Gamete Research 9:145-152 (1984)

Culture of Mouse Preimplantation Embryos as a Quality Control Assay for Human In Vit ro Fertilization Steven 6. Ackerman, R. James Swanson, Gordon K. Stokes, and Lucinda L. Veeck

Andrology Laboratory, Department of Biological Sciences, Old Dominion University, Norfolk, Virginia (S. B.A., R.J.S., G. K.S.) and VIP Human In Vitro Fertilization Program, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk (L. L. K)

Techniques for culturing preimplantation mouse embryos from the two-cell stage to blastocyst are described, and the importance of this system to quality control assay the media and supplements used in human in vitro fertilization (IVF) procedures is discussed. Embryos from B6CBAF, mice were cultured in a commonly used mouse culture medium, modified Krebs-Ringer-bicarbonate (Krebs’), or in a commonly used human culture me- dium, Ham’s FIO nutrient mixture supplemented with human fetal cord serum (FCS), and results were not significantly different. Using the mouse embryo culture system, tests on 174 preparations of FCS resulted in 24.1 % producing less than 75% morula or blastocyst stages after 72 h in culture, compared to 9.5% of the Krebs’ control cultures. Rcsults of the mouse embryo culture system using 98 FCS subsequently used in human IVF were compared to results from the VIP Human In Vitro Program of Eastern Virginia Medical School of Norfolk, Virginia, from June 1982 through June 1983. The data suggest that prescreening of media and supplements using this mouse embryo culture system may indicate sources of factors potentially detrimental to the success of human IVF procedures.

Key words: mouse, embryo, in vitro fertilization, culture

INTRODUCTION

Most human in vitro fertilization (IVF) programs around the world currently supplement their insemination and embryo culture media with either maternal serum or human fetal cord serum (FCS) [Lopata et al, 1980; Edwards et al, 1981; Sundstrom

Received September 8, 1983; accepted November 4, 1983.

Address reprint requests to Dr. Steven Ackerman, Andrology Laboratory, Department of Biological Sciences, Old Dominion University, Norfolk, VA 23508.

0 1984 Alan R. Liss, Inc.

146 Ackerman et a1

et al, 1981; Wentz et al, 1983; Veeck et al, 19831. However, a number of recent studies have indicated that early development of mammalian embryos may be ad- versely affected by substances abnormally present in serum. Klein and co-workers demonstrated teratogenic effects on whole rat embryos cultured with serum collected from rats injected with cadmium chloride or cyclophosphamide [ 19801 or with serum collected from rats or humans prepared after vitamin A ingestion [Steele et a1 19821. These investigators also reported that sera from patients undergoing cancer chemo- therapy or taking anticonvulsant medication were teratogenic or lethal to cultured rat embryos [Chatot et al, 19801. Using a similar assay system, Schmid and associates [ 19821 reported on the teratogenicity of serum collected from procarbazine-treated rats. Although Austin [ 19731 suggested that preimplantation embryos are relatively insensitive to teratogenesis compared to embryos undergoing organogenesis, in our lab we have observed incidences of culture medium preparations which failed to support in vitro development. In most of these incidences the problem has been traced to the source of protein supplement (eg, serum or albumin).

Concentrations of many of the constituents of serum vary dramatically from one source to the next [Stuber and Meszaros, 19791. Such constituents include proteins, carbohydrates, lipids, hormones, enzymes, trace vitamins and minerals, extraneous drugs, etc. Variati0.m in these substances may relate to genetic factors; metabolic, infectious, or immunologic diseases; use of medication; exposure to environmental pollutants; complications during labor; etc. Therefore, it is reasonable to assume that the use of unselected sera to supplement IVF media may produce variations in the results of an IVF program. This paper reports on techniques used in our laboratory to quality-control assay media and supplements prior to their use in the VIP Human IVF Program of Eastern Virginia Medical School in Norfolk, and compares preim- plantation development of mouse and human embryos cultured in media containing samples of the same FCS.

MATERIALS AND METHODS Mice

Previous studies in our laboratory [Ackerman et al, 19831 and by others [see Whittingham, 19751 have indicated that F I hybrid mice yield greater success in preimplantation development than inbred or random-bred mice. Embryos used in this study were obtained from B6CBAFI (C57BL/6 x CBA) virgin mice purchased from the Jackson Laboratory (Bar Habor, ME). Mice were 4-6 weeks old when received and acclimated to our animal facility for at least 2 weeks prior to use. All mice were used within 8 weeks of purchase. Male mice of the CD-1 strain were bred in our animal colony. All mice were maintained on a lighting regime of 14 h light: 10 h dark (lights on at 0500 h) at 21-24°C and had free access to laboratory rodent chow (Ralston Purina, St. Louis) and water.

