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Oral contraceptive compounds and mammalian oocyte meiosis
Oral contraceptive compounds and mammalian oocyte meiosis GEORGIANA J. New
S.
JAGIELLO,
M.D.
LIN
York,
New
York
A meiotic study of the in vitro and in vivo effects of components of currently used oral contraceptives on the oocytes of 6 female mammalian species was made. In vitro inhibition of division and atresia were found in ,several species with both the estrogens and progestogens, as well as a low frequen&y of chromasome breaks and rearrangements, anaphase lags, spindle abnormalities, and polar body suppression. Combined in vivolin vitro testing revealed a possible basis for expecting increased atresia of treated oocytes. Considerable species variation and the concentration of the steroids tested do not permit clear extrapolation to a &aim for a direct action of oral contraceptives on human oocytes. The sensitivity and reproductive endocrinology of the Bovidae in these experiments suggest these as a model for female mammalian meiotic mutagen studies.
A c E RTA I N unrest has been attached to the use of the estrogen-progestogen oral contraceptives since the reports in 1970 of Car+ * that, following their use, the incidence of triploid abortuses was increased. Coupled with scattered papers demonstrating in vivo cytogenetic abnormalities, including breaks and rearrangements33 4 and animal data which document interference of mestranol with embryo survival in dogs5 and impaired egg development in the golden
hamster” treated with the metabolite of mestranol, ethinyl estradiol, study of the effects of the separate components of the currently used oral contraceptives on female mammalian oocyte meiosis seemed overdue. Direct assessment of the chromosomes of 6 species of female mammalian eggs including man as they proceeded through first meiotic division was undertaken. The possibility of developing a model system for extrapolation to a mutagen screening test for human meiosis was present since the reproductive endocrinology of the higher animals used resembles in many ways that of man.
From the Department of Obstetrics and Gynecology, the Department of Human Genetics and Development, and the International Institute for the Study of Human Reproduction, Columbia University College of Physicians and Surgeons. Supported by National FoundationMarch of Dimes Grant CRBS-249, National Institute of Child Health Human Development Grant 5ROl 05862, and the Ford Foundation. Received 1974.
for
publication
Revised February Accepted March
January
Material
and
methods
Cytogenetic analysis of first and second meiotic metaphase chromosomes was carried out on oocytes of mouse, ewe, cow, rhesus and baboon matured in vitro using techniques already described for these species7-” (Fig. 1). Mouse and human oocytes’” were also studied with a combination technique of in viva/in vitro progression. The specific techniques varied from species to species and will thus be described for each. In all cases, resting oocytes were manually removed from
and -HD31,
21, 1974. 6, 1974.
Reprint requests: Dr. Georgiana Jagiello, College of Physicians and Surgeons, 630 W. 168th St., New York, New York 10032.
390
Volume 120 Number 3
Oral
contraceptives
and
oocyte
meiosis
391
b
d
Fig. 1. Normal control sheep Ml; d, cow Ms; ovarian
consisting Diploid” additives, *Grand York.
meiotic preparations: cr, mouse oocyte e, baboon Ml; f, rhesus Ms. (Original
follicles, pooled, incubated in media of McCoy’s 5A modified,* or with or without serum and steroid in a 5 per cent CO,/air atmoIsland Biological
Company,
Grand
Island,
New
Ml; b, mouse oocyte magnification ~1,250.)
M2;
c,
sphere with high humidity and at a constant pH of 7.2. Preliminary experiments provided minimum effective doses of each steroid for each oocyte species. These were initially estimated from the concentrations contained in the commonly used tablets on a weight
392
Jagiello
Table -
IA.
and
Lin
vitro
In
cytologic ~
Media Diploid Ethanol
(@g/cc.) estradiol
Norethynodrel (PP/C.C.) Ethynodiol (PdC.C.)
No. div.
% j div.
74 “7 19 46
66 “1 16 39
89 78 84 84
-
38 33 44 36 14
30 25 17 32 l:!
79 79 39 89 86
-.
0.1 10.0
22 34
19 26
86 76
0.1 10.0
56 25
47 19
84 76
34 41
27 30
39 39 28 27
0.005 0.0005 7.41 ( 1 7a-E2) 0.74 7.41 74.1 0.1 10.0
N
17a-Es 7.41 + norethynodrel 0.1 10.0 (PdC.C.) 17a-Es 7.41 + ethvnodiol diacetate 0.1 10.0 17a-E2 7.41 pg/c.c. + 0.1 norethindrone 10.0 @g/c.c.)
(&c.)
*N
=
tA
= spindle
Normal
in structure abnormalities;
and
number;
B = failure
A =
Raritan, Illinois.
New
Metaphase’” (No. oocytes) N
/
8 2 1 I
-
A -
1 B -
1 C
1 1 1
2 -
1 15 1
-
-~
-
1 D
-
-
3 11
2
2
-
-
-
-
4 4
1
-
-
-
79 73
-
9 -
8 13
-
3 -
1
-
32 32
8’ 82
-
-
4 5
3 -
-
-
1 -
21 19
75 70
-
1
6 4
-
-
-
-
body
Jersey.
rearranged;
formation
(mixture
B
=
clumped;
C
=
fuzzy,
indefinite
_---/ E
-. -
-
basis of 70 kilograms for an adult human female. Other guidelines for concentrations were adapted from the literature on mitotic effects of estrogen. I1 The constituents of 3 commonly used oral contraceptives-OrthoNovum 2,” Ovulen,t and Enovid-Et and their significant metabolites were studied. These were mestranol and its metabolite 17cy-ethinyl estradiol ( 1 7a-E2) ,12-14 norethynodrel,15-zo ethynodiol diacetate (ED) and norethindrone, its metabolite.21 These were dissolved in ethanol, diluted with serum-free media and added in appropriate concentrations to the final culture media. In most instances, both media were studied to eliminate spurious effects resulting from metabolic components of the serum-free media or potential binding of the test steroid to *Ortbo Pharmaceutical Corporation, tG. D. Sea& & Company, Chicago,
rnetaphase
-
fragmented,
of polar
/ Ab
-
diacetate
Norethindrone (cLg/c.c.)
oocyte
diak.
Total eggs
+ additives
(c.c./c.c.)
Mestranol 17ru-ethinyl (PdC.C.)
effect of oral contraceptives on cow --...-~-~. I Early
outlines;
of M* + PB’ chromosomes).
serum. Media selection was based on previous experience of meiotic analysis with each species. Cow and ewe. Ovaries were harvested from random-bred diestrous exsanguinated cows and ewes at a local slaughterhouse, washed in penicillin/streptomycin, and transported rapidly under sterile conditions to the laboratory. Oocytes were manually removed with sterile precautions with a Zeiss dissecting microscope, pooled, and immediately placed in the media. Cultures of cow oocytes were carried out for 28 hours in Diploid media which contained 10 per cent fetal calf serum and in serum-free McCoy’s modified 5A. Harvests at this time interval yielded eggs in both first and second metaphase. Ewe oocyte cultures were made in McCoy’s 5A and in Diploid containing an additional 12 per cent sheep serum, making a total concentration of 22 per cent fetal calf
D
Volume Number
120 3
Oral
contraceptives
and
oocyte
MetaphaseP* (No. ova) N
1
A
1
B
1
meiosis
Othert C
1
D
i
E
A
1
B
58 19 15 34
-
1
3
-
-
-
-
26 17 26 10
2 -
-
-
1
4 -
-
2 -
-
13 9
1
-
-
1 -
2 1
-
-
38 10
-
-
-
-
3
2 -
4
4 10
-
-
2 4
-
-
-
1 2
18 13
1
-
6
-
5
4 2
2 10
-
-
-
-
12 2
1
mlverized;
E = atretic,
advanced
393
1
-
-
1
“C.”
serum (FCS) . These were harvested at 26 hours for first and second metaphase stages. Rhesusand baboon. Restricted availability of ovaries from these species prohibited completely parallel experiments for comparison with the Bovidae and mouse. The evolutionary position of these species made an attempt mandatory and information for selection of effective concentrations of steroids was derived from the other studies. Oophorectomy was carried out rapidly using Sernylan anesthesia on adult, cycling females. Eggs were removed from the ovaries as above and incubated in McCoy’s 5A with 12 per cent FCS. Ten international units of human chorionic gonadotropin (HCG) was added to the cultures of rhesus oocytes. These media and the harvest time of 28 to 30 hours had been previously demonstrated to yield a good division rate for harvesting both desired stages.
Mouse and human female. Extensive studies were carried out with the randombred Camm* strain mouse. For in vitro study, 40 to 60 day old diestrous females were used and oocytes incubated in McCoy’s 5A with 40 per cent FCS for 4.5 to 6 hours to obtain cells in first metaphase and 12 to 15 hours for second metaphase. In addition, 0.05 to 1.0 pg of mestranol per mouse per day was given subcutaneously for 4 days (1 vaginal cycle) followed by in vitro incubation for 14 hours of oocytes recovered 24 hours after treatment. Parenteral administration of 0.01 to 10 mg. of mestranol per mouse per day for 12 to 16 days, 0.02 to 1.0 pg per gram of body weight per day (GBW) of ethynodiol diacetate for 16 days, norethynodrel 0.1 to 4.0 pg per gram body weight for 16 days, 0.05 pg per mouse of mestranol ‘Camm
Research
Institute,
Wayne,
New
Jersey.
394
Jagiello
Table
IB.
and
In
Lin
vitro
effect
of oral
on COW oocytr
contracc.I,ti\rcs
metaphasc
Early/ diak.
Media McCoy’s 5A Ethanol (c.c./c.c.) Mestranol (pg/c.c.)
(cdc.c.)
