Journal uf in l/)tro Fertilization and Emb/3.o Transfer, Vol. 3, No. 6, 1986
The Effect of Pregnant Mare Serum Gonadotropin on Mouse Embryos Fertilized in Vivo or in Vitro FUMIHIKO SATO 2 and RICHARD R MARRS 3,4
Submitted: August 30, 1985 Accepted, June 23, 1986 (North American Edttorial Office)
report demonstrating an increased rate of chromosomal abnormality in the abortuses of hMG-treated women (12). In this report we have studied the effect of PMSG on mouse embryos fertilized in vivo and in vitro. Comparisons of the rate of degenerated embryos, chromosomal aberrations, and sister-chromatid exchange (SCE) are demonstrated in both groups. SCE is utilized as a sensitive measure of deoxyribonucleic acid (DNA) damage, as this has previously been utilized to identify cell toxicity under various conditions (13 - 15).
The effect of increasing doses of exogenous gonadotropin stimulation for ovarian hyperstimulation was studied utilizing mouse embryos fertilized in vivo or in vitro, bwreased rates of embtyo degeneration, fragmentation, and triploidy, increased sister-chomatid exchange, and decreased fertilization rates were observed in high-dose stimulation groups. It appears, therefore, that oocyte and~or emblwo quality may be affected by increased amounts of exogenous gonadotropin stimulation. KEY WORDS: sister-chromatid exchange; chromosomal aberration; triploidy.
MATERIALS AND METHODS INTRODUCTION Female B6C3F1 hybrid mice (8 to l0 weeks old) were maintained on a 12:12 schedule of light-dark and allowed free access to food and water.
Pregnant mare serum gonadotropin (PMSG) has been widely used for induction of ovulation in laboratory animals and cattle, while the administration of human menopausal gonadotropin (hMG: Pergonal, Serono Laboratories, Inc., Randolph, MA) has also been used in humans for ovulation induction (1,2) and for multiple follicle development with in vitro fertilization (IVF) procedures (3,4). In mammalian systems, the administration in highdose PMSG has been correlated with embryonic death (5), an increase in triploidy (6-8), chromosome aberration (9), fertilization failure (10), and embryo fragmentation (11). There has been a single
Experiment 1 Female mice (N = 57) were injected with 1.5, 3.0, or 10.0 IU PMSG (Sigma Chemical Company, St. Louis, MO) intraperitoneally, followed 48 hr later by 5.0 IU human chorionic gonadotropin (hCG; E. R. Squibb and Son, Inc., La Mirada, CA). Thirty-one unstimulated female mice were caged with males and were inspected each morning for vaginal plug formation. Stimulated females were paired with males of the same strain after hCG administration and examined for vaginal plugs on the following morning (18 hr after mating). Two cell embryos were collected 25 hr after visualization of a vaginal plug in both the unstimulated and the stimulated females. Embryos were cultured in modified Ham's F-10 medium with 10 txg/ml of 5-bromodeoxyuridine (BrdU) for 27 hr, followed by 2 hr with Colcemid-supplemented culture medium (16).
Presented in part at the American Fertility Society, New Orleans, Louisiana, April 2-7, 1984. z Section of Endocrinology Infertility, Department of Obstetrics and Gynecology, USC School of Medicine. Los Angeles, California. 3 Department of Obstetrics and Gynecology, USC School of Medicine, Women's Hospital, Los Angeles, California. 4 To whom correspondence should be addressed at 1240 North Mission Road, Los Angeles, California 90033,
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0740-7769/86/1200-035351)5 00/09' 1986PlenumPubhshmgCorporation
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SAI"O AND MARRS
Table I. Effect of P M S G Dose on m Vlvo Ovulation Group
No. of e m b r y o s collected ( m e a n • SD) No. of e m b r y o s degenerated Fragmentation
Unstimulated (N = 31)
1.5 IU (N = 23)
3.0 IU (N = 13)
10.0 IU (N = 21)
229 (7.4 • 1.5) 6 (2.6%) 0
263",** (11.4 • 5.1) 36 d ( 13.7%) 0
2920 (22.5 • 5.8) 43 e (14.7%) 11 * (3.8%)
495 ~ (24.8 • 8.6) 100f (20.2%) 17* (3.4%)
* P < 0.05, significant difference c o m p a r e d with unstlmulated group. *~ ab, P < 0.05: ac, P < 0.05; df, P < 0.05; ef, P < 0.05.
Embryos were fixed by the method of Tarkowski (17) and stained with the modified method of Perry and Wolff (18).
