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through gestation, or at 120 days of gestation in the case of the bovine. This was originally demonstrated with rat fibrosarcoma cells, which secrete large quantities of HA in the presence of HASA (3). If a beneficial effect of HA occurred with mouse embryos, it most likely would have been with FBS from the fourth month of gestation since this is the time when HASA was maximal with these same FBS samples. This was not the case for either onecell or two-cell embryos. Perhaps early mouse embryos produce sufficient HA and cannot respond to additional exogenous sources of stimulation. Another possibility is that heating at 56°C for 45 min may have inactivated or altered the HASA protein. No reference to heat treatment was given in previous reports (3,4) and rat fibrosarcoma cells (3) or human foreskin fibroblasts (4) were used in those studies. Additionally, the volume of culture medium may have had a dilutional effect as reported previously (5,6) and obviated any beneficial effect that increased levels of HA may exert. We are currently planning to repeat these experiments in 10fold less volume. The reason for the increased hatching rate observed with two-cell embryos and FBS from gestational months 6 and 9 is unknown. Results for month 6 barely reached significance and may have been due to chance. For gestational month 9 serum, perhaps other unknown factors or cytokines (7,8) are present in FBS that also vary during gestation. The rather poor overall results with one-ceU embryos was surprising. In previous work in our laboratory with this system, the blastocyst formation rate has been at least 70%. This certainly reinforces the sensitivity of the one-cell versus the two-cell system of evaluating media and disposables in clinical human in vitro fertilization.
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2. Carson DD, Dutt A, Tang JP. Glycoconjugate synthesis during early pregnancy: Hyaluronate synthesis and function. Dev Biol 1987;120:228-235 3. Longaker MT, Harrison MR, Crombleholme TM, Langer JC, Decker M, Verrier ED, Spendlove R, Stern R: Studies in fetal wound healing. I. A factor in fetal serum that stimulates deposition of hyaluronic acid. J Pediatr Surg 1989;24:789792 4. Longaker MT, Harrison MR, Langer JC, Crombleholme TM, Verrier ED, Spendlove R, Stern R: Studies in fetal wound healing: II. A fetal environment accelerates fibroblast migration in vitro. J Pediat Surg 1989;24:793-798 5. Quinn P, Hirayamada T, Marrs RP: Cooperative interaction among mouse zygotes cultured in protein-free medium: Blastocyst development and hatching. Serono Symposium on Preimplantation Embryo Development, 1991, p 68 (abstr) 6. Canseco RS, Sparks AET, Pearson RE, Gwazdauskas FC: Embryo density and medium volume effects on early murine embryo development. J Assist Reprod Genet 1992;9:454--457 7. Rapolee DA, Brenner CA, Schultz R, Mark D, Verb Z: Developmental expression of PDGF, TGFct and TGFI3genes in preimplantation mouse embryos. Science 1988;241:18231825 8. Paria BC, Dey SK: Preimplantation embryo development in vitro: Cooperative interaction among embryos and role of growth factors. Proc Natl Acad Sci 1990;87:4756--4760
Terry T. Olar 1'2 Adele S. Potts Fertility Institute of New Orleans New Orleans, Louisiana 70128 i Also Department of Obstetrics/Gynecology, Division of Reproductive Endocrinology and Infertility, Tulane University School of Medicine, New Orleans, Louisiana 70118. z To whom correspondence should be addressed at 6020 Bullard Avenue, New Orleans, Louisiana 70128.
GRAND
RAPIDS,
MICHIGAN
Effect of Storage of Ham's F-10 Medium on One-Cell Mouse Embryo Development in V i t r o
ACKNOWLEDGMENTS We acknowledge the gift of FBS from Hyclone Laboratories, Logan, Utah. This work was supported in part by a grant from Humana Hospitals, Women's Center of Excellence.
