Journal of in Vitro Fertilization and Embryo Transfer, Vol. 5, No. 5, 1988
Effects of Charcoal-Extracted Serum as a Growth Medium Supplement on in Vitro Development of Mouse Embryos SANTIAGO L. PADILLA, 1'2 ANITA M. HOWE, 1 and J E F F R E Y P. BOLDT 1
Submitted: June 27, 1988 Accepted: July 7, 1988 (North American Editorial Office)
dia are tested by evaluating their ability to support one-cell (11) or two-cell (8) mouse embryo development to blastocysts before they are used for human IVF. From the beginning of our IVF program at the Medical College of Georgia, we have used 15% serum-supplemented media previously tested in the two-cell mouse embryo quality-control model (8). We have tested Ham's F-10 media alone and supplemented with 15% serum from the IVF patients, their husbands, and volunteers individually on the two-cell mouse embryo model and found that, many times, serum samples do not support embryo development. Similar observations have been made by Shirley et al. (2). The purpose of this study was to evaluate if dextran-coated charcoal extraction of the serum used to supplement the Ham's F-10 medium would modify its ability to support mouse embryo growth in vitro.
Two-cell mouse embryos (N = 952) were cultured in modified Ham's F-IO medium supplemented with 15% charcoal-extracted serum or 15% nonextracted serum from 17 patients. Each sample was assayed independently and all experiments run in duplicate. Ten nonextracted samples inhibited development to the blastocyst stage compared to controls in 17-10 alone. Charcoal extraction significantly improved (P < 0.05) development compared to nonextracted serum in eight of these samples. Seven samples supported development and no difference was noted between F-IO alone and extracted and nonextracted serum-supplemented media in this group. In conclusion, charcoal extraction significantly reversed the "embryotoxic" effects of some sera in the two-cell mouse embryo mode~: KEY WORDS: in vitro fertilization; mouse embryo; serum; charcoal extraction; growth media.
INTRODUCTION Serum is commonly used as a medium supplement to support mammalian preimplantation development in the mouse (1,2), rabbit (3), bovine (4,5), sheep (6), and human (7,8). The complexity of serum, with its various protein, lipid, hormonal, and micromolecular factors, makes it difficult to evaluate critically which factors benefit or harm embryo development in vitro. Fetal cord serum or maternal serum, usually 10 to 15% by volume, is utilized for medium supplementation in human in vitro fertilization (IVF) (8-10). These serum-supplemented me-
MATERIALS AND METHODS Culture Medium
Ham's F-10 medium (12) (F-10) (GIBCO, Grand Island, NY) was supplemented with sodium bicarbonate, calcium lactate, and penicillin G (8). Highperformance liquid chromatographic (HPLC)-grade water (Chromar Mallinkrodt, Paris, KY) was used for the stock medium preparation and the stock medium stored at 4~ and used within 2 weeks.
1 Department of Obstetrics and Gynecology, Section of Reproductive Endocrinology, Medical College of Georgia, Augusta, Georgia. 2 To whom correspondence should be addressed at Women's Fertility Center, Greater Baltimore Medical Center, 6701 North Charles Street, Baltimore, Maryland 21204.
0740-7769/88/1000-0286506.00/09 1988PlenumPublishingCorporation
Mouse Embryo Culture Random-bred CFW mice were obtained from Charles River Breeding Labs, Inc. The animals were allowed to reach an age of at least 6 weeks 286
C H A R C O A L - E X T R A C T E D S E R U M AS A G R O W T H M E D I U M S U P P L E M E N T IN I V F
before use and maintained on a 12-hr light/dark cycle. Female mice were injected intraperitoneally at 4--5 PM with 10 IU of pregnant mare serum gonadotropin (PMS; Organon, West Orange, NJ) irrespective of the stage of the estrous cycle. Fortyeight hours after PMS, 10 IU of human chorionic gonadotropin (hCG; Sigma, St. Louis, MO) was given. At the time of the hCG injection, each female was mated with a 10- to 12-week-old CFW male. Females with a vaginal plug the next morning were sacrificed by cervical dislocation 36--40 hr after hCG administration. The oviducts were removed surgically and placed in 35 x 10-ram tissue culturegrade plastic petri dishes (Falcon Plastics, BecktonDickinson and Company, Cockeysville, MD) containing 2 ml ofF-10. Two-cell embryos were flushed from the oviducts with F-10 in a 1-ml syringe with a 30-gauge needle. The embryos were transferred to organ culture dishes (Falcon Plastics No. 3037, Beckton-Dickinson and Company) containing either F-10 alone or F-10 with 15% heat-inactivated serum from the sources described below in lots of 10-20 embryos per dish. All media used for embryo culture had been sterilized by passage through a 0.2-txm Gelman filter and equilibrated overnight in 5% CO2 in air and at 37~ prior to use. The organ culture dishes contained 0.9 ml of culture medium in the inner well and 1.5 ml of medium in the outer well. Cultures were maintained at 37~ in a waterjacketed incubator (Forma No. 3158) with 5% CO2 in air as the gas phase. Two cultures were established for each of the sera to be tested. Embryo development was assessed at 24, 48, and 72 hr of incubation.
