Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 3, 1987

The Effect of Serum Fractions on Single-Cell Mouse Embryos in Vitro I TETSUJI OGAWA, 2 T S U T O M U ONO, 2 and RICHARD E MARRS 3,4

Submitted: November 26, 1986 Accepted: January 22, 1987 (North American Editorial Office)

seen with Fraction D. In conclusion, HCS contains embryo growth inhibitory properties in the high (>30,000) and low (<1000) molecular weight components, while growth promoting factors are found in the 1000-5000 M W fraction. It also seems that there are some factors in the 70,000-I00,000 M W fraction which may promote the ability o f the embryo to hatch.

To examine the effect o f various fractions of human fetal cord serum (HCS) on mouse embryos cultured in vitro, heat-inactivated HCS was separated by ultrafiltration into five distinct fractions: Fractions A, M W >30,000; B, M W 30,000-10,000; C, M W 10,000-5000; D, M W 5000-1000; and E, M W <1000. Seven hundred twentyeight single-cell embryos were cultured in TYH-280 medium supplemented with 8 mg/ml bovine serum albumin (BSA) and a 20% concentration of Fraction A, B, C, D, or E, whole HCS, or BSA alone. Embryos cultured with Fraction A or E or whole HCS demonstrated a significantly reduced growth rate (P < 0.01), while embryos cultured with Fraction D demonstrated a significantly increased growth rate (P < 0.01). Additionally, 649 singlecell embryos were cultured in medium which was supplemented with 8 mg BSA/ml and a O, I, 2, or 5% concentration of Fraction A or E. Fraction E displayed toxicity even at a 1% concentration (P < 0.01), while Fraction A demonstrated growth inhibition at a 5% concentration (P < 0.05) but increased the hatching rate at a 1% concentration (P < 0.01). Finally, 635 single-cell embryos were cultured with four distinct fractions of HCS obtained from a Sephacryl S-200 column: Fractions I, M W 100,000; H, M W 70,000-100,000; III, M W 30,00070,000; and IV, low molecular weight (<5000). Fraction I or III significantly reduced the embryo growth rate as seen with Fraction A (P < 0.01) and Fraction H significantly increased only the hatching rate (P < 0.01), while Fraction IV significantly increased the growth rate as

KEY WORDS: serum; fractions; effects; in vitro fertilization.

INTRODUCTION Protein supplementation of medium utilizing human fetal cord serum (HCS) or preovulatory serum is generally used for in vitro fertilization (IVF) and embryo transfer (ET) (1-4). In previous experiments from our laboratory, we have demonstrated an improved rate of blastocyst development from in vivo fertilized two-cell mouse embryos when HCS or the high molecular weight fraction of HCS was utilized as a protein supplement (5,6). Most recently we have demonstrated significant toxicity in certain components of HCS which was demonstrated only when in vivo fertilized one-cell mouse embryos were cultured in vitro (7). The purpose of this study is to examine more closely the effects of distinct fractions of HCS separated by Amicon ultrafiltration or a Sephacryl S-200 column on singlecell mouse embryo development in vitro.

MATERIALS AND METHODS

i Presented in part at the American Fertility Society meeting, March 1985. 2 Department of Obstetrics and Gynecology, Yamagata University School of Medicine, Yamagata, Japan. 3 Department of Obstetrics and Gynecology, University of California at Los Angeles, Cedars Sinai Medical Center, Los Angeles, California. 4 To whom correspondence should be addressed at 8700 Beverly Boulevard, Los Angeles, California 90048.

Single-Cell Mouse Embryo Cultures B6C3Fj hybrid mice 5 to 6 weeks old were primed with an intraperitoneal injection of 7.5 IU pregnant mare's serum gonadotropin (PMSG) (Sigma Chem15 3

