel-impregnated glass fiber paper3 and uniformly sprayed with concentrated sulfuric acid and charred at 180’ C. for 20 minutes. Percentages were calculated with the use of a varicord densitometer48 5 equipped with a recorder. The phospholipid patterns were determined by thin-layer chromatography. The
Volume Number
112 2
Oral
contraceptive
effect
on
cervical
mucus
A CONTROL OVULEN 0 OVRAL n C-QUENS l
a2
30-
i P
9
11
13
15
17
CYCLE Fig. 2. The percentage amount treated cycles, respectively.
Table II. Distribution menstrual
- 21
of fatty acids of human
12:o (Laurie acid) 13:o (Tridecanoic acid) 14:o (My&tic acid) 15:o (Pentadecanoic acid) 16:0 (Palmitic acid) 16:l (Palmitoleic acid) 17:o (Heptadecanoic acid) 17:l (Heptadecenoic acid) 18:O (Stearic acid) 18:l (Oleic acid) 18:2 (Linoleic acid) 30:o ( Arachidic acid) 2O:l (cis-5-Eicosenoic acid) 22:o (Behenic acid) 20~4 ( Arachidonic acid) 24:o (Lignoceric acid) 22:6 (Docosahexaenoic acid) Saturated Unsaturated Monounsaturated Polyunsaturated Total Total
of phospholipid
19
21
23
25
27 28
DAYS (in
cervical
micrograms
per
cent)
mucus phospholipids
in the
control
and
in
cycle
Fatty acid (Acid: No. of bond)
T =
287
25-
7
normal
phospholipids
odd even
Postmenstrual cycle (5-9 cycle days) (% +- S.D.) 3.1 0.5 3.9 1.3 23.2 1.6 1.1 0.5 21.0 17.6 7.8 1.3 1.6 3.1 5.9 6.5 T 65.0 35.0 21.3 13.7 3.4 96.6
+ + 2 +_ * 2 + 2 + 2 2 + + + t t +
0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.0
Mid-cycle (12-15 cycle days) (%’ + SD.) 3.6 2.0 9.0 2.0 26.5 7.2 2.0 2.0 12.5 14.2 5.4 0.3 2.0 2.0 5.4 3.7 T 63.6 36.2 25.4 10.8 8.0 91.8
+ k t + + -L t +_ 2 + k 2 c ” I t +
0.1 0.1 0.2 0.1 0.3 0.1 0.1 0.1 0.2 0.; 0.1 0.1 0.1 0.1 0.1 0.1 0.0
Premenstrual cycle (20-25 cycle days) ‘(70 z?z S.D.) 4.3 2.2 8.4 2.1 29.3 6.3 2.2 2.2 10.5 12.6 6.3 0.1 2.2 2.2 6.3 2.6 T 63.9 35.9 23.3 12.6
+ 2 + t + + 2 + t .k + t f ? t + 2
0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.t 0.2 0.2 0.1 0.0 0.1 0.1 0.2 0.1 0.0
8.7 91.1
trace.
extraction procedures, preparation of the plates and solvents, the procedure for phosphatide determination, and a typical thinlayer chromatography of phospholipids have been previously reported.6
A Barber Colman Model 5000 gas chromatograph with a dual hydrogen flame detector was used. The two U-shaped glass columns measured 6 feet by I/Z inch outside diameter and contained 3 per cent SE-30 on
288 Singh, Swartwout,
January Am. J. Obstet.
and Boss
Table IV. Distribution of fatty acids of human cervical mucus phospholipids menstrual cycle with contraceptive ( C-Quens)
Table III. Distribution of fatty acids of human cervical mucus phospolipids in menstrual cycle with oral contraceptive (Ovral)
12:o 13:o
14:o 15:o 16:O
2.1 -+ 0.1
3.5 _+ 0.1
2.4+
1.0+0.1 6.3 2 0.1 2.1 + 0.1
1.7 + 0.1
1.2 + 0.1
8.92 0.2 3.5 20.1
5.9kO.l 2.45
17.9 f 0.2 5.42 0.1 1.0 c 0.1 1.720.1 l.O? 0.1 1.8 + 0.1
26.0 ? 0.3 7.3 s 0.1
16:l 17:o 17:l 18:O ia:1
14.6 +_ 0.2 11.4kO.l 4.2 _+0.1
ia:2 2O:l
12.5 t 0.2 8.9 + 0.1 5.4? 0.1
2.1 +-0.1
3.6tO.l 2 0.1 8.9 2 0.1 9.3 2 0.1 12.5 + 0.2 T +_0.0 T fO.O 3.1 2 0.1 8.3 2 0.2
22:o 20:4 24:0 22:6
Saturated Unsaturated Monounsaturated Polyunsaturated
0.1
0.1 29.4 ?I0.3 3.5-+ 0.1 1.2 + 0.1 1.220.1 12.92 0.1 8.2 to.1 4.7-+ 0.1 2.45 0.1 4.7 5 0.1 7.0 2 0.1 12.9-+ 0.1 T + 0.0
65.5 34.3
65.8 34.0
73.0 27.0
21.8
19.7
15.3
12.5
14.3
11.7
Total odd 5.1 Total even 94.7 T =
3.6
15, 1972 Gynecol.