Experiment 2 Female mice (N = 127) were injected wih 1.5, 3.0, or 10.0 IU of PMSG, followed by 5 IU of hCG 48 hr later. The mice were sacrificed 12 to 14 hr after hCG injection and cumulus masses were flushed from the oviducts. Spermatozoa were obtained from male mice of the B6C3F- 1 hybrid strain by removal of the testicular tissue after the animal was sacrificed. The testicles were minced in a 35-mm dish in 2 ml of Tyrode's solution and the solution was centrifuged at 160g for 10 rain. The sperm pellet was resuspended in Tyrode's solution containing a 0.5 mM concentration of sodium pyruvate and incubated for 90 rain. Motile sperm were collected from the supernatant for insemination. Oocytes were inseminated with a final sperm concentration of 0.5-1.0 x 106 motile sperm/ml in an organ culture dish containing Ham's F-10 medium. After 24 hr of culture fertilized oocytes were identified by the presence of a second polar body or a two-cell-stage embryo. Forty-eight hours from insemination, BrdU (10 txg/ml) was added to the culture medium, and embryos were cultured for another 27 hr, followed by 2 hr with Colcemid supplementation. Embryos were fixed and stained as in Experiment 1. In both experiments blastomere number, sisterchromatid exchange, and chromosome aberration were observed after staining by a single observer in a blinded fashion. Statistical analysis was performed by Student's t and chi-square test where appropriate.
RESULTS Experiment 1 A total of 1279 two-cell embryos was collected from the 88 mice in the four treatment groups. The number of embryos (mean + SD) increased with increasing doses of PMSG, from 11.4 _+ 5.1, to 22.5 _+ 5.8, to 24.8 _+ 8.6 embryos/mouse in the stimulation groups, compared to 7.4 _+ 1.5 embryos/mouse in the unstimulated group, and was significantly different among groups (P < 0.05) (Table I). The number of degenerated embryos was significantly increased in all PMSG-treated groups compared to that in unstimulated animals (P < 0.05). Moreover, there was a significant increase in degenerated embryos between the group receiving 10 IU PMSG and the 1.5 and 3.0 IU PMSG-treated animals (P < 0.05). Fragmented embryos were observed only in the 3.0 IU and 10.0 IU PMSG groups and this was a significant increase compared to unstimulated or 1.5 IU PMSG groups (P < 0.05). Table II. Effect of P M S G Dose on C h r o m o s o m a l A b e r r a u o n with in Vivo Fertilized O v a Group
No. of metaphases examined No of metaphases with 2n Hyperploidy l>40) Hypoploidy (<40) Triploidy
Unstimulated
1.5 IU
3.0 IU
10.0 IU
65
61
51
45
63
57
43
43
l
1
l
1
1 0
3 0
1 6*
1 0
* P < 0.05 compared to um;timulated control and I 5-1U group
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PMSG EFFECTS ON MOUSE EMBRYOS
Table III. Effect of PMSG Dose on SCE N u m b e r and B l a s t o m e r e N u m b e r in in Vlvo Fertilized O va Group Unstimulated SCE n u m b e r (mean • SD) Blasto mere n u m b e r (mean +_ SD)
20.8 (N 8.36 (N
-= _+ =
4.3 61) 3.3 143)
1,5 IU 19.5 (N 8.80 (N
• = • =
3.0 IU
4.3 ~,* 68) 2.5 ~ 151)
24.7 (N 8.38 (N
• = • =
10. l IU
5.44 57) 2.8 17)
26 4 (N 8.13 (N
• = • =
5.8 b 53) 3.2 c 147)
* a, P < 0.05: ab. P < 0.05: c, NS.
Chromosomal aberration was studied in 222 metaphase preparations in these four groups, and no significant changes were observed except for a significan~ increase in triploidy seen in the group treated with 3.0 IU PMSG (P < 0.05) (Table IF). Two hundred thirty-nine (239) metaphase preparations were studied for SCE determination. In the 3.0 IU ?MSG group a SCE rate of 24.7 _+ 5.4 was observed, which was significantly higher than that in the unstimulated group (20.8 -+ 4.3) or the 1.5 IU PMSG group (19.5 ___ 4.3) (P < 0.05). Moreover, the 10 IU PMSG group had a significant elevation in the SCE rate (26.4 + 5.8) compared to the unstimulated and 1.5 IU PMSG animals (P < 0.05). The blastomere number was not significantly different in PMSG-treated animals compared to spontaneously ovulating animals (Table Ill). Experiment 2 Eighteen hundred ninety-three oocytes were recovered in the three treatment groups. A significant increase in ovulations per animal correlated with increasing PMSG dose, with 9.7 -+ 2.4 in the 1.5 IU PMSG group. 16 +_ 4.9 in the 3 IU PMSG group, and 22.4 _+ 4.7 in the 10 IU PMSG group (P < 0.05) (Table IV). Twenty-four hours following insemination, 91.5% of the oocytes in the 1.5 IU PMSG
group fertilized, while 85% in the 3.0 IU PMSG group and 80% in the l0 IU PMSG group fertilized (P < 0.002 and P < 0.001, respectively) (Table IV). At 6 and 24 hr after sperm addition the 10 IU PMSG group demonstrated a significant increase in fragmented embryos (P < 0.02 and P < 0.05, respectively) (Table V). One hundred seventy-seven (177) embryos were investigated for chromosome aberrations. Although no significant increase in aneuploidy was seen in any group, triploid metaphase was observed in all groups. The rate of triploid metaphase was increased in the 3.0 and 10.0 IU PMSG groups, with a significant increase in triploidy between the 1.5 and the 10.0 IU PMSG groups (P < 0.05) (Table VI). One hundred forty-six (I46) metaphases were prepared for SCE determination. There was a significant increase in SCE rate in the 3 IU and i0 IU PMSG-stimulated groups compared to the 1.5 IU PMSG treatment group (P < 0.05 and P < 0.001, respectively) (Table VII).