0740-7769/87/0600-0153505.00/0 9 1987PlenumPublishingCorporation

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OGAWA, ONO AND MARRS

ical Company, St. Louis, MO) followed 48 hr later by 5.0 IU human chorionic gonadotropin (hCG) (Sigma). The injections of PMSG and hCG were given at 6 PM. The mice were mated with adult males immediately after the injection of hCG and sacrificed by cervical dislocation 15 hr later. The in vivo fertilized early single-cell embryos were collected from the tubal ampulla and the embryos were cultured in organ tissue culture dishes (Falcon Plastic, Becton-Dickinson, Oxnard, CA) which contained 1 ml of medium with the various serum fractions. The dishes were incubated in an atmosphere of 5% CO2 and air at 37~ The medium used for culture was the TYH-280 medium described by Kasai e t a/.(8), with slight modifications. Bovine serum albumin (BSA) (Fraction V, Sigma) was increased to 8 mg/ml and phenol red was reduced to 1.0 i~g/ml. Embryos were observed every evening for 5 days. The control range for normal growth was two-cell stage or greater on Day 1 (48 hr after hCG), four-cell stage or greater on Day 2 (72 hr), compaction stage or greater on Day 3 (96 hr), early blastocyst stage or better on Day 4 (120 hr), and late blastocyst or hatching stage on Day 5 (144 hr). The final growth rates were calculated based on the number of embryos which were twocell stage or greater on Day I. Preparation of Human Fetal Cord Serum Human fetal cord blood was collected at the time of vaginal delivery and, after clotting, was centrifuged at 3000 rpm for 15 min. The serum was separated and serum with hemolysis was discarded. Multiple aliquots of sera were pooled into a 500-ml sample which was heat inactivated at 56~ for 30 rain, filtered with a 0.22-p.m filter, and stored at -

4oc.

Embryo Culture with Ultrafiltration Fractions Amicon Daiflo ultrafiltration membranes (YM30, >30,000 MW; YM10, >10,000 MW; YM5, >5000 MW; and UM2, >1000 MW) (Amicon, Lexington, MA) were floated in a beaker of distilled water for at least I hr before use in order to remove coated glycerin and sodium. The rinsed membranes were placed in an ultrafiltration cell, Model 12. First, 20 ml of heat-inactivated HCS was passed through a YM30 under N 2 gas pressure of 50 psi. Sixteen milliliters of this ultrafiltrate was separated to be further filtered through a YMI0 membrane. Seven

milliliters of TYH-280 medium without BSA and phenol red was added to the first retentate from the YM30 ultrafiltration retentate, the ultrafiltration was continued in order to refine the retentate, and the filtrate was d i s c a r d e d . The w a s h i n g with TYH-280 medium was repeated three times during this process. The final 4-ml retentate was diluted to 20 ml with TYH-280 medium. This solution was designated Fraction A (MW 30,000). The 16 ml of the first ultrafiltrate from the YM30 was further ultrafiltered through the YM10, YM5, and UM2 under a pressure of 30 psi in the same manner as a b o v e , and then F r a c t i o n B (MW 10,000-30,000), Fraction C (MW 5000-10,000), Fraction D (MW 1000-5000), and Fraction E (MW <1000) were obtained (Fig. 1). All ultrafiltrations were performed in a cold room at 4~ Seven hundred twenty-eight single-cell mouse embryos were cultured in medium supplemented with 8 mg BSA/ml and a 20% (v/v) concentration of Fraction A, B, C, D, or E, whole HCS, or no serum fraction supplement (BSA only). This volume/ volume concentration was based on the ultrafiltrate volume. The medium was checked for changes in osmolarity and pH after protein addition and no significant differences were observed. An additional 641 single-cell embryos were cultured in medium supplemented with 8 mg BSA/ml and 0, l, 2, and 5% concentrations of Fraction A or E. Embryo Culture with Gel Filtration Fractions Nine milliliters of heat-inactivated H C S was eluted through a Sephacryl S-200 column (80 x 3 cm) (Pharmacia, Piscataway, N J) which was previously equilibrated with 0.05 M Tris-HCl, pH 7.0, 0.1 M NaCl. Elution was performed over 8 hr at a flow rate of 60 ml/hr at 4~ controlled by a P-3 peristaltic pump (Pharmacia). The eluent was monitored at 280 nm using a UV absorption detector (ISCO Model UA5, Lincoln, NE) and 5-ml fractions were collected with an ICSO Model 328 fraction collector. The molecular weight elution profile of the column was established using blue dextran (2,000,000 MW), BSA (67,000 MW), and ribonuclease A (13,700 MW). Fractions were pooled as indicated in Fig. 2, dialyzed against double-distilled water, and lyophilized. Each lyophilized fraction was dissolved in 9 ml of TYH-280 medium. These solutions were designated Fractions I, II, III, and IV. Six hundred thirty-five single-cell embryos were cultured in medium supplemented with 8 mg BSA]

Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 3, 1987

EFFECT OF SERUM FRACTIONS ON MOUSE EMBRYOS

I 20 ml of heat-inactivated HCS

155

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Fig. 1. HCS fractions by Amicon ultrafiltration: 20 ml of heat-inactivated HCS was separated by Amicon ultrafiltration (YM30, YMI0, YM5, and UM2) into five distinct fractions: Fractions A (MW < 30,000), B (30,000 > MW > 10,000), C (10,000 > MW > 5000), D (5000 > MW > 1000), and E (MW < t000).

to the percentage of embryos reaching normal growth parameters and the data were analyzed by chi-square analysis.

ml and a 20% concentration of Fraction I, II, III, or IV or no serum fraction supplement (BSA alone). Statistical Analysis

RESULTS

Each experiment was conducted in at least three separate trials. The data were compared according

1.0

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FRACTION NUMBER Fig. 2. Elution profile of heat-inactivated HCS from a column of Sephacryl S-200: Fractions I, II, III and IV were pooled as indicated by the arrows.

Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 3, 1987

156

OGAWA, ONO AND MARRS

fraction on embryo development is demonstrated in Fig. 3. In the control group, 97.1% of the embryos were four-cell stage on Day 2, 96.2% were compaction stage on Day 3, 84.6% were blastocyst stage on Day 4, 73.1% were late blastocyst stage, and 32.7% were hatching. Embryos cultured with Fraction A, Fraction E, or whole HCS showed significantly reduced growth rates at all stages of development (P < 0.01), and less than 80% reached the four-cell stage and only 10% reached late blastocyst. Embryos cultured with Fraction B or C did not demonstrate any difference in growth rate compared with controls. On the other hand, embryos cultured with Fraction D demonstrated a significantly higher growth rate, with 93.0% early blastocyst (P < 0.05), 91.0% late blastocyst (P < 0.01), and 70.0% hatching (P < 0.01). Figure 4 illustrates the effect of Fraction A or E at a 0, I, 2, or 5% concentration. With Fraction E, there was no difference with any concentration at the four-cell stage, however, embryos cultured even with a concentration of 1% demonstrated a significantly lower number reaching the compaction stage (78.5%), early bias-

tocyst stage (49.5%), and late blastocyst stage (43.0%) (P < 0.01). Embryos cultured with Fraction A showed a significantly reduced number (69.6%) at only the late blastocyst stage with a concentration of 5%. There was no significant growth reduction at the other stages with a 5% concentration or any .of the developmental stages with lower concentrations, but on the contrary, the hatching rate with a concentration of 1% was 51%, which was significantly higher than controls (P < 0.01). The results of culture with HCS fractions produced from Sephacryl S-200 separation are demonstrated in Fig. 5. Embryos cultured with Fraction I or II had a significantly reduced growth rate at every stage compared with controls (P < 0.01) and only 28.7 or 4.7% of the embryos developed to late blastocyst, respectively, compared to 85.3% in the control group. Fraction II significantly increased (P < 0.01) the hatching rate (60.7%) compared with controls (38.8%), although growth rates at the fourcell stage and compaction stage were significantly lower than controls (P < 0.05). On the other hand, embryos cultured with Fraction IV demonstrated

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Fig. 3. Single-ceU embryo culture with HCS fractions separated by Amicon ultrafiltration: single-cell embryos were cultured in medium supplemented with 8 mg BSA/ml and 20% of Fraction A, B, C, D, or E, whole serum, or BSA alone (control). The numbers of embryos cultured were 111, 111, 110, 100, 73, 119, and 104, respectively.

Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 3, 1987

EFFECT OF SERUM FRACTIONS ON MOUSE EMBRYOS

157

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Fig. 4. Single-cell embryo culture with low concentrations of Fraction A or E: single-cell embryos were cultured in medium supplemented with 8 mg BSA/ml and 0 (control), 1, 2, or 5% of Fraction A or E. The numbers of embryos cultured were 74 for the control; 100, 84, and 92 for Fraction A; and 93, 115, and 91 for Fraction E.