A/BiCIDjE
+ additive-s 0.0005 0.001 0.005 7.41
17cu-ethinyl estradiol ( 17a-E2) 0.74 (Fg/c.c.) 7.41 74.1 Norethynodrel 0.1 10.0 (ag1c.c.) Ethynodiol diacetate 0.1 (PdC.C.) 10.0 Norethindrone 0.1 (PdC.C.) 10.0 17a-E2 7.41 fig/cc. + norethynodrel 0.1 10.0 (cg1c.c. ) 17~~E2 7.41 &c.c. + ethynodiol diacetate 0.1 10.0 (Ilg/c.c.) 17sE2 7.41 cg/c.c. norethindrone
Metaphase’” (No. imytes)
:
+ 0.1 10.0
* and t: For explanation
of abbreviations,
194 45 “3 45 30
164 39 1x 38 22
8.5 87 79 84 73
64 122 28 69 85
53 88 27 63 63
82 72 83 91 74
106 149
87 128
82 86
33 ‘1 1
28 34
85 83
61 70
48 6.5
79 93
5L’ 62
49 51
94 82
43 40
39 31
90 77
see footnote
to Table
combined with norethynodrel 0.5 or 4 pg per gram body weight for 17 days, and mestranol 0.1 pg per mouse plus 0.02 lug per gram body weight of ethynodiol diacetate for 17 days followed 24 hours later by in vitro incubation of oocytes for 14 hours to obtain second metaphase ova was carried out. The parenteral schedules were designed to treat one complete developmental cycle of mouse oocytes as determined by Bake? and to parallel by weight in mouse the in vivo doses used in human subjects. Human female oocytes were recovered from ovarian material obtained from patients aged 24 to 51 as part of the course of indicated pelvic operation, principally hysterectomy. Initially all surgical material was studied, oocytes removed from all usable specimens, placed in Diploid medium with added 40 per cent fetal calf serum and harvested after 30 to 35 hours of culture.
/I
-
-
1 .~ .-
_~ -
..-
-
.-
-
1 18 -. 1
-
2 2
.
9 12 11 8
4 3
5 1 .-
1 29
17 ‘10
2 1
1
1 2
1
4 4
-
-
-
3
1 19
35 30
1 2
-
2 4
-
3 1
13 33
24 -
3 -
7
2 6
-
-
31 16
-
6 6
11 -~ 3 -
-
-
1 15 26
40 9 6 8 6
-
-
IA.
Those ovarian specimens subsequently shown to be without histologically detectable pathology were assessed for meiotic complements. The use of oral contraceptives by the patient prior to operation was determined from a retrospective examination of the chart and depended entirely on the clinical history. Type, dose and duration varied widely. Techniques for cytogenetic harvest and preparations were uniform for all species. A modification of the method of Tarkowski2” was used to prepare the oocytes and ova for cytologic examination of the chromatids and chromosomes. Initial staining was made with 1 per cent toluidine blue in resin and permanent preparations were made with 10 per cent Giemsa stain. Microscopic assessment of number and structure of both first and second metaphase was made at ~1,250 with oil immersion using a Zeiss photomicroscope
Volume Number
120 3
Oral
contraceptives
Metaphase”+ (No. ova)
and
Ana.
N(AIB(C)DIE -
-
1
.-
27 20 44 27
-
1 :
-. 1 8
-
52 53
1 -
4 8
-
23 10
-
-
3 2
-
1
1 1
‘9
5 1 1 2
-
‘> -
1 2
1
7
1 3
-
2 5
1 3
.-
-
10
1
3 -
-
1
1 -
.-
-
-
-
: -
1
meiosis
395
Other?
NIAb
116 29 11 28 10
oocyte
A/B -
-
-
8 1 -
1
2 2
4 2
3
-
1
1 5
1 2
-
-
-
2 -
-
1
-
-
-
-
-
-
-
-
-
1
-
and optics. Confirmation of these observations was made with photomicrographs taken at x1,250. Results The cytologic effects of the constituents of 3 commonly used oral contraceptives on the meiosis of the mammalian oocytes studied have been assessed in terms of (a) numbers of oocytes maturing (per cent) to the first or second metaphase stages at the appropriate harvest time, (b) normalcy of number and structure of bivalents (Ml) or chromosomes (M*) , and (c) types of induced structural abnormalities annotated by the number of eggs observed at each meiotic stage in the tables under the headings A = fragmented and/or rearranged; B = clumped but with sufficient structural integrity to be recognizable as to meiotic stage; C = fuzzy, indefinite outlines to either bivalents or
chromosomes but with a normal number of each; D = pulverized or shattered into innumerable parts and reminiscent of structural damage seen with virus infections in mitotically dividing cells, and E = atretic, an advanced stage of “C,” all shown in Fig. 2. Anaphase 1 was assessed as normal with complete separation of the 2 chromosome complements from the equatorial plate or abnormal with 1 or more chromosomes distinctly lagging on the mid-body (Fig. 2). In addition, multipolar divisions seen following first metaphase connoting spindle abnormalities were classed under “other” as “A” and failure of first polar body formation with resultant mixture of the second metaphase and polar body chromosomes was scored as “B” throughout (Fig. 3). Cow and ewe. The in vitro cytologic effects on cow oocytes of mestranol, 17~E’, norethynodrel, ethynodiol diacetate and
396
Jagiello
Table
and
II.
In
Media
Lin
vitro
cytologic
effect
(Pg/c.c.) (P&.C.)
t:
Far
contraceptives
on
~111’
metaphase
oocyte
+ additives
Diploid + sheep serum 12% Ethanol 0.0005 (c.c./c.c.) Mestranol 7.41 (Pg/c.c.) 17a-ethinyl estradiol ( 1 7a-E2) 0.74 (PdC.C.) 7.41 74.1 Norethynodrel 0.1 10.0 b4dc.c.) Ethynodiol diacetate 0.1 (/Lg/c.c.) 10.0 Norethindrone 0.1 (PdC.C.) 10.0 17a-E2 7.41 + norethynodrel 0.1 10.0 (adc.c.) 17~E’ 7.41 + ethynodiol diacetate 0.1 @g/c.c.) 10.0 17~2-E” 7.41 + 0.1 norethindrone 10.0 (&/C.C.) McCoy’s 5A 0.0005 Ethanol (c.c./c.c.) 7.41 Mestranol (ELg/c.c.) 17cu-ethinvl estradiol i 17a-E2) 0.54 ' 7.41 74.1 0.1 Norethvnodrel 1.0 10.0 Ethynodiol diacetate 0.1 (Pg/c.c.) 1.0 10.0 0.1 Norethindrone 10.0 (PLp/C.C.) 17a-EZ 7.41 f norethynodrel 0.1 10.0 k/c.c.) 17a-E2 7.41 + ethynodiol diacetate 0.1 (Bg/c.c.) 10.0 17a-E2 7.41 + norethindrone 0.1 (pg1c.c.) 10.0 ‘* and
of oral
rxplanation
of abbreviations,
79 38
55 :10
70 79
14
12
86
34 43 5 24 "3
'6 26 1 17 16
76 60 20 71 69
46 40
19 29
41 7"
29 34
13 19
45 56
29 43
"4 34
3" is
-
-
__~
-
4 5r,
-
3
-
-
-
-
-~
-
-
9 3 -
1
-
-
-
2
-
-
1 -
-_ .-
14 1 3
-
-
4 -.
-
1 2
2
-
1 11
-
2 -
1
-
2
I
.-
3 1
83 79
-
-
1 2
2 -
-
4 -
.~ -
1 5
25 19
79 73
-
-
4 1
-
-
2 -
-
4 2
28 33 66 39
18 16 62 28
64 48 94 72
-
4 3
-
-
-
: 7 1
23
16
70
-
2
2
-
-
44 42 19 18 28 32
31 28 18 16 17 24
71 50 95 89 61 75
4 2 1
-
-
'7 30 33 35 30
22 2' 21 28 23
81 73 64 80 77
1 8 1
30 32
24 23
27 31 25 33 see footnote
-
1 3
1
-
11 10 -
3 -
-
2 2
1 1 4
5
-
-
1 4 -
_-
5 2
19 1 1
1
1 1 -
-. -
2 4
80 72
1 10
15 2
1 1
1
2 4
-
2 3
20 23
74 74
10 16
5 -
4
-
1
-
L' -
10 23
40 70
1 3
-
1 -
3 .-
2
-
1 19
to Table
IA
9 18 1
-
1
-
Oral
contraceptives
MetaphaseP* (No. ova) N
B
E
N
49 25
-
-
-
-
2 1
-
-
9
-
-
-
-
-
-
-
-
-
-
-
-
-
I 1
1 -
-
-
-
-
__
12 6
1
D
(
1 1 8
-
(
397
Other+
Ana.’ C
meiosis
A
15 9
1
oocyte
-
16 I -
(
and
Ab.
-
-
1
-
5 3
-
-
-
-
1 2
-
6 18
-
1 -
2 2
-
1 1
-
8 13
1
-
1
1
1
-
-
5 3 48 25
2 -
-
4 1 -
1 1 2
7
-
-
-
3
-
-
-
-
-
4 -
-
-
-
-
2
1 1
3 -
-
3
4 4
-
1 -
-
-
-
-
-
1 -
-
-
-
-
14 -
-
9 IO 9 1 17 6 13 6
1 2 1
-
2 -
-
-
A
(
B
-
-
4 -
2 -
-
1
-
2
-
-
1 -
-
-
2
1 -
1
3 -
-
1 -
-
3 1
-
-
-
-
-
-
-
-
-
-
__ -
-
1 -
398
Jagiello
and
III.
In vitro
Table
Lin
cytologic
effect of oral contraceptives
on rhesus
and baboon
oocyte
metaphas~
-Media
+ additives
A. Rhesus data McCoy’s 5A + fetal calf serum 12% + HCG IO I.U. 0.005 Ethanol (PdC.C.) Mestranol 7.41
22 7
,42 58
1 -
-
10 ‘,
-
._
12 21
7
33
-
-
1
-
-
( 17a-E2) 7.41 0.1 10.0
“0 6 3
9 4 3
45 67 100
-
-
4 1 1
1
0.1 10.0 10.0
21 20 18
7 6 11
33 30 60
-
1 -
2 2 1
4 8
3 4
75 50
-
1 2
8 18
2 5
-
-
8
3
38
-
-
24 11 6 9 18
17 5 4 2 3
71 45 67 22 16
-
14 14
4 5
28 36
12 21
5 12
42 57
5’J
-
-
.-
1
-
2
-
2 -
-
2
-
-
4
3
3 -
-
-
-
-
-
-
-
(pg/c.c.) 17sethinyl (Pg1c.c.) Norethvnodrel
estradiol
(P3YC.C.) Ethynodiol (P!dC.C.)