REFERENCES 1. Toole BP: Glycosaminoglycans in morphogenesis. In Cell Biology of Extra-cellular Matrix, ED Hay (ed). New York, Plenum, 1981, pp 259-294
Submitted: February 26, 1993 Accepted: May 6, 1993
INTRODUCTION In vitro fertilization/embryo transfer (IVF/ET) has become an important aspect of the treatment for Journal of Assisted Reproduction and Genetics, Vol. 10, No. 3, 1993
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many infertile couples. One of the most critical issues to consider when setting up an IVF laboratory is how gametes and embryos are going to be maintained in the laboratory until they are transferred to the patient. Therefore, it is very important that a culture medium of optimum quality is available. Preparation and quality control of the culture medium on a weekly basis are time-consuming and expensive. Many feel that the culture medium is unsuitable for embryo cultures, when it is more than 2 weeks old. One study (1) showed that storing Ham's F-10 medium for up to 425 days at 4°C did not affect two-cell mouse embryo development to blastocysts in culture. However, the two-cell mouse embryo may not be sensitive (2,3) to subtle changes in the culture medium and, therefore, may not have detected any differences between the freshly prepared and the stored media. In a separate study, German investigators (4) reported that Ham's F-10 medium could be frozen without affecting two-cell mouse embryo development in vitro or affecting pregnancy rates in women undergoing IVF. However, in that study, Ham's F-10 and sodium bicarbonate solutions were frozen separately and mixed prior to use. Sometimes, there was a need to adjust the osmolarity. With frozen media, there is the possibility that errors may occur, due to improper thawing and osmolarity adjustments. It would be ideal if a culture medium, once prepared, could be simply stored at 4°C and used for embryo cultures without compromising the development of the embryo. In the present experiment, one-cell mouse embryos were cultured in Ham's F-10 medium freshly prepared (Fresh) or stored in a refrigerator at 4°C (Old), to determine if storing the media at 4°C affects embryo development in vitro. MATERIALS AND METHODS Animals Virus antibody-free B6C3F1 female mice, 28 days old, and breeding-age male mice, 8-10 weeks old, were obtained from Charles River Laboratories (Wilmington, MA). They were allowed free access to feed and water. Lighting was provided for 12 hr daily. Collection of One-Cell Embryos Female mice were injected with 5 IU pregnant mare's serum gonadotropin (PMSG; Sigma ChemiJournal of Assisted Reproduction and Genetics, Vol. 10, No. 3, 1993
239
cal, St. Louis, MO) intraperitoneally (ip) at 1400 on a random day of their estrous cycle. Forty-eight hours later, they were injected with 5 IU human chorionic gonadotropin (hCG; Sigma Chemical) ip (5) and each female was immediately paired with a male mouse. About 18-20 hr after hCG, mice were checked for vaginal plugs and killed by cervical dislocation. Oviducts were collected using sterile techniques and the swollen ampullae were gently incised with a fine pair of forceps and a 26-G hypodermic needle to allow escape of the one-cell embryos. The embryos were always in clumps surrounded by a cumulus mass. Preparation of Ham's F-10 Media Ham's F-10 nutrient mixture (GIBCO, Grand Island, NY) in powder form was dissolved in highperformance liquid chromatography (HPLC)-brand water (American Chemical, Providence, RI) and was supplemented with Ca-lactate (0.2452 g; Mallinckrodt, Paris, KY), sodium bicarbonate (2.1 g; Mallinckrodt, Paris, KY), streptomycin sulfate (0.075 g; Sigma Chemical), and penicillin G (benzylpenicillin, 0.075 g; Sigma Chemical). After the contents were brought up to a