Collection of Embryos

Female mice selected at random with regard to their estrus were induced to superovulate by IP injections of 5 IU pregnant mare’s serum (Sigma, St. Louis) and 5 IU human chorionic gonadotropin (hCG, Sigma) 48 h apart. At the time of hCG injection each female mouse was placed in an individual cage with one provenfertile male (3-10 months old). The following morning female mice were inspected for

Culture of Mouse Embryos 147

vaginal plugs and the results of the males’ copulatory performances were recorded. Any male mouse failing to mate on more than two consecutive attempts was culled from the colony. At 40-44 h after hCG injection plug-positive females were killed by cervical dislocation and oviducts and a small segment of the distal uteri were excised and placed in a sterile 35 X 15 mm polystyrene culture dish containing 2 ml medium. Using microdissecting tools and a dissecting microscope the oviducts were separated from the uteri at the uterotubal junction. Embryos were flushed from the oviducts by inserting into the ampulla a 30-guage needle attached to a 1-cc syringe filled with medium. Normal two-cell embryos were collected and dispensed into the various media using 15 or 20 pl micropipettes fitted with sterile tips (5-10 embryos/culture tube). At least five embryos from each mouse were cultured in one tube containing control medium. Each test medium was evaluated in triplicate cultures containing at least 15 embryos from three different mice.

Media

A modified Krebs-Ringer-bicarbonate (Krebs’) solution supplemented with 4 mg BSA/ml as described by Ackerman et a1 [ 19831 was used for collecting all embryos and as a control culture medium. Human fetal cord sera (15% v/v) were added to Ham’s nutrient mixture (F 10, Gibco Laboratories, Grand Island, NY) supplemented with 25 mg% Ca lactate, 100 IU penicillin/ml, and 100 IU streptomycin/ml as described by Ham [1963]. Media were prepared; adjusted to pH 7.3 and 280 mOsm; filter-sterilized; dispensed into Nunculon 110 X 16 mm flat-side, tissue culture tubes (GIBCO, I mlltube); and incubated at 37°C in 5% CO2 in air for at least 18 h prior to use.

Fetal Cord Sera Preparation

Cord bloods from normal vaginal or cesarian-section births (healthy mothers and babies) were collected into 50-ml syringes by the Labor and Delivery Unit of Norfolk General Hospital, Norfolk, Virginia. Bloods were stored at 4°C for 1-24 h prior to separation of sera by centrifugation. Serum specimens were inactivated at 56°C and sterilized by negative pressure filtration. An aliquot of each serum was prepared for mouse system testing and all samples were stored as -20°C until used.

Embryo Monitoring

The number of one-cell, two-cell, and fragmented embryos collected from each female was recorded; however, only two-cell embryos are routinely used for testing media. Cultures were observed daily using a Leitz Diavert inverted microscope at 125 magnification and embryos were categorized as two-cell, three- to four-cell, six- to eight-cell, 16- to 32-cell, morula, blastocyst, degenerative (ie, condensed, granu- lated blastomeres), or fragmented (numerous, uneven cytoplasmic blebs).

RESULTS

These results represent tests on over 500 mice conducted weekly from June 1982 until June 1983. In a random sample of 63 female mice which were superovu- lated and caged with males 79.4% possessed vaginal mucus plugs the morning following hCG stimulation. Although occasionally mice without plugs were found to yield two-cell embryos, we routinely only take the time to dissect these mice and

148 Ackerman et a1

flush their oviducts if we have not recovered enough two-cell embryos from mice with plugs. Furthermore, on rare occassions (< 1 %) we observed mice possessing vaginal plugs but recovered only one-cell embryos. We have also observed several mice without plugs on the morning following hCG injection, but with motile sperma- tozoa in the oviducts upon flushing. Ova from these mice were in the one-cell stage and were usually degenerative.

Results of superovulation are shown in Table 1 for a random sample of 78 mice with vaginal plugs on the morning following hCG injection. These mice produced a mean (SD) of 41.7 (9.1) total embryos per mouse. Although the number of two-cell embryos recovered varied even among mice of identical genetic makeup and treated identically, an average of 33.3 two-cell embryos were collected from each mouse. Two-cell embryos represented approximately 80 % of the total embryos recovered. Almost all of these two-cell embryos are normal in appearance; however, occasionally (< 1 %) embryos were observed that were obviously abnormal (eg, devoid of zona pellucida, enlarged perivitelline space, shrunken and granulated blastomeres, blasto- meres of grossly different sizes). Only normal-appearing two-cell embryos were transferred to media for quality control cultures.