Norethindrone (pg,‘c.c.) 17n-E2 7.41 + norethynodrel 0.1 10.0 (P.5dC.C.) 17sE2 7.41 + ethynodiol 0.1 diacetate 10.0 (&/C.C.) 17~~E” 7.41 + 10.0 norethindrone (Pg/c.c.) B. Baboon data McCoy’s 5A t fetal calf serum 0.1 Norethynodrel 10.0 (PLs/C.C.) Norethindrone 0.1 10.0 (!%/c.c. ) 17a-ethinyl estradiol ( 17a-E2) 7.41 + norethynodrel 0.1 (PdC.C.) 10.0 17sE3 7.41 + norethindrone 0.1 10.0 (pg/c.c.) * and
t:
-
2
diacetate
For
explanation
25%
of abbreviations,
see footnote
norethindrone are shown in Tables IA and IB. Diploid medium which contains 10 per cent fetal calf serum supported a high rate of normal meiotic maturations (89 per cent), as did McCoy’s 5A modified medium which yielded 85 per cent divisions. Maximal concentrations of ethanol solvent did not interfere with this in either series. Statistically significant restriction of division was seen in Diploid with 7.41 pg per cubic centimeter of 17~E” with norethynodrel 10 pg per cubic centimeter and 17~EZ 7.41 pg per
-
to Table
-
1
1 1
1 2 2 3
-
-
-
-
1 -
-
-
‘I
-
-
-
-
-
8
-
-
-
-
-
IA.
cubic centimeter plus norethindrone 10 t% per cubic centimeter (p value = < 0.05, chi square test), and in McCoy’s with 7.41 pg per cubic centimeter 17~~E” and norethynodrel 10 pg per cubic centimeter (p value = < 0.05). In addition there was a restriction of development from M1 to M2 seen minimally in the Diploid but markedly in the McCoy’s 5A with the 17a-E2 and combinations of 17a-E” with all 3 progestogens. Very high concentrations (74.1 pg per cubic centimeter) of 17~E2 severely limited mei-
1
1 3 5 4
volume Number
120 3
Oral
contraceptives
Metaphase”* (No. oua) N
10 5
/
A
2 -
1
-
1
-
3
1
‘C
/
D
(
E
1
1
N
1
oocyte
/
meiosis
399
Othert
I Ab
A
/‘B
-
-
-
-
-
-
1
15 3 2 2
B‘
Ana.’
-
1 1
1
and
-
.-
-
-
-
1
-
-
-
-
-
-
2
-
-
-
-
T
1 1
-
otic progression in Diploid (39 abnormal Ml’s) or to abnormal diakinesis in McCoy’s 5A. Scattered instances of fragmentation and rearrangement of bivalents were seen in both experiments at M1 but were without significant incidence of cytogenetic sequelae such as nondisjunction or anaphase bridging in later stages. Fuzziness of bivalents at Ml, particularly marked with 17a-E2 in both media did not have later detectable effects and cell death characterized by clumping or obvious atresia predominated among the
abnormalities at M’. Of particular interest from the genetic viewpoint were the 2 instances of induction of lagging at anaphase’ by norethynodrel (0.1 pg per cubic centimeter) and ethynodiol diacetate (10 pg per cubic centimeter) in McCoy’s as well as production of spindle abnormalities and failure of polar body1 formation, particularly with the progestogens (Tables IA and IB) . Utilizing ewe oocytes as the test cell in both media systems (Table II), it was noted that the control division rate varied from 70
400
Jagiello
and Lin
Table IVA. In vitro cytologic
effect of oral contraceptives
on mouse oocyte first
metaphase
McCoy’s 5A + fetal calf serum 40% Ethanol 0.0005 (c.c./c.c.) 0.0025 Mestranol 7.76 (PgYC.C.) 17cu-ethinyl estradiol ( 17a-E*) 0.74 (&/C.C.) 7.41 74.1 Norethynodrel 0.1 10.0 (cg/c.c. ) Ethynodiol diacetate 0.1 hg/c.c.) 10.0 Norethindrone 0.1 10.0 ( pg1c.c. ) 17~~E2 7.41 + norethynodrel 0.1 10.0 h!x/c.c.) 17~E2 7.41 + ethynodiol diacetate 0.1 (PdC.C.) 10.0 17a-E2 7.41 + norethindrone 0.1 10.0 Wc.c.) *For
explanation
of abbreviations,
-
5
66 19 13 16
-
I
-
--
1 5
-
-
1 -
-
-
4 -
-
-
24 52 10 12
-
61 67
-
23 19 16 17
77 76 50 39
-
3
22 14 12 5
-
-
1 -
4 ‘) -
19 62
3 32
47 52
-
-
24
6
-
9 -
0
-
2:! 57
9 32
41 56
-
-. -
-
5
28
4 -
3
39 19
18 6
46 31
-
13 1
-
-
-
5 1
69
26 51
67 19 23 27
25 73 16 18 21
25 56 11 14
100 77
30 “5 32 44
97 30
see Table
63 88 53
4
1 -
2 9
1 ?i
IA.
tFragmerited.
per cent in the Diploid with added 12 per cent sheep serum to 94 per cent in McCoy’s 5A (Table II). The division rate in Diploid was significantly reduced by 74.1 pg per cubic centimeter of 17~E’, ethynodiol diacetate 0.1 pg per cubic centimeter, norethindrone 0.1 pg per cubic centimeter, and 17~E? 7.41 pg per cubic centimeter plus norethindrone 10 pg per cubic centimeter (p values = < 0.05). Restriction of development to M1 was noted primarily with the combination of 17a-E* and norethindrone 10 pg per cubic centimeter. Atresia was the predominant effect noted in Diploid but a few spindle and polar body abnormalities were seen in the combination trials. In contrast, the same study conducted with McCoy’s 5A with serum (Table II) showed a severe restriction of division with almost
all compounds. The largest dose of permitted dissolution of the germinal vesicle and reappearance of the diplotene state but this progressed only to badly damaged diakinesis. The smallest concentrations of norethynodrel and ethynodiol diacetate did not inhibit progression. Breaks with rearrangements and considerable atresia were seen at Ml, and clumping and atresia in M2. As with cow oocytes, spindle and polar body abnormalities were noted with the progestogens. Rhesus and baboon. Rhesus data (Table III) demonstrated no statistically significant inhibition of division in the presence of any components of the oral contraceptives. Atresia was the predominant effect at M’ and M2. Only 1 instance of suppression of PB1 was seen with ethynodiol diacetate.
Volume Number
120 3
Oral
contraceptives
and
oocyte
meiosis
40’
b
c
f
a, fragmented and rearranged M’ Fig. 2. Representative abnormal cytologic configurations: (arrows indicate breaks and rearranged bivalants) (sheep) ; b, clumped M2 (sheep) ; c, fuzzy Ml with sticky bivalents (mouse); d, pulverized M2 (cow) ; e, atretic M1 (cow) ; f, late anaphasel (arrow indicates lagging chromosome) (sheep). (Original magnification ~1,250.)
The baboon oocytes (Table III) were more sensitive than the rhesus in terms of restriction of division. Norethindrone 0.1 pg per cubic centimeter, or 10 pg per cubic centimeter and 1 7a-E2 with norethynodrel 0.1 and 10 pg per cubic centimeter depressed progression (p = < 0.05). Again atresia was the prime abnormality produced and 3
examples of spindle misbehavior and PB’ suppression were found. Mouse and human female. In mouse the study of short incubation progression to M’ in the presence of oral contraceptive compounds (Table IVA) revealed restriction of division with 17a-E2 (0.74 and 74.1 pg per cubic centimeter), norethindrone 10 pg per
402
Jagiello
Table IVB.
and
Lin
In vitro
cytologic
effect of oral
contraceptives
I Media
*For
explanation
Early disk:
+ additives
McCoy’s 5A + fetal calf serum Ethanol 0.001 0.0005 (c.c./c.c.) Mestranol 0.74 7.74 b%/c.c.) 17sethinyl estradiol ( 17a-E2) 0.007 (pg1c.c. ) 0.074 0.74 7.41 Norethynodrel 0.1 (pg1c.c.) 1.0 10.0 Ethynodiol diacetate 0.1 (PdC.C.) 10.0 Norethindrone 0.1 (BdC.C.) 10.0 17cr-E* 7.41 + norethinodrel 0.1 10.0 (/Jg/c.c.) 17ru-E* 7.41 + ethynodiol diacetate 0.1 (Pg./C.C. ) 10.0 17&-E* 7.41 + norethindrone 0.1 10.0 (fig/=) of abbreviations,
N 40%
see Table
on mouse
-
oocyte
second
mrtaphasc
i Metaphn.re’*
1 Ab
/ N
i
A
73 78 1nn 59 70
.-
13 17 33 104
13 17 24 66
100 100 73 63
-
18
6 9 10 24
-. -
18 42 20
12 32 14
67 76 70
-
” 11 5
3
36 53
26 40
72 75
-
-
16 43
11 28
69 65
-
“8 15
25 8
89 53
31 49
24 31
25 i5
20 12
.~ 1
i
R
1 C
1 D
16 1 9 Iz-
I
-.~.
3 ‘I
-
-
-
7
3
-
--
-
-
6
-
2 7
1
-
-. -
1 5
-
1 10
-
-
-
1 4
5 4
-
1
-
-.