liter, osmolarity was checked and adjusted to 280 mOsm/kg. Ham's F-10 medium was filter-sterilized using 0.2-p~m Nalgene filters (Nalge Company, Rochester, NY), and a portion of it was kept tightly capped in the filter itself, inside a refrigerator at 4°C. A small portion of the remainder was used for embryo cultures the following day. Fresh or Old medium was preequilibrated for 1820 hr inside a Forma Incubator (Forma Scientific Inc, Marietta, OH) at 37°C under a humidified atmosphere of 5% CO2 and 95% air. Preequilibration was achieved by placing 1 ml of the respective medium in the center well of an organ culture dish (Becton Dickinson, Lincoln Park, N J) with 4-5 ml of the medium placed in the moat to achieve a humid environment. A small amount of each type of the medium was preequilibrated in tubes to check the pH the following day. Experimental Protocol One-cell embryos from five or six mice were pooled. Embryos were treated with freshly prepared hyaluronidase (300 IU/ml; type II from sheep testes; Sigma Chemical) to strip the cumulus masses. The embryo dish with hyaluronidase was
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kept in the incubator for 5 min. Within 5 min, the hyaluronidase was easily able to strip off the cumuli. The naked embryos were washed in Fresh Ham's F-10 medium and then cultured in Fresh or 1-, 2-, 3-, 4-, 5-, or 6-month Old medium (four to six replicates for each month of comparison). For each replicate, within each month of comparison, a new batch of Fresh medium was compared with a different batch of Old medium. Fresh and Old Ham's F-10 media were prepared by the same technician, using the same source of water and ingredients.
Percentage development of one-ceU embryos to two cells was calculated. Percentage development to small, expanded/collapsed, hatched, and All (small, expanded/collapsed, and hatched stages combined) blastocysts by 96 and 120 hr after culture was calculated based on the number of two cells. Angular transformations of percentages were performed prior to statistical analyses. Differences in percentage development to two cells and blastocysts were evaluated using an analysis of variance for a completely randomized block design. In this analysis, replicates were considered blocks, Fresh or Old medium was considered the main effect (6). Table I. D e v e l o p m e n t of One-Cell M o u s e E m b r y o s to Blastoc y s t s W h e n Cultured for 96 hr in F r e s h (F) or Old (O) H a m ' s F-10 M e d i u m % Blastocysts ( m e a n - SE) ~ Expanded/ collapsed
Comparison
Small
F ( n = 124) c vs 1 - m o O ( n = 122)
19-+3
53±5
2±
14-+ 3
43---7
2-+ 1
59-
F ( n = 188)
Hatched
10-+ 3
57±4
3-
70---6
67±6
2-2
72+--6
1
2
All b 74±7
3±
1
F ( n = 142) vs 3 - m o O ( n = 123)
15---4
58~-4
2--- 1
75+-5
10± 3
70-4
3 ±2
83+-2
F ( n = 114) vs 4 - m o O ( n = 93)
12-+5
45± 1
2±2
59---6
9±4
43 +--6
3±2
55+-6
F ( n = 136) vs 5-mo O (n = 131)
10---4
55±4
6---2
71+--6
10 +- 4
52 ±- 8
2 -+ 1
64 --- 10
F ( n = 141) vs 6 - m o O ( n = 128)
12±4
57-+4
8±5
77-+6
6±2
54±9
1 2 ± 10
Expanded/ collapsed
Comparison
Small
Hatched
All b
F ( n = 124) c vs 1 - m o O ( n = 122)
6---3
42-7
24-7
7 2 ± 10
8-3
32-9
23+-9
63 ± 12
F ( n = 188) vs 2 - m o O ( n = 140)
2--- 1
34+-5
36---8
72---9
3-2
27-7
44-7
74+-7
5+--2
48±8
28± 1
81±6
4 - 3
49 +- 10
31 --- 9
84 --- 4
F ( n = 114) vs 4 - m o O ( n = 93)
6~3
35±5
17±6
58±7
5+--3
27---8
24---9
56+-5
F ( n = 136) vs 5 - m o O ( n = 131)
3 22
46±8
24±7
73-+5
5±2
35-+ 11
29-
F ( n = 141) vs 6 - m o O ( n = 128)
2±2
48-9
20---9
70--- 10
5-2
38+6
27+-6
70±-9
10
69±9
a M e a n s not different (P > 0.05) b e t w e e n c o m p a r i s o n s . b All--small, expanded/collapsed, and h a t c h e d stages combined. ¢ Total n u m b e r of one-cell e m b r y o s in all replicates.