significantly higher growth rates at the late blastocyst stage (94.1%) and hatching stage (79.8%) (controls, 85.3 and 38.8%, respectively) (P < 0.01). DISCUSSION In a recent report we demonstrated that protein supplementation in culture medium is very important for single-ceU mouse embryo cultured in vitro, while protein is not required for a two-cell mouse embryo to reach late blastocyst in culture. We also reported that BSA is much more beneficial as a supplement than HCS, and moreover, HCS has, in the low molecular weight fraction (MW 10,000), a potentially toxic effect on single-cell embryos in vitro when HCS is separated into high or low molecular weight fractions by Amicon ultra.filtration (YM10) (7). In the present study HCS was separated into more precise fractions according to molecular weight, and the effect of each fraction on single-ceU mouse embryo developed in vitro was examined. Fraction A (MW >30,000) and Fraction E (MW <1000) of HCS demonstrated a significant reduction of embryo growth. Fraction E displayed toxicity even at a concentration of 1% in the culture

medium. The minimum concentration of Fraction A necessary to display toxicity was 5%; however, hatching rates of embryos cultured with a I% concentration of Fraction A were increased, although growth rates up to the late blastocyst stage were the same as controls. This suggests that there are some beneficial factors in the MW >30,000 fraction which support embryo growth and hatching. This is also supported by the finding that Fraction II (70,000-100,000 MW) from Sephacryl S-200 column separation increased the embryo hatching rate. However, the embryo growth rates at the four-cell stage and compaction stage with Fraction II were significantly lower. This may be due to overlapping components of Fraction I or III to Fraction II. It was found with interest that Fraction D (10005000 MW) significantly increased the e m b r y o growth rate. This factor(s) may be similar to Fraction IV (low molecular weight fraction) from the S-200 column, which also promoted embryo development. These findings further elucidate earlier reports from this laboratory which demonstrated the benefit of whole HCS (5,6). Although these recent data may appear contradictory, they are not. The earlier reports utilized in vivo fertilized mouse two-

Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 3. 1987

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OGAWA, ONO AND MARRS

/

_> 4 cell

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Fig. 5. Single-cell embryo culture with HCS fractions from a Sephacryl S-200 column: single-cell embryos were cultured in medium supplemented with 8 mg BSA/ml and 20% of Fraction I, II, III, or IV or BSA alone (control). The numbers of embryos cultured were 136, 124, 127, 119, and 129, respectively.

cell embryos cultured in vitro. In our recent report utilizing HCS and high and low molecular weight fractions of HCS, the single-cell in vitro cultured mouse embryo demonstrated a greater ability to differentiate optimal and suboptimal in vitro culture conditions (7). Therefore the results seen here with the mouse one-cell embryo should more closely conform to the human in vitro fertilization model. In summary, it is likely that HCS contains emb r y o growth inhibitory p r o p e r t i e s in the M W >100,000, MW 70,000-30,000, and MW <1000 components, while growth promoting factors are found in the MW 1000-5000 fraction. Embryo hatching factors seem to be found in the MW 70,000-30,000 fraction. It is concluded that HCS has embryo growth inhibitory and also promoting components, and these findings cast doubt on using whole serum as protein supplement in human embryo culture in vitro. It also seems that selected serum fractions may improve the culture environment so that improved embryo quality may occur with human in vitro fertilization procedures.

REFERENCES 1. Caro CM, Trounson A: The effect of protein on preimplantation mouse embryo development in vitro. J Vitro Fert Embryo Transfer 1984;1:183 2. Menezo Y, Testart J, Perrone D: Serum is not necessary in human in vitro fertilization, early embryo culture, and transfer. Fertil Steril 1984;42:750 3. Kemeter P, Feichtinger W: Pregnancy following in vitro fertilization and embryo transfer using pure human serum as culture and transfer medium. Fertil Steril 1984;41:936 4. Marrs RP, Saito H, Yee B, Sato F, Brown J: Effect of variation of in vitro culture techniques upon oocyte fertilizationembryo development in human in vitro fertilization procedures. Fertil Steril 1984;41:519 5. Saito H, Berger T, MisheU DR Jr, Marrs RP: Effect of variable concentration of serum on mouse embryo development. Fertil Steril 1984;41:460 6. Saito H, Berger T, Mishell DR Jr, Marts RP: The effect of serum fractions on embryo growth. Fertil Steril 1984;41:761 7. Ogawa T, Marrs RP: The effect of protein supplementation on single cell m o u s e e m b r y o in vitro. F e r t i l Steril 1987;47:156 8. Kasai K, Minato Y, Toyoda Y: Fertilization and develop-. merit in vitro of mouse eggs from inbred strains and F~ hybrids. Jap J Anim Reprod 1978;24:19

Journal of in Vitro Fertilization and Embryo Transfer, Vol. 4, No. 3, 1987