77 63
-
22
5 5
-
‘1
-
-
80 80
-
-
2
-
1 -
-
-
-
7
IA
cubic centimeter, and 17~E* with norethynodrel 10 pg per cubic centimeter, ethynodiol diacetate 0.1 pg per cubic centimeter, and norethindrone 0.1 and 10 pg per cubic centimeter (p = < 0.05). Longer incubation, however, allowed the oocytes to reach M” despite the presence of some of the steroids, but particularly with 17~E’ and norethynodrel or combinations, progress was limited to abnormal diakinesis. Structural abnormalities were infrequent at M’ with a predominance of clumping and atresia. Two examples of a fragmented M’ in short-term culture were seen with norethynodrel 10.0 pg per cubic centimeter. Table IVB demonstrates that long-term culture merely increased the number of cells capable of reaching M’ with failure at this stage seen cytologically as clumping and atresia. Many cells proceeded to a normal
i
-
47 7 28 13 37
M2 but again clumping and atresia were seen. No anaphase, spindle or polar body abnormalities were observed. The cytologic effect on oocytes incubated for 14.5 hours after removal from female mice treated for only 4 days with 0.05 to 1.0 pg per mouse per day subcutaneously of mestranol is shown in Table V. No significant effect on ability to re-enter meiosis or on structure was noted, although a greater number of M’ stages were seen in oocytes from animals given 0.1 pg per day than controls. The chronic administration of each progestogen alone and in combination with mestranol was followed by assessment of in vitro meiotic progression (Table VI). Suppression of the ability to divide was noted in oocytes removed from mice treated with mestranol 10.1 to 10 mg. per day’!, ethyno-
-
6
Volume Number
120 3
Oral
contraceptives
and
oocyte
Metaphase”* N 31 6 28 13
1
A
1
1
C -
1
D -
Ab -
-
7 8 14 4
-
-
-
-
4
-
-
8 13 8
-
-
-
-
1 1 -
-
-
13 17
-
-
-
9 9
1 -
-
-
6 4
-
2 9
-
-
1 -
-
-
4 -
-
14 -
-
-
-
-
-
10 3
-
7 -
-
-
-
-
-
16 6
-
-
1 3
-
-
Me 17 11 17 6
p value
-
Table V. The cytologic effect of in vitro mouse oocyte meiosis studied after 4 days of parenteral mestranol treatment Dose ~g/SC/M/D 0.0 0.05 0.1 1.0
r
N
E -
Ana.l 1
403
B -
-
1
meiosis
Total eggs 23 18 46 18
No. div. 19 15 33 10
% division 83 83 72 56
diol diacetate 1.0 pg, norethynodrel 1 .O and 4.0 pg, and mestranol 0.5 pg per mouse plus norethynodrel 4.0 pg (p = < 0.05). A shift to the M’ stage was noted with the prior administration of mestranol alone or with norethynodrel. A few significant structural abnormalities were detected and atresia was marked with mestranol plus norethynodrel. The only instances of suppression of polar body were seen in these experiments where
iu’ 2 3 16 4
3
24 hours
A’ 1
< 0.9 0.35 0.062
very large doses of mestranol (5 mg. per mouse per day) were given. With regard to the human female, the results from this small retrospective experiment revealed that 57 out of 174 oocytes divided (32.8 per cent) compared with 35.4 per cent of oocytes from an age matched group from the same pool of surgical patients who by history had not taken oral contraceptives. There were no structural or
404
Jagiello
Table
and
VI.
Effect on in vitro
Contraceptive incubation duration No therapy 0.08 c.c./M Mestranol (ms.)
Ethynodiol (ag/GBW)
Octobrl 1, I+74 Am. J Obstet. Gynml
Lin
ethanolt 0.01 0.1 1.0 5.0 10.0 diacetate
M/12D M/12-l6D M/12-16D M/16D MA2D 0.02 0.25 1.0
16D 16D 16D
0.1-0.5 1.0 2.0 4.0 Mestranol 0.1 pg/M + ethynodiol diacetate 0.02 Irg/GBW 17D MestranolO.05 ag/M + norethynodrel 0.5 ( ag/GBW ) 4.0 ~Solvent
t:
For
16D 16D 16D 16D
17D 17D
explanation
for injected
Fig. 3. “Other” formation ~1,250.)
progression of mouse oocytes recovered ~~_ -__..___-.-.._ ___-... Early diak.
therapy before (SC dose + in days, D)
Norethyriodrel (&GBW)
* and
meiotic
with
of abbreviations
N
!
20 54
19 48
95 89
-. -
22 62 134 84 28
16 42 58 56 19
73 68 43 67 68
.-
14 20 57
14 15 37
100 75 65
-
16 35 14 49
15 22 14 23
93 63 100 47
-
41
33
81
60 62
45 34
75 55
see footnote
to Table
parenteral
Metaphase” (No. oocytes)
---
/ Ab.
after chronic
N 7 5 8 8 43 35 6
/
A
-. -. _. -
/
B
-. -. 1 -
12
-.
5 3
-
-
-
6
-
-
2 3
20 13
-
-
7 1
/
C
1
D
-
-
1
-
-
-
-
-
-
-
IA
steroids.
classes resultant
of abnormalities: a, tripolar spindle mixture of Ms/PBl chromosomes
disjunctional abnormalities seen in the oocytes or ova of either group. One cell with a mixture of M2/PB1 was seen in the control
grOUP* Comment There was considerable species variation in the in vitro response to the contraceptive
(cow) ; b, failure (sheep). (Original
of polar body1 magnification
compounds studied. Since the basic question concerns the relevance of the data to the production of viable abnormal progeny, the evaluation of the structural abnormalities such as fragments with rearrangements, spindle effects in which a single chromosome was lagging and suppression of first polar body formation must be considered prime.
/
E
Volume Number
120 3
Oral
contraceptives
-
N
and oocyte
meiosis
405
administration of contraceptive compounds Metaphase** (No. ova) N
1
12 43
c
A
)
B
)
Ana. C
-
-
308 -15
-
14 3
-
3
7 :“3
5
z
15 17 14 19
-
27
-
22 17
-
-
-
1
D
)
E
-
-
-
-4
-
2 5
_
-_
/
Other? Ab.
A
/
B
-
-
-
-
-
3 -
-
--
-
2
-
--
2
-
-
-
-
-
r
-
-
-
-
Clearly, effects of contraceptives which result in clumping, atresia or other cytologic appearances immediately preceding or consistent with nonviability are of no concern. The genetical literature supports the concept that each of the other cytologic alterations can have significant effects on derived progeny. The induced breakage with reunion and rearrangement of gamete chromesomeshas predictable consequencesfor the transmission of balanced and unbalanced gametes. These are well documented for many species including mouse,24 sheep,25 COW,~~ and man. 27For man, the pedigreesof D/G and D/D translocation families as well as X-autosomal translocations well exemplify such consequences. Chromosome lagging at anaphasealso has a demonstrated base in classical genetic thought= as a prime mechanism of nondisjunction with production of monosomy for 1 chromosome complement. That these can be abnormal but viable human progeny is well documented in the instance of the
-
1
human X0 complement.2g The significance of polar body suppressionwith the production of triploid and tetraploid embryos has been pointed out by Carr,l Hamerton,29 and others30 These 3 types of abnormalities were seenin a low frequency in the in vitro studies with cow, sheep, rhesus and baboon, and even fewer in the in vitro or combined in viva/vitro studies with mouse and human oocytes. But even this must be considered in the light of the internal inconsistencies (i.e., variations of effects for a single speciesin the 2 media) and the magnitude of the concentrations of each compound compared to estimated concentrations in the total organism achieved by ingestion of the currently available pharmaceutical compounds. Combined with the other observations on mouse, rhesus and human female oocytes it cannot be accurately claimed that use of oral contraceptives will result in direct changes in the chromosome constitution of oocytes known to produce abnormal progeny,
406
Jagiello
and
Lin
A consideration of the in vitro data of cow, ewe, and baboon, which demonstrate inhibition of division in the presence of some of the steroids and the in viva/in vitro data of mouse both coupled with atresia, would suggest that an acceleration of the natural rate of oocyte atresia in these species might be expected. As a screening system for detecting chromosome damage during meiosis, the cow or ewe oocyte was more vulnerable to changes other than simple inhibition of progression or atresia than the others tested and this coupled with the similarity of their normal reproductive endocrine environment (luteal
phase. LH-induced ovulation, human female suggest their purposes.
etc.) to the use for such
The authors are grateful to the G. D. Searle Company and the Ortho Pharmaceutical Corporation for generous supplies of rnestranol, ethinyl estradiol, norethynodrel, ethynodiol diacetate and norethindrone, and to the Laboratory for Experimental Medicine and Surgery in Priniates for baboon ovaries. We are also grateful to the attending, resident, and nursing staffs of the Department of Obstetrics and Gynecology of Columbia University, College of Physicians and Surgeons, for permitting study of human material.
REFERENCES
1.
Carr, D. H.: Canad. Med. Ass. J. 103: 343,
1970. 2: 830, 1967. 2. Carr, D. H.: Lancet rewrt. Medical Division. 3. Goh. K.: Research Oakridge Associated cnivkrsities, Springfield, 1967, Clearing House for Federal Science and Technological Information, p. 97. 4. McQuarrie, H. G., Scott, C. D., Ellsworth, H. S., Harris, J. W., and Stone, R. A.: AM. J. OBSTET. GYNECOL. 108: 659, 1970. 5. Kennelly, J. J.: Biol. Reprod. 1: 282, 1969. 6. Noske, I., and Davis, B. K.: Acta Endocrinol. 69: 617, 1972. G.: Cytogenetics 4: 245, 1965. 7. Jagiello, 8. Jagiello, G. M., Miller, W. A., Ducayen, M. B., and Lin, J. S.: Biol. Reprod. In Press, 1974. G., Ducayen, M., Miller, W., Lin, 9. Jagiello, J. S., and Fang, J. S.: Humangenetik 18: 117, 1973. 10. Jagiello, G., Karnicki, J., and Ryan, R. J.: Lancet 1: 178, 1968. 11. Rao, P. N., and Engelberg, J. Exp. Cell Res. 48: 71, 1967. K. I. H.: Steroids 13: 539, 1969. 12. Williams, 13. Abdel-Aziz, M. T., and Williams, K. I. H.: Steroids 13: 809, 1969. 14. Lee, S., and Chen, C.: Steroids 18: 565, 1971. 15. Freudenthal, R. I., Cook, Cl. E., Twine, M., Rosenfeld, R., and Wall, M. E.: Biochem. Pharmacol. 20: 1507, 1971. 16. Baggett, B., Hall, R. H., Boegli, G., Palmer,
17.