12:o
4.0 + 0.1 1.9to.1 7.8f0.2 2.8f. 0.1 23.8f0.3 6.9 -+0.1 0.72 0.0 0.7 +_ 0.0
13:o
14:o 15:o
16:0 16:l
17:o 18:0 18:l 18:2 20:o PO:1 22:o 20:4 24~0 22:6
1.6kO.l 7.4 -i 0.2
1.9 to.1
3.1 c 0.1
30.5kO.3 5.6 + 0.1 0.4-f 0.0
21.920.3 6.02 0.1 0.22 0.0
68.5 31.3
4.8 ko.1 4.5 2 0.1 T 50.0 66.9 32.9
20.8
21.2
22.1
13.5
10.1
10.8
Total odd 6.1 Total even 94.1
5.1 94.4
93.3
T =
trace.
4.5 r 0.2
10.7 k 0.2 4.3 to.1 T to.0 65.7 34.3
Saturated Unsaturated Monounsaturated Polyunsaturated
91.1
0.1 0.1
1.OkO.l 1.6 + 0.1 15.3 t 0.2 15.2 50.1 19.0 t 0.2 11.9 -+0.2 13.5 + 0.2 13.3 i 0.1 2.8+ 0.1 5.5 + 0.1 6.0 F 0.1 0.3 50.1 0.42 0.0 0.2to.o 1.3 + 0.1 1.1 r 0.1 1.2 20.1 4.8? 0.1 3.6kO.l 4.5 to.1
17:l
8.7
4.5 r 1.85 7.4kO.l
in
4.620.1
2.8 + 0.1 T + 0.0
6.5
trace.
Table V. Distribution of total fatty acids of human cervical mucus phospholipids in entire menstrua Fatty acid (Acid:
No.
of
bond)
12:o
C-Quens
0.1 1.5+ 0.1 7.1+ 0.2
4.0 2 0.2 2.o‘L 0.1 8.02 0.3 4.0 + 0.2 24.0r 0.4 6.0 2 0.1 2.0 r0.2 2.0? 0.1
3.6+
13:o 14:o 15:o
16:0 16:l 17:o
17:l 18:0 18:l 18:2 20:o '2o:l 22:o 20:4 24:0
Saturated Unsaturated T =
Control (%)
trace.