Sources and Processing of Serum Serum was collected from (a) normal cycling women referred for in vitro fertilization (IVF) to the Medical College of Georgia, (b) normal cycling female volunteers, (c) husband of IVF patients, and (d) normal male volunteers. All samples were collected in sterile red-topped Vacutainer tubes (Beckton-Dickinson, Rutherford, N J) and allowed to clot at 4~ overnight. Serum was then separated by centrifugation at 3000g for 10 min. The serum was then heat inactivated at 55-60~ for 30 min, filtered through a 0.45-txm filter, and stored at -20~ in 2-ml aliquots until used. Dextran-Coated Charcoal Extraction Serum was extracted for 20-24 hr with dextrancoated charcoal (10%, w/v) at 4~ under constant
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low vortex. The charcoal was then removed by high-speed centrifugation (12,000g) for 5 min.
Study Design Mouse embryo cultures were established with (a) F-10 alone, (b) F-10 supplemented with 15% nonextracted serum, and (c) F-10 supplemented with 15% charcoal-extracted serum. Extracted and nonextracted samples from each individual were tested simultaneously with two cultures each. An F-10 control was used in every experiment. All experiments were done in duplicate. Results were analyzed by chi-square analysis. A P value less than 0.05 was considered significant.
RESULTS Serum samples from 17 individuals were tested for embryotoxic effects in this study. During the course of these experiments, embryos were cultured in Ham's F-10 alone as a control in each experiment and consistently showed >65% development of two-cell embryos to blastocysts. As shown in Table I, 10 of the 17 serum samples (numbers 1-10) tested prior to charcoal extraction were embryotoxic, i.e., they supported <40% development of two-cell embryos to the blastocyst stage when used as a medium supplement. The embryotoxic effect of serum did not appear related to the sex of the serum donor. Of the 10 embryotoxic sera examined, 7 samples were from female donors and 3 were from male donors. The level of toxicity of the serum appeared to vary between samples. In samples 1 and 8, for example, all embryos degenerated within 48 hr of culture. In comparison, samples 4, 7, and 9 did not demonstrate embryotoxic effects until 72 hr, where many embryos either degenerated or did not complete the morula-to-blastocyst transition. Extraction of the serum for 24 hr with dextrancoated charcoal caused a significant improvement in the ability of eight (80%) of the embryotoxic sera to support embryonic development. In 2 of 10 cases (samples 6 and 8), extracted sera still demonstrated embryotoxic effects. Table I also provides data on the effects of charcoal extraction on nonembryotoxic serum (samples 11-17). Each of these sera supported >55-60% development of two-cell embryos to the blastocyst stage prior to extraction. Extraction with dextran-coated charcoal had no stimulatory or inhibitory effect on the ability of
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 5, No. 5, 1988
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PADILLA, HOWE, AND BOLDT
Table I. Effects of Charcoal Extraction of Serum Prior to Medium Supplementation on Mouse Embryo Development Incubation time 72 hr
Serum sample (sex)
Number of embryos
24 hr, No. of embryos degenerated
48 hr, No. of embryos degenerated
No. of embryos degenerated
No. of morulae
No. of blastocysts
(1)
S.B. (F)
Nonextracted Extracted
34 41
24 2
10 0
0 0
0 8
0 (0%) 31 (76%)
P < 0.0001
(2)
K.C. (F)
Nonextracted Extracted
33 34
2 0
3 0
7 3
10 8
11 (33%) 23 (68%)
P < 0.0005
(3)
B.C. (M)
Nonextracted Extracted
27 29
3 2
15 2
9 4
0 6
0 (0%) 15 (52%)
P < 0.0001
(4)
M.Be. (F) Nonextracted Extracted
24 24
0 0
0 0
7 2
10 7
7 (29%) 15 (63%)
P < 0.05
(5)
B.B. (M)
Nonextracted Extracted
20 29
0 0
0 1
0 0
13 3
7 (35%) 25 (86%)
P < 0.001
(6)
R.D. (M)
Nonextracted Extracted
23 25
0 1
0 1
17 10
5 8
1 (4%) 5 (20%)
NS
(7)
D.N. (F)
Nonextracted Extracted
42 35
2 1
1 0
14 6
8 7
17 (41%) 21 (60%)
(8)
P.H. (F)
Nonextracted Extracted
22 29
10 10
12 16
0 1
0
0 (0%)
1
1 (3.4%)
(9) J.E.C. (F) Nonextracted Extracted
24 23
0 1
1 2
7 0
7 4
9 (38%) 16 (69%)