18.
19. 20.
21.
22. 23. 24. 25. 26. 27.
28.
29. 30.
K. H., and Wall, M. E. : Fertil. Steril. 21: 68, 1970. Palmer, K. H., Ross, F. T., Rhodes, L. S., Baggett, B., and Wall, M. E.: J. Pharmacol. Ther. 167: 207, 1969. Cook, Cl. E., Twine, M. E., Tallent, C. R., Wall, M E., and Bressler, R. C.: J. Pharmacol. Ther. 183: 197, 1972. Laumas, K. R., Murugesan, K., and Hingorani, V.: Acta Endocrinol. 88: 385, 1971. Laumas, V., Malkani, P. K., and Laumas, K. R.: AM. J. OBSTET. GYNECOL. 109: 457, 1971. Cook, C. E., Karim, A., Forth, J., Wall, M. E., Ranney, R. E., and Bressler, R. C.: J. Pharmacol. Ther. 185: 696, 1973. Baker, T. G. : Advances Biosci. 6: 7, 1971. Tarkowski, A. K.: Cytogenetics 5: 394, 1966. Cattanach, B. M.: Zeit. Vererbunglehre 92: 165, 1961. Brutre, A. N.: Cytogenetics 8: 209, 1969. Gustavsson, I., Fraccaro, M., Tiepolo, L., and Lindsten, J.: Nature 218: 183, 1968. Cohen, M. M., Lin, C. C., Sybert, V., and Orecchio, E.: Am. J. Hum. Genet. 24: 585, 1972. John, B., and Lewis, K. R.: The Meiotic System, Vienna and New York, 1965, SpringerVerlag. Hamerton, J. L.: Human Cytogenetics, New York, 197 1, Academic Press, Inc., vol. II. Rothe, D. J., and Simpson, J. L.: Lancet 2: 1391, 1973.
S.
JAGIELLO,
M.D.
LIN
York,
New
York
A meiotic study of the in vitro and in vivo effects of components of currently used oral contraceptives on the oocytes of 6 female mammalian species was made. In vitro inhibition of division and atresia were found in ,several species with both the estrogens and progestogens, as well as a low frequen&y of chromasome breaks and rearrangements, anaphase lags, spindle abnormalities, and polar body suppression. Combined in vivolin vitro testing revealed a possible basis for expecting increased atresia of treated oocytes. Considerable species variation and the concentration of the steroids tested do not permit clear extrapolation to a &aim for a direct action of oral contraceptives on human oocytes. The sensitivity and reproductive endocrinology of the Bovidae in these experiments suggest these as a model for female mammalian meiotic mutagen studies.
A c E RTA I N unrest has been attached to the use of the estrogen-progestogen oral contraceptives since the reports in 1970 of Car+ * that, following their use, the incidence of triploid abortuses was increased. Coupled with scattered papers demonstrating in vivo cytogenetic abnormalities, including breaks and rearrangements33 4 and animal data which document interference of mestranol with embryo survival in dogs5 and impaired egg development in the golden
hamster” treated with the metabolite of mestranol, ethinyl estradiol, study of the effects of the separate components of the currently used oral contraceptives on female mammalian oocyte meiosis seemed overdue. Direct assessment of the chromosomes of 6 species of female mammalian eggs including man as they proceeded through first meiotic division was undertaken. The possibility of developing a model system for extrapolation to a mutagen screening test for human meiosis was present since the reproductive endocrinology of the higher animals used resembles in many ways that of man.
From the Department of Obstetrics and Gynecology, the Department of Human Genetics and Development, and the International Institute for the Study of Human Reproduction, Columbia University College of Physicians and Surgeons. Supported by National FoundationMarch of Dimes Grant CRBS-249, National Institute of Child Health Human Development Grant 5ROl 05862, and the Ford Foundation. Received 1974.
for
publication
Revised February Accepted March
January
Material
and
methods
Cytogenetic analysis of first and second meiotic metaphase chromosomes was carried out on oocytes of mouse, ewe, cow, rhesus and baboon matured in vitro using techniques already described for these species7-” (Fig. 1). Mouse and human oocytes’” were also studied with a combination technique of in viva/in vitro progression. The specific techniques varied from species to species and will thus be described for each. In all cases, resting oocytes were manually removed from
and -HD31,
21, 1974. 6, 1974.
Reprint requests: Dr. Georgiana Jagiello, College of Physicians and Surgeons, 630 W. 168th St., New York, New York 10032.
390
Volume 120 Number 3
Oral
contraceptives
and
oocyte
meiosis
391
b
d
Fig. 1. Normal control sheep Ml; d, cow Ms; ovarian
consisting Diploid” additives, *Grand York.
meiotic preparations: cr, mouse oocyte e, baboon Ml; f, rhesus Ms. (Original
follicles, pooled, incubated in media of McCoy’s 5A modified,* or with or without serum and steroid in a 5 per cent CO,/air atmoIsland Biological
Company,
Grand
Island,
New
Ml; b, mouse oocyte magnification ~1,250.)
M2;
c,
sphere with high humidity and at a constant pH of 7.2. Preliminary experiments provided minimum effective doses of each steroid for each oocyte species. These were initially estimated from the concentrations contained in the commonly used tablets on a weight
392
Jagiello
Table -
IA.
and
Lin
vitro
In
cytologic ~
Media Diploid Ethanol
(@g/cc.) estradiol
Norethynodrel (PP/C.C.) Ethynodiol (PdC.C.)
No. div.
% j div.
74 “7 19 46
66 “1 16 39
89 78 84 84
-
38 33 44 36 14
30 25 17 32 l:!
79 79 39 89 86
-.
0.1 10.0
22 34
19 26
86 76
0.1 10.0
56 25
47 19
84 76
34 41
27 30
39 39 28 27
0.005 0.0005 7.41 ( 1 7a-E2) 0.74 7.41 74.1 0.1 10.0
N
17a-Es 7.41 + norethynodrel 0.1 10.0 (PdC.C.) 17a-Es 7.41 + ethvnodiol diacetate 0.1 10.0 17a-E2 7.41 pg/c.c. + 0.1 norethindrone 10.0 @g/c.c.)
(&c.)
*N
=
tA
= spindle
Normal
in structure abnormalities;
and
number;
B = failure
A =
Raritan, Illinois.
New
Metaphase’” (No. oocytes) N
/
8 2 1 I
-
A -
1 B -
1 C
1 1 1
2 -
1 15 1
-
-~
-
1 D
-
-
3 11
2
2
-
-
-
-
4 4
1
-
-
-
79 73
-
9 -
8 13
-
3 -
1
-
32 32
8’ 82
-
-
4 5
3 -
-
-
1 -
21 19
75 70
-
1
6 4
-
-
-
-
body
Jersey.
rearranged;
formation
(mixture
B
=
clumped;
C
=
fuzzy,
indefinite
_---/ E
-. -
-
basis of 70 kilograms for an adult human female. Other guidelines for concentrations were adapted from the literature on mitotic effects of estrogen. I1 The constituents of 3 commonly used oral contraceptives-OrthoNovum 2,” Ovulen,t and Enovid-Et and their significant metabolites were studied. These were mestranol and its metabolite 17cy-ethinyl estradiol ( 1 7a-E2) ,12-14 norethynodrel,15-zo ethynodiol diacetate (ED) and norethindrone, its metabolite.21 These were dissolved in ethanol, diluted with serum-free media and added in appropriate concentrations to the final culture media. In most instances, both media were studied to eliminate spurious effects resulting from metabolic components of the serum-free media or potential binding of the test steroid to *Ortbo Pharmaceutical Corporation, tG. D. Sea& & Company, Chicago,
rnetaphase
-
fragmented,
of polar
/ Ab
-
diacetate
Norethindrone (cLg/c.c.)
oocyte
diak.
Total eggs
+ additives
(c.c./c.c.)
Mestranol 17ru-ethinyl (PdC.C.)
effect of oral contraceptives on cow --...-~-~. I Early
outlines;
of M* + PB’ chromosomes).
serum. Media selection was based on previous experience of meiotic analysis with each species. Cow and ewe. Ovaries were harvested from random-bred diestrous exsanguinated cows and ewes at a local slaughterhouse, washed in penicillin/streptomycin, and transported rapidly under sterile conditions to the laboratory. Oocytes were manually removed with sterile precautions with a Zeiss dissecting microscope, pooled, and immediately placed in the media. Cultures of cow oocytes were carried out for 28 hours in Diploid media which contained 10 per cent fetal calf serum and in serum-free McCoy’s modified 5A. Harvests at this time interval yielded eggs in both first and second metaphase. Ewe oocyte cultures were made in McCoy’s 5A and in Diploid containing an additional 12 per cent sheep serum, making a total concentration of 22 per cent fetal calf
D
Volume Number
120 3
Oral
contraceptives
and
oocyte
MetaphaseP* (No. ova) N
1
A
1
B
1
meiosis
Othert C
1
D
i
E
A
1
B
58 19 15 34
-
1
3
-
-
-
-
26 17 26 10
2 -
-
-
1
4 -
-
2 -
-
13 9
1
-
-
1 -
2 1
-
-
38 10
-
-
-
-
3
2 -
4
4 10
-
-
2 4
-
-
-
1 2
18 13
1
-
6
-
5
4 2
2 10
-
-
-
-
12 2
1
mlverized;
E = atretic,
advanced
393
1
-
-
1
“C.”
serum (FCS) . These were harvested at 26 hours for first and second metaphase stages. Rhesusand baboon. Restricted availability of ovaries from these species prohibited completely parallel experiments for comparison with the Bovidae and mouse. The evolutionary position of these species made an attempt mandatory and information for selection of effective concentrations of steroids was derived from the other studies. Oophorectomy was carried out rapidly using Sernylan anesthesia on adult, cycling females. Eggs were removed from the ovaries as above and incubated in McCoy’s 5A with 12 per cent FCS. Ten international units of human chorionic gonadotropin (HCG) was added to the cultures of rhesus oocytes. These media and the harvest time of 28 to 30 hours had been previously demonstrated to yield a good division rate for harvesting both desired stages.