1.8kO.l 26.3 kO.3 5.OkO.l 1.7kO.l
1.9-fO.l 14.6I! 0.3 14.8 -to.4 6.5 _+ 0.2 0.5 ? 0.1 1.9 10.1 2.4+ 0.1 5.850.2 4.3 2 0.3 63.8
35.9
(%)
Control (%)
0.1 1.520.1
3.or
6.3 2 0.2 1.520.1 26.0+ 0.3 4.5 + 0.2 1.5 t 0.1
C-Quens f%)
?I0.1 1.920.1 4.0
7.7 20.2 2.4 + 0.1 26.5 k0.4 5.7 + 0.1 0.5 t 0.1
10.0k 0.2
1.5+_0.1 15.0+ 0.3
8.1 50.2 6.0+ 0.2
16.5 + 0.2 6.02 0.1
10.3 to.2 4.0 + 0.2 0.8 2 0.1
2.8 2 0.2 6.0-c 0.2 5.OkO.l 63.5 36.5
5.0t 0.2 5.1 20.2 6.02 0.2 72.7 27.3
T kO.0 2.0 !: 0.1 6.0+ 0.2 a.o-+ 0.3 a.0 ko.3 68.0 32.0
0.9+ 0.1 2.0 kO.1
0.9kO.l 17.9t 0.3
1.3kO.l
Oral
contraceptive
60 to 80 mesh Gas Chrom P. The column, injector, and detector temperatures were 260, 260, and 270’ C., respectively. Helium was the carrier gas at 60 ml. per minute. With the system containing 15 per cent DEGS on 60 to 80 mesh Gas Chrom P, the column, injector, and detector temperatures were 210, 210, and 240° C. in the order stated. Helium flow was the same. Gas chromatographic analysis was performed directly on the methyl ester mixtures? of various components. The methyl ester fatty acid samples were dissolved in ether, and volumes of 2 ~1 were injected. The samples were hydrogenated as described elsewhere.8 Peaks were identified by use of references and the logarithmic plots of relative retention times vs. chain length and degree of unsaturation.” Results Figs. I and 2 show the variation of human cervical mucus and phospholipids during the itrenstrual cycle with and without oral contraceptives. It is noted from Fig. 1 that the amount of mucus decreased with oral contraceptives. It is observed from Fig. 2 that 1he percentage amount of phospholipids in mucus increased with contraceptives, and this increase remains almost constant in the cycle. The variation of phospholipid is most probably due to the higher and lower content of cycle with
and without
effect on cervical
mucus phospholipids
the other components in the total lipids. ‘Table I shows the distribution of various phosphatides in cervical mucus phospholipids. The main components of phospholipids are phosphatidyl ethanolamine, and lecithin, cardiolipin. Thin-layer chromatography indicates that lysolecithin is present in about 2.0 per cent concentration in mucus. Tables II to IV show the cyclic variations of fatty acid composition of phospholipids with and without contraceptives. In Table II. the noteworthy acid is palmitoleic; it varies from 1.6 to 7.2 per cent in the cycle. Stearic acid is 21.0 per cent in the postmenstrual cycle, while in the premenstrual cycle it is I03 per cent. The fatty acid composition also shows differences in the cycle. Table III presents the cyclic variations of fatty acid conq”‘sitions in the cycle with Ovral. The palmitic acid goes down in the mid-cycle while stearic acid almost remains constant. Table IV shows the cyclic variation of fatty acid compositions, with the USC of C-Quens as the oral contraceptive. Here, palmitic acid goes up in the mid-cycle, while linolcic acid varies from 2.8 to 6.0 per cent in the cycle. Table V presents the total fatty acid composition of phospholipids in the entire cycle with and without contraceptives. It is clear front the table that the fatty acid composition in phospholipids becomes more saturated with