P < 0.05
P < 0.05
NS
(10)
L.P. (M)
Nonextracted Extracted
50 50
2 0
13 2
16 2
10 10
9 (18%) 36 (72%)
e < 0.0001
(11)
M.B. (M) Nonextracted Extracted
21 18
0 1
0 0
2 0
7 3
12 (57%) 14 (78%)
NS
(12)
J.S. (F)
Nonextracted Extracted
24 25
0 0
1 3
1 1
6 7
16 (67%) 14 (56%)
NS
(13)
J.B. (M)
Nonextracted Extracted
24 23
1 0
0 1
5 3
3 2
15 (63%) 17 (74%)
NS
(14)
J.P. (M)
Nonextracted Extracted
22 24
0 1
0 0
1 0
1 2
20 (91%) 21 (88%)
NS
(15)
N.B. (F)
Nonextracted Extracted
21 20
1 0
0 2
0 0
0
20 (95%)
NS
0
18 (90%)
(16)
M.R. (F)
Nonextracted Extracted
24 30
1 0
7 3
0 2
0 3
16 (67%) 22 (73%)
NS
(17)
J.C. (M)
Nonextracted Extracted
29 29
0 1
0 0
0 0
9 4
20 (69%) 24 (83%)
NS
these sera to support development. As with the embryotoxic sera, the sex of the donor did not appear to influence the results (three female donors vs four male donors).
DISCUSSION The results of our study indicate that the embryotoxic effects of certain human serum samples, as
assayed by the two-cell mouse embryo culture system, can be reversed by extraction with dextrancoated charcoal. Extraction of serum that supported embryo development did not affect the ser u m ' s subsequent p e r f o r m a n c e . T h e s e results therefore suggest that charcoal extraction removes particular serum components from embryotoxic serum that are responsible for their toxic properties. The extraction process using d e x t r a n - c o a t e d charcoal is known to remove a variety of lipid corn-
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 5, No. 5, 1988
CHARCOAL-EXTRACTED SERUM AS A GROWTH MEDIUM SUPPLEMENT IN IVF
ponents from serum, including steroids and fatty acids (14-16). Although steroid levels were not obtained on each serum sample studied, it is unlikely that the steroid profile of the sera was related to embryotoxicity, since (i) embryotoxic and nonembryotoxic sera were obtained from both male and female patients and (ii) previous studies have demonstrated no correlation between estradiol or progesterone levels and support of two-cell mouse embryo development (2). The embryotoxic factor could be a low molecular weight component or components that are bound to steroids or fatty acids and, thereby, extracted when treated with charcoal. Since the level of serum embryotoxicity appears to differ in the various sera examined, the concentration and/or biochemical nature of the embryotoxic "factor" might differ between different samples. It is of interest that a recent report has indicated a detrimental effect of serum (collected as in the present report) that is mediated by platelet-derived factors such as adenosine diphosphate produced during the clotting reaction (17). Such a factor, or other as yet unknown substances, may be present in serum and contribute to embryotoxic effects. Further experiments will be needed to identify specific factors present in embryotoxic serum that, when extracted, allow normal development to occur. The benefits of serum supplementation of IVF culture media have not been thoroughly explored. Human embryos can be cultured under serum-free conditions and lead to pregnancies (18); however, experiments employing mouse embryos have suggested that certain fractions of fetal cord serum augment embryonic development (11,13). Most IVF programs continue to use serum, from either the patient, the donor, or the fetal cord blood, as a supplement for human embryo cultures. Ideally, however, serum from the patient should be used in order to limit the possibility of spread of infectious diseases such as hepatitis, cytomegalovirus, and acquired immune deficiency syndrome through the use of serum from affected individuals. Charcoal extraction of patients' serum which is embryotoxic may allow the use of such samples that would normally not have been utilized. Further experiments toward evaluating effects of charcoal-extracted serum on the development of mammalian embryos in vitro, including the human, will be required to evaluate properly the use of charcoal extraction in a clinical IVF setting.
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Journal of in Vitro Fertilization and Embryo Transfer, Vol. 5, No. 5, 1988