Mouse and human female. Extensive studies were carried out with the randombred Camm* strain mouse. For in vitro study, 40 to 60 day old diestrous females were used and oocytes incubated in McCoy’s 5A with 40 per cent FCS for 4.5 to 6 hours to obtain cells in first metaphase and 12 to 15 hours for second metaphase. In addition, 0.05 to 1.0 pg of mestranol per mouse per day was given subcutaneously for 4 days (1 vaginal cycle) followed by in vitro incubation for 14 hours of oocytes recovered 24 hours after treatment. Parenteral administration of 0.01 to 10 mg. of mestranol per mouse per day for 12 to 16 days, 0.02 to 1.0 pg per gram of body weight per day (GBW) of ethynodiol diacetate for 16 days, norethynodrel 0.1 to 4.0 pg per gram body weight for 16 days, 0.05 pg per mouse of mestranol ‘Camm
Research
Institute,
Wayne,
New
Jersey.
394
Jagiello
Table
IB.
and
In
Lin
vitro
effect
of oral
on COW oocytr
contracc.I,ti\rcs
metaphasc
Early/ diak.
Media McCoy’s 5A Ethanol (c.c./c.c.) Mestranol (pg/c.c.)
(cdc.c.)
A/BiCIDjE
+ additive-s 0.0005 0.001 0.005 7.41
17cu-ethinyl estradiol ( 17a-E2) 0.74 (Fg/c.c.) 7.41 74.1 Norethynodrel 0.1 10.0 (ag1c.c.) Ethynodiol diacetate 0.1 (PdC.C.) 10.0 Norethindrone 0.1 (PdC.C.) 10.0 17a-E2 7.41 fig/cc. + norethynodrel 0.1 10.0 (cg1c.c. ) 17~~E2 7.41 &c.c. + ethynodiol diacetate 0.1 10.0 (Ilg/c.c.) 17sE2 7.41 cg/c.c. norethindrone
Metaphase’” (No. imytes)
:
+ 0.1 10.0
* and t: For explanation
of abbreviations,
194 45 “3 45 30
164 39 1x 38 22
8.5 87 79 84 73
64 122 28 69 85
53 88 27 63 63
82 72 83 91 74
106 149
87 128
82 86
33 ‘1 1
28 34
85 83
61 70
48 6.5
79 93
5L’ 62
49 51
94 82
43 40
39 31
90 77
see footnote
to Table
combined with norethynodrel 0.5 or 4 pg per gram body weight for 17 days, and mestranol 0.1 pg per mouse plus 0.02 lug per gram body weight of ethynodiol diacetate for 17 days followed 24 hours later by in vitro incubation of oocytes for 14 hours to obtain second metaphase ova was carried out. The parenteral schedules were designed to treat one complete developmental cycle of mouse oocytes as determined by Bake? and to parallel by weight in mouse the in vivo doses used in human subjects. Human female oocytes were recovered from ovarian material obtained from patients aged 24 to 51 as part of the course of indicated pelvic operation, principally hysterectomy. Initially all surgical material was studied, oocytes removed from all usable specimens, placed in Diploid medium with added 40 per cent fetal calf serum and harvested after 30 to 35 hours of culture.
/I
-
-
1 .~ .-
_~ -
..-
-
.-
-
1 18 -. 1
-
2 2
.
9 12 11 8
4 3
5 1 .-
1 29
17 ‘10
2 1
1
1 2
1
4 4
-
-
-
3
1 19
35 30
1 2
-
2 4
-
3 1
13 33
24 -
3 -
7
2 6
-
-
31 16
-
6 6
11 -~ 3 -
-
-
1 15 26
40 9 6 8 6
-
-
IA.
Those ovarian specimens subsequently shown to be without histologically detectable pathology were assessed for meiotic complements. The use of oral contraceptives by the patient prior to operation was determined from a retrospective examination of the chart and depended entirely on the clinical history. Type, dose and duration varied widely. Techniques for cytogenetic harvest and preparations were uniform for all species. A modification of the method of Tarkowski2” was used to prepare the oocytes and ova for cytologic examination of the chromatids and chromosomes. Initial staining was made with 1 per cent toluidine blue in resin and permanent preparations were made with 10 per cent Giemsa stain. Microscopic assessment of number and structure of both first and second metaphase was made at ~1,250 with oil immersion using a Zeiss photomicroscope
Volume Number
120 3
Oral
contraceptives
Metaphase”+ (No. ova)
and
Ana.
N(AIB(C)DIE -
-
1
.-
27 20 44 27
-
1 :
-. 1 8
-
52 53
1 -
4 8
-
23 10
-
-
3 2
-
1
1 1
‘9
5 1 1 2
-
‘> -
1 2
1
7
1 3
-
2 5
1 3
.-
-
10
1
3 -
-
1
1 -
.-
-
-
-
: -
1
meiosis
395
Other?
NIAb
116 29 11 28 10
oocyte
A/B -
-
-
8 1 -
1
2 2
4 2
3
-
1
1 5
1 2
-
-
-
2 -
-
1
-
-
-
-
-
-
-
-
-
1
-
and optics. Confirmation of these observations was made with photomicrographs taken at x1,250. Results The cytologic effects of the constituents of 3 commonly used oral contraceptives on the meiosis of the mammalian oocytes studied have been assessed in terms of (a) numbers of oocytes maturing (per cent) to the first or second metaphase stages at the appropriate harvest time, (b) normalcy of number and structure of bivalents (Ml) or chromosomes (M*) , and (c) types of induced structural abnormalities annotated by the number of eggs observed at each meiotic stage in the tables under the headings A = fragmented and/or rearranged; B = clumped but with sufficient structural integrity to be recognizable as to meiotic stage; C = fuzzy, indefinite outlines to either bivalents or
chromosomes but with a normal number of each; D = pulverized or shattered into innumerable parts and reminiscent of structural damage seen with virus infections in mitotically dividing cells, and E = atretic, an advanced stage of “C,” all shown in Fig. 2. Anaphase 1 was assessed as normal with complete separation of the 2 chromosome complements from the equatorial plate or abnormal with 1 or more chromosomes distinctly lagging on the mid-body (Fig. 2). In addition, multipolar divisions seen following first metaphase connoting spindle abnormalities were classed under “other” as “A” and failure of first polar body formation with resultant mixture of the second metaphase and polar body chromosomes was scored as “B” throughout (Fig. 3). Cow and ewe. The in vitro cytologic effects on cow oocytes of mestranol, 17~E’, norethynodrel, ethynodiol diacetate and
396
Jagiello
Table
and
II.
In
Media
Lin
vitro
cytologic
effect
(Pg/c.c.) (P&.C.)
t:
Far
contraceptives
on
~111’
metaphase
oocyte
+ additives
Diploid + sheep serum 12% Ethanol 0.0005 (c.c./c.c.) Mestranol 7.41 (Pg/c.c.) 17a-ethinyl estradiol ( 1 7a-E2) 0.74 (PdC.C.) 7.41 74.1 Norethynodrel 0.1 10.0 b4dc.c.) Ethynodiol diacetate 0.1 (/Lg/c.c.) 10.0 Norethindrone 0.1 (PdC.C.) 10.0 17a-E2 7.41 + norethynodrel 0.1 10.0 (adc.c.) 17~E’ 7.41 + ethynodiol diacetate 0.1 @g/c.c.) 10.0 17~2-E” 7.41 + 0.1 norethindrone 10.0 (&/C.C.) McCoy’s 5A 0.0005 Ethanol (c.c./c.c.) 7.41 Mestranol (ELg/c.c.) 17cu-ethinvl estradiol i 17a-E2) 0.54 ' 7.41 74.1 0.1 Norethvnodrel 1.0 10.0 Ethynodiol diacetate 0.1 (Pg/c.c.) 1.0 10.0 0.1 Norethindrone 10.0 (PLp/C.C.) 17a-EZ 7.41 f norethynodrel 0.1 10.0 k/c.c.) 17a-E2 7.41 + ethynodiol diacetate 0.1 (Bg/c.c.) 10.0 17a-E2 7.41 + norethindrone 0.1 (pg1c.c.) 10.0 ‘* and
of oral
rxplanation
of abbreviations,
79 38
55 :10
70 79
14
12
86
34 43 5 24 "3
'6 26 1 17 16
76 60 20 71 69
46 40
19 29
41 7"
29 34
13 19
45 56
29 43
"4 34
3" is
-
-
__~
-
4 5r,
-
3
-
-
-
-
-~
-
-
9 3 -
1
-
-
-
2
-
-
1 -
-_ .-
14 1 3
-
-
4 -.
-
1 2
2
-
1 11
-
2 -
1
-
2
I
.-
3 1
83 79
-
-
1 2
2 -
-
4 -
.~ -
1 5
25 19
79 73
-
-
4 1
-
-
2 -
-
4 2
28 33 66 39
18 16 62 28
64 48 94 72
-
4 3
-
-
-
: 7 1
23
16
70
-
2
2
-
-
44 42 19 18 28 32
31 28 18 16 17 24
71 50 95 89 61 75
4 2 1
-
-
'7 30 33 35 30
22 2' 21 28 23
81 73 64 80 77
1 8 1
30 32
24 23
27 31 25 33 see footnote
-
1 3
1
-
11 10 -
3 -
-
2 2
1 1 4
5
-
-
1 4 -
_-
5 2
19 1 1
1
1 1 -
-. -
2 4
80 72
1 10
15 2
1 1
1
2 4
-
2 3
20 23
74 74
10 16
5 -
4
-
1
-
L' -
10 23
40 70
1 3
-
1 -
3 .-
2
-
1 19
to Table
IA
9 18 1
-
1
-
Oral
contraceptives
MetaphaseP* (No. ova) N
B
E
N
49 25
-
-
-
-
2 1
-
-
9
-
-
-
-
-
-
-
-
-
-
-
-
-
I 1
1 -
-
-
-
-
__
12 6
1
D
(
1 1 8
-
(
397
Other+
Ana.’ C
meiosis
A
15 9
1
oocyte
-
16 I -
(
and
Ab.