contraceptives __-
Control (%) 3.0 r 0.1 1.7kO.l 6.5 +_ 0.2
--
1.7 to.1 L’i.5 2 0.3 4.5 2 0.2 1.7iO.l 1.5 + 0.1 1.3.6 + 0.3 17.0 -+ 0.3 5..5 5 0.1 0.4 + 0.1 ‘) ““0.1 -._23 + 0.2 6.” ? 0.1 4 5 2 0.2 63:; 36.9
Ovral mi 3.0 + 0.1 1.5 -e 0.1 6.5 +_ 0.2 2.5 k 0.2 26.0 _+ 0.4 i.5 t 0.2 1.:!+01 1.8-fO:l 14.0 -c 0.2 9.0 f 0.2 4.5 t 0.1 o..i + 0.1 2.5 + 0.1 3.0 -c 0.2 7.0 i- 0.3 9.5 + 0.” 69.7 30.3
Control (%) 3.5 + 0.2 1.5 + 0.1 7.0 t 0.2 1.5 + 0.1 26.3 k 0.5 5.0 f 0.2 1.5 + 0.1 1.8 im 0.1 14.5 +_ 0.2 16.9 + 0.3 6.5 + 0.1 0.5 r 0.1 2.040.1 2.5 5 0.1 6.0 5 0.2 3.0+ 0.1 61.8 38.2
289
Omal (%J 4.0 f 0.2 1.6 + 0.1 6.8 + 0.1 2.5 rt 0.1 30.0 t 0 L’ 5.0 * 0:1 1.5 i 0.1 1.7 f 0.1 14.0 + 0.3 11.2’O.L’ 5.5 t 0.2 0.2 + 0.1 1.5 5 0.1 2.5 5 0.1 6.0 + 0.1 6.0 _C0.1 69.1 30.9
Control (%J 4.0 t I.52 7.0 + 1.32 25.0 2 5.5 5 1.5 2 1.4 t 1”.9+0:1 16.8 t 7.0 5 0.3 It 1 ..i k 3.0 2 3.0 t 6.0 + 63.7 37.2
0.1 1 0.2 0.1 0.3 0.2 0.1 0.1 0:3 0.3 0.1 0.1 0.1 0.1 0.1
-_Ui? 01 (%.I
4.0 f 0.1 1.8 -4. 0.1 6.0 -+ 0.2 3.0 ?: 0.2 29.0 ? 0.2 4.5 -! 0.1 1 .n + 0.1 1.O.kO.l Ii.0 i- 0.2 12.0 + 0.2 5.0 IO.1 0.3 kO.1 1 i -t I). 1 3.0 + 0.1 7.nto.2 5.7 t 0.2 69.0 31.0
290
Sigh,
Swartwout,
and
Boss
Table VI. Fatty acid compositions Fatty acid (Acid: No. of bond)
SPH
4.0 t 0.2 0.6 2 0.1 7.4 i: 0.2 3.02 0.1 18.8 +_0.2 6.8 + 0.1 1.820.1 1.7 + 0.1 9.4 + 0.2 11.8 + 0.2 5.0 _+0.2 1.7 2 0.1
12:o
13:o l4:O 15:o 16:0 16:l 17:o 17:l la:o la: i 18:2 20:o 2O:l 21:ot 22:o 20:4 23:0$ ‘24:O Saturated Odd Even Unsaturated Monoenes Polyenes Even No. of double bond/ molecule
2.3 + 0.1 5.8 i 0.2 3.OkO.l 16.9 + 0.3 74.7 10.7 64.0 25.3 20.3 5.0 23.6 0.3
CL, Diphosphatidyl phosphatidyl inositol; *Entire
pool
tHeneicosan& $Tricosanoic
sample
glycerol; GPC, PS, phosphatidyl of 3 normal
of phosphntides CL + GPC
LPC
2.8 + 0.2 1.2CO.l 5.6 -+ 0.2 1.7kO.l 24.6 + 0.3 5.3 2 0.2 1.2ro.1 2.9 + 0.1 9.2 + 0.2 13.9 + 0.3 4.2 + 0.1 2.3 +_0.1 5.8 2 0.2
4.1 2 0.1 0.5 2 0.1 7.5 2 0.2 1.6” 0.1 24.8 It 0.3 6.8 2 0.2 1.450.1 1.6 + 0.1 12.9 + 0.2 22.5 _+0.3 7.2 + 0.2 1.4 + 0.1 79+0.1 -.- 0.8 +- 0.1 1.6kO.l 2.9 +_0.1 57.9 3.5 54.4 41.9 33.1 8.8 40.3
a.7 +_0.3 3.5 + 0.1 7.0 +_0.2 64.3 4.1 60.2 35.6 27.9 7.7 32.7 0.5
0.5
glycerophosphoryl serine; SPH, cycles
(cycles
were
of human
PS
cervical
muws+ ------. PE
PI
3.7 2 0.1 0.6 -+0.1 6.5 + 0.2 1.2 2 0.1 26.4 5 0.2 6.2 + 0.1 1.2 + 0.1 1.4kO.l 17.9 _+0.2 20.3 + 0.3 6.2fO.l 1.420.1 1.5 * 0.1
4.4 !: 0.2 0.5 + 0.1 8.2 _+0.1 1.5 k 0.1 26.9 + 0.3 7.2 _+0.2 1.5 + 0.1 2.0 + 0.1 11.7fO.Z 13.8 + 0.2 5.9 2 0.1 1.0%0.1 3.2 _+0.1
2:8+0.1 1.2 _+0.1
3.2 +_0.1 0.5 _+0.1 6.6 + 0.2 1.6 + 0.1 25.8 + 0.3 8.1 _+0.2 2.2 5 0.1 3.2 + 0.1 9”?0 1 9:s -+ 0:1 2.7 f 0.1 1.020.1 2.7 k 0.1 1.6f-0.1 8.6 _+0.2
0.6 2 0.1 3.1 2 0.1
3.3 2 0.1 2.0-+0.1
5.2 * 0.1 69.3 6.0 63.3 30.6 25.2 5.4 28.2
13.4 + 0.2 65.1 4.3 60.8 34.9 23.6 11.3 31.7
1.7 +_0.1 61.3 3.0 58.2 38.7 29.4 9.3 37.3
6.9 2 0.2 65.9 3.5 62.4 34.1 36.2 7.9 32.1
3.5 2 0.1 1.2 -+ 0.1 12.5 2 0.2 2.4 + 0.1 29.4 5 0.4 5.4 2 0.2 2.4 2 0.1 2.4 i: 0.1 9.9 f: 0.2 13.2 _+0.2 4”tol i- k 0:o 4.2 _+0.1
0.4
PC
~~.