The development of preimplantation mouse embryos cultured in Krebs’ medium was compared to culture in Ham’s plus FCS medium (Table 2). Twenty-one different preparations of Krebs’ and 174 different FCS in several lots of Ham’s were tested. After 48 and 72 h in vitro no significant differences (95% confidence level, Student’s t-test) were noted between mean embryo development in the two media. Table 2 depicts ranges for the percentages of morula and blastocyst development after 72 h in vitro culture using the two media. Embryo development was less than 75% for only two of 21 (9.5%) preparations of Kreb’s medium compared to 42 of 174 (24.1 %) preparations of Ham’s plus FCS. Furthermore, in four of 174 preparations of Ham’s plus FCS mouse embryo development was less than 50%, with many of the embryos

TABLE 1. Embryo Recovery Following Superovulation of 78 B6CBAFl Mice*

Embryo stage at recovery Total One-cell Two-cell Fragmented embryos

No. embryos per mouse (Mean k SD) 4.6 f 3.2 33.3 * 3.7 3.8 k 3.7 41.7 k 9.1

% Total embryos 11.0 79.9 9.1 -

‘Mice received 5 IU PMS and 51U hCG via intraperitoneal injection 48 h apart and embryos flushed from oviducts 40-44 h after hCG injection.

TABLE 2. In Vitro Development of Two-Cell Mouse Embryos: Comparison of Two Media’

Mean k SD % Morula + blastocysts

Medium No. tests 48 h 72 h

Krebs’ 21 89.9 k 13.6 86.8 & 12.1 Ham’s f FCS 174 83.2 k 19.3 77.1 f 15.0

‘Krebs’, modified Krebs-Ringer-bicarbonate; Ham’s FCS, Ham’s F 10 nutrient mixture.

Culture of Mouse Embryos 149

remaining in the two-cell stage during the 72 h in culture. This was observed on only one occasion with the Kreb’s medium, when a new lot of bovine serum albumin was tested (data not in Tables 2 or 3).

Each time a new lot of Ham’s was prepared it was tested with several FCS and simultaneously with an old lot of Ham’s previously shown to support embryo devel- opment. On only one occasion was the Ham’s medium found to be detrimental to embryo development; however, this was later found to be due to technician error during preparation.

Ninety-eight of the FCS tested in the mouse system were subsequently used for human IVF. These 98 FCS supported the development of a mean (SD) of 86.8% (14.3) of two-cell embryos to the morula or blastocyst stage (compare to Table 2). The results of mouse embryo development after 72 h in culture are compared to the results of human IVF in Table 4. Ninety-five of the 98 FCS were used in insemination media in which successful fertilization occurred (ie, presence of two pronuclei 14 h after insemination), and three of the FCS were used in media in which fertilization was unsuccessful. Successful cleavage during approximately 46 h in culture occurred in media incorporating 91 of the 98 FCS and pregnancies occurred following transfer of embryos cultured in media incorporating 34 of the 98 FCS. The mean values for mouse embryo development did not differ significantly for any of these parameters (fertilization, cleavage, or pregnancy) between successful and unsuccessful.

DISCUSSION

In our facilities using the mouse strains and techniques described in this study, approximately 75430% of the mice will successfully mate following superovulation. These mice will yield approximately 30 two-cell embryos per mouse. Since we establish triplicate cultures each containing 5-10 embryos per culture for each test medium and one control culture for every mouse producing usable embryos, we routinely superovulate 0.8 mice for each medium to be tested (rounded up to the nearest whole mouse). For example, if we plan to quality-control assay 10 media in a given week, we would need a minimum of 150 two-cell embryos (= 10 media x 3 cultures/medium X 5 embryos/culture) for the test media. To produce 150 embryos for the test media we would need six successfully superovulated and mated mice (= 6 mice X 25 embryos/mouse for the test media + 5 embryos/mouse for control medium). Considering that mating only occurs in approximately 75 % of the supero- vulated mice, to assure recovery of two-cell embryos from six successfully mated mice we would initiate superovulation in eight mice.