-
-
1
-
5 3
-
-
-
-
1 2
-
6 18
-
1 -
2 2
-
1 1
-
8 13
1
-
1
1
1
-
-
5 3 48 25
2 -
-
4 1 -
1 1 2
7
-
-
-
3
-
-
-
-
-
4 -
-
-
-
-
2
1 1
3 -
-
3
4 4
-
1 -
-
-
-
-
-
1 -
-
-
-
-
14 -
-
9 IO 9 1 17 6 13 6
1 2 1
-
2 -
-
-
A
(
B
-
-
4 -
2 -
-
1
-
2
-
-
1 -
-
-
2
1 -
1
3 -
-
1 -
-
3 1
-
-
-
-
-
-
-
-
-
-
__ -
-
1 -
398
Jagiello
and
III.
In vitro
Table
Lin
cytologic
effect of oral contraceptives
on rhesus
and baboon
oocyte
metaphas~
-Media
+ additives
A. Rhesus data McCoy’s 5A + fetal calf serum 12% + HCG IO I.U. 0.005 Ethanol (PdC.C.) Mestranol 7.41
22 7
,42 58
1 -
-
10 ‘,
-
._
12 21
7
33
-
-
1
-
-
( 17a-E2) 7.41 0.1 10.0
“0 6 3
9 4 3
45 67 100
-
-
4 1 1
1
0.1 10.0 10.0
21 20 18
7 6 11
33 30 60
-
1 -
2 2 1
4 8
3 4
75 50
-
1 2
8 18
2 5
-
-
8
3
38
-
-
24 11 6 9 18
17 5 4 2 3
71 45 67 22 16
-
14 14
4 5
28 36
12 21
5 12
42 57
5’J
-
-
.-
1
-
2
-
2 -
-
2
-
-
4
3
3 -
-
-
-
-
-
-
-
(pg/c.c.) 17sethinyl (Pg1c.c.) Norethvnodrel
estradiol
(P3YC.C.) Ethynodiol (P!dC.C.)
Norethindrone (pg,‘c.c.) 17n-E2 7.41 + norethynodrel 0.1 10.0 (P.5dC.C.) 17sE2 7.41 + ethynodiol 0.1 diacetate 10.0 (&/C.C.) 17~~E” 7.41 + 10.0 norethindrone (Pg/c.c.) B. Baboon data McCoy’s 5A t fetal calf serum 0.1 Norethynodrel 10.0 (PLs/C.C.) Norethindrone 0.1 10.0 (!%/c.c. ) 17a-ethinyl estradiol ( 17a-E2) 7.41 + norethynodrel 0.1 (PdC.C.) 10.0 17sE3 7.41 + norethindrone 0.1 10.0 (pg/c.c.) * and
t:
-
2
diacetate
For
explanation
25%
of abbreviations,
see footnote
norethindrone are shown in Tables IA and IB. Diploid medium which contains 10 per cent fetal calf serum supported a high rate of normal meiotic maturations (89 per cent), as did McCoy’s 5A modified medium which yielded 85 per cent divisions. Maximal concentrations of ethanol solvent did not interfere with this in either series. Statistically significant restriction of division was seen in Diploid with 7.41 pg per cubic centimeter of 17~E” with norethynodrel 10 pg per cubic centimeter and 17~EZ 7.41 pg per
-
to Table
-
1
1 1
1 2 2 3
-
-
-
-
1 -
-
-
‘I
-
-
-
-
-
8
-
-
-
-
-
IA.
cubic centimeter plus norethindrone 10 t% per cubic centimeter (p value = < 0.05, chi square test), and in McCoy’s with 7.41 pg per cubic centimeter 17~~E” and norethynodrel 10 pg per cubic centimeter (p value = < 0.05). In addition there was a restriction of development from M1 to M2 seen minimally in the Diploid but markedly in the McCoy’s 5A with the 17a-E2 and combinations of 17a-E” with all 3 progestogens. Very high concentrations (74.1 pg per cubic centimeter) of 17~E2 severely limited mei-
1
1 3 5 4
volume Number
120 3
Oral
contraceptives
Metaphase”* (No. oua) N
10 5
/
A
2 -
1
-
1
-
3
1
‘C
/
D
(
E
1
1
N
1
oocyte
/
meiosis
399
Othert
I Ab
A
/‘B
-
-
-
-
-
-
1
15 3 2 2
B‘
Ana.’
-
1 1
1
and
-
.-
-
-
-
1
-
-
-
-
-
-
2
-
-
-
-
T
1 1
-
otic progression in Diploid (39 abnormal Ml’s) or to abnormal diakinesis in McCoy’s 5A. Scattered instances of fragmentation and rearrangement of bivalents were seen in both experiments at M1 but were without significant incidence of cytogenetic sequelae such as nondisjunction or anaphase bridging in later stages. Fuzziness of bivalents at Ml, particularly marked with 17a-E2 in both media did not have later detectable effects and cell death characterized by clumping or obvious atresia predominated among the
abnormalities at M’. Of particular interest from the genetic viewpoint were the 2 instances of induction of lagging at anaphase’ by norethynodrel (0.1 pg per cubic centimeter) and ethynodiol diacetate (10 pg per cubic centimeter) in McCoy’s as well as production of spindle abnormalities and failure of polar body1 formation, particularly with the progestogens (Tables IA and IB) . Utilizing ewe oocytes as the test cell in both media systems (Table II), it was noted that the control division rate varied from 70
400
Jagiello
and Lin
Table IVA. In vitro cytologic
effect of oral contraceptives
on mouse oocyte first
metaphase
McCoy’s 5A + fetal calf serum 40% Ethanol 0.0005 (c.c./c.c.) 0.0025 Mestranol 7.76 (PgYC.C.) 17cu-ethinyl estradiol ( 17a-E*) 0.74 (&/C.C.) 7.41 74.1 Norethynodrel 0.1 10.0 (cg/c.c. ) Ethynodiol diacetate 0.1 hg/c.c.) 10.0 Norethindrone 0.1 10.0 ( pg1c.c. ) 17~~E2 7.41 + norethynodrel 0.1 10.0 h!x/c.c.) 17~E2 7.41 + ethynodiol diacetate 0.1 (PdC.C.) 10.0 17a-E2 7.41 + norethindrone 0.1 10.0 Wc.c.) *For
explanation
of abbreviations,
-
5
66 19 13 16
-
I
-
--
1 5
-
-
1 -
-
-
4 -
-
-
24 52 10 12
-
61 67
-
23 19 16 17
77 76 50 39
-
3
22 14 12 5
-
-
1 -
4 ‘) -
19 62
3 32
47 52
-
-
24
6
-
9 -
0
-
2:! 57
9 32
41 56
-
-. -
-
5
28
4 -
3
39 19
18 6
46 31
-
13 1
-
-
-
5 1
69
26 51
67 19 23 27
25 73 16 18 21
25 56 11 14
100 77
30 “5 32 44
97 30
see Table
63 88 53
4
1 -
2 9
1 ?i
IA.
tFragmerited.
per cent in the Diploid with added 12 per cent sheep serum to 94 per cent in McCoy’s 5A (Table II). The division rate in Diploid was significantly reduced by 74.1 pg per cubic centimeter of 17~E’, ethynodiol diacetate 0.1 pg per cubic centimeter, norethindrone 0.1 pg per cubic centimeter, and 17~E? 7.41 pg per cubic centimeter plus norethindrone 10 pg per cubic centimeter (p values = < 0.05). Restriction of development to M1 was noted primarily with the combination of 17a-E* and norethindrone 10 pg per cubic centimeter. Atresia was the predominant effect noted in Diploid but a few spindle and polar body abnormalities were seen in the combination trials. In contrast, the same study conducted with McCoy’s 5A with serum (Table II) showed a severe restriction of division with almost
all compounds. The largest dose of permitted dissolution of the germinal vesicle and reappearance of the diplotene state but this progressed only to badly damaged diakinesis. The smallest concentrations of norethynodrel and ethynodiol diacetate did not inhibit progression. Breaks with rearrangements and considerable atresia were seen at Ml, and clumping and atresia in M2. As with cow oocytes, spindle and polar body abnormalities were noted with the progestogens. Rhesus and baboon. Rhesus data (Table III) demonstrated no statistically significant inhibition of division in the presence of any components of the oral contraceptives. Atresia was the predominant effect at M’ and M2. Only 1 instance of suppression of PB1 was seen with ethynodiol diacetate.
Volume Number
120 3
Oral
contraceptives
and
oocyte
meiosis
40’
b
c
f
a, fragmented and rearranged M’ Fig. 2. Representative abnormal cytologic configurations: (arrows indicate breaks and rearranged bivalants) (sheep) ; b, clumped M2 (sheep) ; c, fuzzy Ml with sticky bivalents (mouse); d, pulverized M2 (cow) ; e, atretic M1 (cow) ; f, late anaphasel (arrow indicates lagging chromosome) (sheep). (Original magnification ~1,250.)
The baboon oocytes (Table III) were more sensitive than the rhesus in terms of restriction of division. Norethindrone 0.1 pg per cubic centimeter, or 10 pg per cubic centimeter and 1 7a-E2 with norethynodrel 0.1 and 10 pg per cubic centimeter depressed progression (p = < 0.05). Again atresia was the prime abnormality produced and 3
examples of spindle misbehavior and PB’ suppression were found. Mouse and human female. In mouse the study of short incubation progression to M’ in the presence of oral contraceptive compounds (Table IVA) revealed restriction of division with 17a-E2 (0.74 and 74.1 pg per cubic centimeter), norethindrone 10 pg per
402
Jagiello
Table IVB.
and
Lin
In vitro
cytologic
effect of oral
contraceptives
I Media
*For
explanation
Early disk:
+ additives
McCoy’s 5A + fetal calf serum Ethanol 0.001 0.0005 (c.c./c.c.) Mestranol 0.74 7.74 b%/c.c.) 17sethinyl estradiol ( 17a-E2) 0.007 (pg1c.c. ) 0.074 0.74 7.41 Norethynodrel 0.1 (pg1c.c.) 1.0 10.0 Ethynodiol diacetate 0.1 (PdC.C.) 10.0 Norethindrone 0.1 (BdC.C.) 10.0 17cr-E* 7.41 + norethinodrel 0.1 10.0 (/Jg/c.c.) 17ru-E* 7.41 + ethynodiol diacetate 0.1 (Pg./C.C. ) 10.0 17&-E* 7.41 + norethindrone 0.1 10.0 (fig/=) of abbreviations,
N 40%
see Table
on mouse
-
oocyte
second
mrtaphasc
i Metaphn.re’*
1 Ab
/ N
i
A
73 78 1nn 59 70
.-
13 17 33 104
13 17 24 66
100 100 73 63
-
18
6 9 10 24
-. -
18 42 20
12 32 14
67 76 70
-
” 11 5
3
36 53
26 40
72 75
-
-
16 43
11 28
69 65
-
“8 15
25 8
89 53
31 49
24 31
25 i5
20 12
.~ 1
i
R
1 C
1 D
16 1 9 Iz-
I
-.~.