0.7
choline; PE, phosphatidyl ethanolamine; sphingomyelin; LPC, Iysophosphatidyl choline.
0.6 I%,
phosphatidyl
0.5 choline;
PI.
28 days).
acid acid.
the use of oral contraceptives. In general, it changes from 63.0 to 69.0 per cent in saturation. The total composition of phospholipid fatty acids, however, did not differ significantly between the control or treated groups. Table VI illustrates the composition of phosphatide fatty acids. Sphingomyelin contains the highest percentage amount of odd acids as compared to other components. It is noted that lecithin-, phosphatidyl ethanolamineand cardiolipid-type fractions are most unsaturated, while the sphingomyelin is most saturated.
3. The lecithin and phosphatidyl ethanolamine are the main components of phospholipid. 4. Cyclic variations in fatty acid composition were observed in the cycle. 5. With contraceptives the fatty acid becomes more saturated. 6. Among phosphatides, the numbers of double bonds per molecule in diphosphatidyl glycerol + glycerophosphoryl choline, phosphatidyl ethanolamine, lecithin, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, and lysolecithin are 0.5, 0.6, 0.7, 0.5, 0.5: 0.3, and 0.5, respectively.
Results
1. The amount of total mucus decreased with the use of oral contraceptives. 2. The percentage amount of phospholipids increased with contraceptives.
Comment
Cervical mucus is a complex mixture of inorganic and organic substances and enzymes, bearing a similarity to a dual phase gel, with
Oral
contraceptive
hydration during the proliferative phase and dehydration during the secretory phase.‘O Spem migration was accelerated in hydrolyzed cervical mucus.l’ Contraceptives affect the cervical mucus viscosity, making it difficult for the spermatozoa to penetrate it and ascend.‘” Recently, it has been suggestedthat phospholipids take part in viscosity13 in the mucus and hence are responsible for sperm migration as one of the components. Viscosity is very important in the mid-
REFERENCES
1. Folch, J., Lees, M., and Stanley, G. H. S. : J, Biol. Chem. 226: 497, 1957. 2. Carroll, K. K.: J. Lipid Res. 2: 135, 1961. 3. Hamilton, J. G., Swartwout, J, R., and Miller, OTT.: Biochem. Biophys. Res. Commun. 5: 226, 1961. 4. Blank, M. L., Schmit, J. A., and Privett, 0. S.: J. Am. Oil Chem. Sot. 41: 371, 1964. .5. Privett, 0. S., and Blank, M. L.: J. Am. Oil Chem. Sot. 40: 70, 1963. 6. Singh, E. J., and Gerishbein, L. L.: J. Chromatogr. 31: 20, 1967.
effect on cervical
mucus phospholipids
291
cycle. It has been demonstrated that pills have complex effects on the secretion of the cervical mucus. It is noted that with the contraceptives the phospholipids increased in the cycle. These drugs presumably act lo inhibit ovulation either directly or indirectly, and they also change the amount and cornposition of phospholipid fatty acids. It may be possible that phospholipid takes part in the elasticity of mucus.
7.
8. 9. 10. 11. 12. 13.
Luddy, F. E., Barford, R. A., and RiemenSchneider, R. W.: J. Am. Oil Chem. Sot. 37: 447, 1960. Singh, E. J., Gerishbein, L. L., and O’Neill. H. J.: Lipids 1: 274, 1966. Woodford, F. P., and Van Gent, C. M.: J. Lipid Res. 1: 188, 1960. Platt, H. A.: J. Reprod. Med. 3: 131, 1969. Moghissi, K. S., and Syner, F. N.: Frrtil. Steril. 21: 234, 1970. Sten. J., and Sting. K.: Fertil. Steril. 21: 307. 1970. Lewis, R. W.: Lipids 5: 917. 1970.