Superovulation rates in this study compared favorably to previous reports [Fowler and Edwards, 1957; Gates, 1971; Ackerman et al, 19831. Many factors may affect responses to gonadotropic hormones [McLaren, 1967; Gates, 19711 and other aspects of fertilization and early embryogenesis [Desjardins, 19791. We have noted that female mice, used within 2 weeks of shipment from the supplier to our lab, produced dramatically reduced number of two-cell and total embryos. Often these mice, and rarely acclimated mice, will produce embryos primarily in the one-cell stage or fragmented, yet with some normal-looking two-cell embryos. It is possible that some of the one-cell embryos may be fertilized ova which, if cultured, would undergo normal mitotic cleavage. However, in several preliminary experiments this was not observed. Therefore, we recommend an acclimation period of at least 2

150 Ackerman et a1

weeks prior to superovulation induction to overcome the stress of travel and introduc- tion to a new environment, and only the use of normal-appearing two-cell embryos to quality-control assay media and supplements.

In a previous study we demonstrated that Ham's plus FCS could be used as an insemination medium for fertilization of mouse ova in vitro [Ackerman et al, 19831. This investigation indicates that for the B6CBAF I strain of mouse, preimplantation embryo development from the two-cell stage to blastocyst can be supported by Ham's plus FCS with mean results comparable to the use of Krebs', a medium commonly used for the culture of mouse embryos (Table 2). However, over 20% of the FCS tested produced less than 75 % morula or blastocyst development over the 72-h culture period (Table 3). The reason for this impairment was not delineated. We have observed significant impairment in embryogenesis when hemolyzed FCS was tested, which is consistent with the report of Hsu [1978] that hemolyzed FCS inhibited growth of the inner cell mass of mouse blastocysts cultured in vitro. In our program FCS that do not support at least 75% morula or blastocyst development from two-cell stages after 72 h in culture are not used for human IVF, although we have no direct proof that human fertilization or embryogenesis would be impaired by the use of these FCS. In the current study 98 FCS, which produced a mean 72-h morula or blastocyst value of approximately 87%, were used in human IVF procedures for 326 patients and 400 ova (Table 4). No significant differences were noted in mouse values between FCS used during successful or unsuccessful fertilizations, in vitro cleavage, or established pregnancies.

Several groups have reported on the effects of serum supplementation on development in culture of human [Steptoe et al, 19761 and rodent [Saito et al, 1983;

TABLE 3. In Vitro Development of Two-Cell Mouse Embryos: Ranges of % Morula + Blastocysts After 72 H in Culture

No. (%) of cultures Krebs' Ham's + FCS

Range (2 I tests) (174 tests)

80- 100 17 (81.0%) 129 (74. I %) 60-79 3 (14.370) 31 (17.8%) < 75 2 (9.5%) 42 (24. I %) < 50 0 (0%) 4 (2.3%)

TABLE 4. Comparison of Mouse Embryo Development in Cultures Supplemented With Human Fetal Cord Sera (FCS) and Human In Vitro Fertilization Using the Same Sera

Mouse values Mean k SD

70 Morula + blastocyst after 72 h

Human IVF parameter Successful Unsuccessful

Fertilization" Cleavageh Pregnancy

"Succeshful fertilization determined by the presence of two pronuclei 14 h after insemination. hErnbryos cultured for approximately 46 h after insemination,

(95 FCS) 86.6 5 14.4 (3 FCS) 90.4 & 11.4 (91 FCS) 86.5 f 14.5 (7 FCS) 89.5 +_ 11.4 (34 FCS) 88.9 & 14.9 (64 FCS) 85.6 +_ 13.9

Culture of Mouse Embryos 151

Skreb et al, 19831 embryos. Each time it was evaluated supplementation of culture media with serum (eg, cord serum or homologous or autologous blood serum) was found superior to medium alone, supplementation with albumin or supplementation with fetal calf serum. Attempts have been made to identify factors in serum supple- ments that may influence in vitro embryogenesis [Cockroft, 1979; Hsu, 1980; Fuchs and Green, 198 11 or implantation [Sherman, 19781. We are currently investigating the role of medium supplements added to cultures of mouse embryos on inhibition and promotion of implantation following embryo transfer. Furthermore, we also use the mouse embryo culture system to investigate the effects on in vitro mammalian embryogenesis of other factors (eg, cultureware, equipment, cleaning, packaging, sterilization products and procedures, etc) used in human IVF programs.

ACKNOWLEDGMENTS

The authors express gratitude to Lynn Laurell, Edward Sismour, and Sherri Taylor for their technical assistance.

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