Mean percentages and standard errors were calculated for purposes of illustration (Tables I and II).
RESULTS There was no difference (P > 0.05) in embryo development to two cells, whether embryos were cultured in Fresh or Old media. Differences were not observed (P > 0.05) in the development to small, expanded/collapsed, hatched, or All blastocysts either at 96 or 120 hr, after initiation of cultures (Tables I and II).
12
VS
= 140)
% Blastocysts (mean ± SE) a
F ( n = 142) vs 3-mo O (n = 123)
Statistical Analyses
2-moO(n
Table II. D e v e l o p m e n t of One-Cell M o u s e E m b r y o s to Blastocysts W h e n Cultured for 120 hr in F r e s h (F) or Old (O) H a m ' s F-10 M e d i u m
72-+9
a M e a n s not different (P > 0.05) b e t w e e n comparisons. b A l l - - s m a l l , expanded/collapsed, and h a t c h e d stages combined. c Total n u m b e r of one-cell e m b r y o s in all replicates.
DISCUSSION In a human IVF program, preparation and quality control of the embryo culture medium can be very time-consuming and expensive. Many feel that the culture medium is unsuitable for embryo cultures when it is more than 2 weeks old. The present experiment was conducted to understand further the possibility of storing media at 4°C for embryo cultures. The findings indicate that Ham's F-10 medium can be stored in a refrigerator at 4°C without compromising one-cell mouse embryo development in vitro. These findings are in complete agreement with two previous studies (1,4) showing that Ham's Journal of Assisted Reproduction and Genetics, Vol. I0, No. 3, 1993
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F-10 medium can be stored refrigerated (4°C) or frozen (-20°C) without affecting two-cell mouse embryo development in vitro. An advantage to using stored media as opposed to making media every week would be that two or three batches of the culture medium can be made and tested at the beginning of an IVF cycle. Those batches that perform well when tested with the one-cell mouse embryo bioassay can be properly stored in a refrigerator at 4°C and subsequently used for human embryo cultures during that cycle. This would certainly minimize the variability that is often encountered when culture medium is prepared on a weekly basis. It would also cut down technician time and expenses. More importantly, it would enable gametes and embryos from a group of patients to be exposed to the same batch of culture medium. REFERENCES 1. Naz RK, Janousek JT, Moody T, Stillman RJ: Factors influencing murine embryo bioassay: Effects of proteins, aging
Journal of Assisted Reproduction and Genetics, Vol. 10, No. 3, 1993
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2.
3.
4.
5.
6.
of medium, and surgical glove coatings. Fertil Steril 1986; 46:914--919 Davidson A, Vermesh M, Lobo RA, Paulson RJ: Mouse embryo culture as quality control for human in vitro fertilization: The one-cell versus the two-cell model. Fertil Steril 1988;49:516--52l Silverman IH, Cook CL, Sanfilippo JS, Yussman MA, Schultz GS, Hilton FH" Ham's F-10 constituted with tap water supports mouse conceptus development in vitro. J Vitro Fert Embryo Transfer 1987;4:185-187 Bernart W, Hauff B, Malez-Kehry W, Kunz G, Leyendecker G: Frozen storage of Ham's F-10 medium for human in-vitro fertilization. Hum Reprod 1990;5:6t0-61:2 Biggers JD, Whitten WK, Whittingham DG: The culture of mouse embryos in vitro. In Methods in Mammalian Embryology, JC Daniel (eds). San Francisco, Freeman, 1968, pp 86-115 Steele RGD, Torrie JH: Principles and Procedures of Statistics. New York, McGraw-Hill, 1960
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