3 ‘I
-
-
-
7
3
-
--
-
-
6
-
2 7
1
-
-. -
1 5
-
1 10
-
-
-
1 4
5 4
-
1
-
-.
77 63
-
22
5 5
-
‘1
-
-
80 80
-
-
2
-
1 -
-
-
-
7
IA
cubic centimeter, and 17~E* with norethynodrel 10 pg per cubic centimeter, ethynodiol diacetate 0.1 pg per cubic centimeter, and norethindrone 0.1 and 10 pg per cubic centimeter (p = < 0.05). Longer incubation, however, allowed the oocytes to reach M” despite the presence of some of the steroids, but particularly with 17~E’ and norethynodrel or combinations, progress was limited to abnormal diakinesis. Structural abnormalities were infrequent at M’ with a predominance of clumping and atresia. Two examples of a fragmented M’ in short-term culture were seen with norethynodrel 10.0 pg per cubic centimeter. Table IVB demonstrates that long-term culture merely increased the number of cells capable of reaching M’ with failure at this stage seen cytologically as clumping and atresia. Many cells proceeded to a normal
i
-
47 7 28 13 37
M2 but again clumping and atresia were seen. No anaphase, spindle or polar body abnormalities were observed. The cytologic effect on oocytes incubated for 14.5 hours after removal from female mice treated for only 4 days with 0.05 to 1.0 pg per mouse per day subcutaneously of mestranol is shown in Table V. No significant effect on ability to re-enter meiosis or on structure was noted, although a greater number of M’ stages were seen in oocytes from animals given 0.1 pg per day than controls. The chronic administration of each progestogen alone and in combination with mestranol was followed by assessment of in vitro meiotic progression (Table VI). Suppression of the ability to divide was noted in oocytes removed from mice treated with mestranol 10.1 to 10 mg. per day’!, ethyno-
-
6
Volume Number
120 3
Oral
contraceptives
and
oocyte
Metaphase”* N 31 6 28 13
1
A
1
1
C -
1
D -
Ab -
-
7 8 14 4
-
-
-
-
4
-
-
8 13 8
-
-
-
-
1 1 -
-
-
13 17
-
-
-
9 9
1 -
-
-
6 4
-
2 9
-
-
1 -
-
-
4 -
-
14 -
-
-
-
-
-
10 3
-
7 -
-
-
-
-
-
16 6
-
-
1 3
-
-
Me 17 11 17 6
p value
-
Table V. The cytologic effect of in vitro mouse oocyte meiosis studied after 4 days of parenteral mestranol treatment Dose ~g/SC/M/D 0.0 0.05 0.1 1.0
r
N
E -
Ana.l 1
403
B -
-
1
meiosis
Total eggs 23 18 46 18
No. div. 19 15 33 10
% division 83 83 72 56
diol diacetate 1.0 pg, norethynodrel 1 .O and 4.0 pg, and mestranol 0.5 pg per mouse plus norethynodrel 4.0 pg (p = < 0.05). A shift to the M’ stage was noted with the prior administration of mestranol alone or with norethynodrel. A few significant structural abnormalities were detected and atresia was marked with mestranol plus norethynodrel. The only instances of suppression of polar body were seen in these experiments where
iu’ 2 3 16 4
3
24 hours
A’ 1
< 0.9 0.35 0.062
very large doses of mestranol (5 mg. per mouse per day) were given. With regard to the human female, the results from this small retrospective experiment revealed that 57 out of 174 oocytes divided (32.8 per cent) compared with 35.4 per cent of oocytes from an age matched group from the same pool of surgical patients who by history had not taken oral contraceptives. There were no structural or
404
Jagiello
Table
and
VI.
Effect on in vitro
Contraceptive incubation duration No therapy 0.08 c.c./M Mestranol (ms.)
Ethynodiol (ag/GBW)
Octobrl 1, I+74 Am. J Obstet. Gynml
Lin
ethanolt 0.01 0.1 1.0 5.0 10.0 diacetate
M/12D M/12-l6D M/12-16D M/16D MA2D 0.02 0.25 1.0
16D 16D 16D
0.1-0.5 1.0 2.0 4.0 Mestranol 0.1 pg/M + ethynodiol diacetate 0.02 Irg/GBW 17D MestranolO.05 ag/M + norethynodrel 0.5 ( ag/GBW ) 4.0 ~Solvent
t:
For
16D 16D 16D 16D
17D 17D
explanation
for injected
Fig. 3. “Other” formation ~1,250.)
progression of mouse oocytes recovered ~~_ -__..___-.-.._ ___-... Early diak.
therapy before (SC dose + in days, D)
Norethyriodrel (&GBW)
* and
meiotic
with
of abbreviations
N
!
20 54
19 48
95 89
-. -
22 62 134 84 28
16 42 58 56 19
73 68 43 67 68
.-
14 20 57
14 15 37
100 75 65
-
16 35 14 49
15 22 14 23
93 63 100 47
-
41
33
81
60 62
45 34
75 55
see footnote
to Table
parenteral
Metaphase” (No. oocytes)
---
/ Ab.
after chronic
N 7 5 8 8 43 35 6
/
A
-. -. _. -
/
B
-. -. 1 -
12
-.
5 3
-
-
-
6
-
-
2 3
20 13
-
-
7 1
/
C
1
D
-
-
1
-
-
-
-
-
-
-
IA
steroids.
classes resultant
of abnormalities: a, tripolar spindle mixture of Ms/PBl chromosomes
disjunctional abnormalities seen in the oocytes or ova of either group. One cell with a mixture of M2/PB1 was seen in the control
grOUP* Comment There was considerable species variation in the in vitro response to the contraceptive
(cow) ; b, failure (sheep). (Original
of polar body1 magnification
compounds studied. Since the basic question concerns the relevance of the data to the production of viable abnormal progeny, the evaluation of the structural abnormalities such as fragments with rearrangements, spindle effects in which a single chromosome was lagging and suppression of first polar body formation must be considered prime.
/
E
Volume Number
120 3
Oral
contraceptives
-
N
and oocyte
meiosis
405
administration of contraceptive compounds Metaphase** (No. ova) N
1
12 43
c
A
)
B
)
Ana. C
-
-
308 -15
-
14 3
-
3
7 :“3
5
z
15 17 14 19
-
27
-
22 17
-
-
-
1
D
)
E
-
-
-
-4
-
2 5
_
-_
/
Other? Ab.
A
/
B
-
-
-
-
-
3 -
-
--
-
2
-
--
2
-
-
-
-
-
r
-
-
-
-
Clearly, effects of contraceptives which result in clumping, atresia or other cytologic appearances immediately preceding or consistent with nonviability are of no concern. The genetical literature supports the concept that each of the other cytologic alterations can have significant effects on derived progeny. The induced breakage with reunion and rearrangement of gamete chromesomeshas predictable consequencesfor the transmission of balanced and unbalanced gametes. These are well documented for many species including mouse,24 sheep,25 COW,~~ and man. 27For man, the pedigreesof D/G and D/D translocation families as well as X-autosomal translocations well exemplify such consequences. Chromosome lagging at anaphasealso has a demonstrated base in classical genetic thought= as a prime mechanism of nondisjunction with production of monosomy for 1 chromosome complement. That these can be abnormal but viable human progeny is well documented in the instance of the
-
1
human X0 complement.2g The significance of polar body suppressionwith the production of triploid and tetraploid embryos has been pointed out by Carr,l Hamerton,29 and others30 These 3 types of abnormalities were seenin a low frequency in the in vitro studies with cow, sheep, rhesus and baboon, and even fewer in the in vitro or combined in viva/vitro studies with mouse and human oocytes. But even this must be considered in the light of the internal inconsistencies (i.e., variations of effects for a single speciesin the 2 media) and the magnitude of the concentrations of each compound compared to estimated concentrations in the total organism achieved by ingestion of the currently available pharmaceutical compounds. Combined with the other observations on mouse, rhesus and human female oocytes it cannot be accurately claimed that use of oral contraceptives will result in direct changes in the chromosome constitution of oocytes known to produce abnormal progeny,
406
Jagiello
and
Lin
A consideration of the in vitro data of cow, ewe, and baboon, which demonstrate inhibition of division in the presence of some of the steroids and the in viva/in vitro data of mouse both coupled with atresia, would suggest that an acceleration of the natural rate of oocyte atresia in these species might be expected. As a screening system for detecting chromosome damage during meiosis, the cow or ewe oocyte was more vulnerable to changes other than simple inhibition of progression or atresia than the others tested and this coupled with the similarity of their normal reproductive endocrine environment (luteal
phase. LH-induced ovulation, human female suggest their purposes.
etc.) to the use for such
The authors are grateful to the G. D. Searle Company and the Ortho Pharmaceutical Corporation for generous supplies of rnestranol, ethinyl estradiol, norethynodrel, ethynodiol diacetate and norethindrone, and to the Laboratory for Experimental Medicine and Surgery in Priniates for baboon ovaries. We are also grateful to the attending, resident, and nursing staffs of the Department of Obstetrics and Gynecology of Columbia University, College of Physicians and Surgeons, for permitting study of human material.
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