DISCUSSION In this study abnormal or fragmented embryos were observed at the higher doses (3 and 10 IU PMSG) in the in vivo fertilized embryos, while only
Table IV. Effect of PMSG Dose on O vul a t i on and Fertilization Rate in Vitro Group
No. of mice No. of collected o v a (mean _~ SD) Fertilization rate
1.5 IU
3.0 IU
10,0 IU
58 565 (9.7 _+ 2.4/mouse) 91.5%
34 5a5' (16 • 4.9/mouse) 85%**
35 783* (22.4 -- 4.7/mouse) 80% **'~
* P < 0.05. "~* P < 0.02. • P < 0,001.
Journal o f in Vitro Fertilization and Embtwo Transfer, Vol. 3, No. 6, 1986
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SATO AND MARRS
Table V. Effect of PMSG Dose on Embryo Fragmentation
Table VII. Effect of PMSG Dose on SCE Rate Group
Group Embryos fragmented
1.5 IU
3.0 IU
10.0 1U
6 hr after in semination 24 hr after insemination
9/656 ",* (1.6%) 5/516 ~ (1.0%)
15/545 (2.8%) 5/494 (1.0%)
29/783 b (3.7% / 17/675d (2.5%)
No. of SCE (mean + SD)
1.5 IU (N -- 47)
3.0 IU (N = 45)
10.0 IU (N = 54)
20.5 _+ 3.9"*
22.9 _+ 4.1 b
26.8 + 4.2 c
* ab, P < 0.05; bc, P < 0.001: ac, P < 0.001. * ab, P < 0.02; cd. p < 0.05.
the 10 IU PMSG group demonstrated increased fragmentation with in vitro fertilized oocytes. This significant increase in fragmentation does not appear to be a factor induced by the IVF technique since the incidence of fragmentation occurred in the vivo fertilized embryos as well. Increasing amounts of PMSG may be the causative factor for the increased rate of fragmentation seen in these experiments. Several authors (6-8) have reported a significant increase in triploidy in the embryos generated by PMSG stimulation and fertilized in vitro. The significant increase in triploid embryos seen in the 3 IU and 10 IU PMSG stimulation groups fertilized in vitro coincides with these previous reports. Moreover, because triploid embryos generated in vivo were a rare observation, it appears that increased triploidy is due most often to the IVF process (probably polyspermia). The insemination of the oocytes was carried out at about the time in vivo mating and fertilization would be expected to occur, and therefore the age of the oocytes in vitro probably did not account for the increased triploidy rate. Moreover, it occurred only in the 3 IU and 10 IU groups. Since an adverse effect on fertilization in vitro was observed with increasing PMSG dosage, a direct effect on the oocyte may be a factor in decreasing the rate of normal fertilization Table VI. Effect of PMSG Dose on Numerical Aberrations of Chromosome Number
in vitro. This has also been seen in our human egg fertilization rate when Pergonal induction is compared with combination stimulation for multiple follicle development. In both in vitro and in vivo generated embryos there was an increase in SCE rate with increasing PMSG dosage. The SCE rate of in vivo fertilized oocytes was not different from the rate observed in the in vitro fertilized oocytes; therefore, the toxic insult is most likely due to the PMSG dose rather than the environment in which fertilization takes place. This increase in SCE rate may be due to changes in the steroid mileau within the follicle prior to oocyte release, or the high LH stimulus throughout the growth and development of the oocyte follicle complex may be the initiating cause of the increased SCE rate. These facts remain unknown at present. In conclusion, the results of this study demonstrate an adverse effect of high-dose PMSG on oocyte and embryo quality whether fertilization occurs in vivo or in vitro in the mouse systems. These findings must be considered when exogenous administration of gonadotropins is being utilized for ovulation induction or for multiple oocyte collections for in vitro fertilization procedures. The relevance of these findings with respect to the human system will be known only when enough clinical data are accumulated.
REFERENCES
Group 1.5 IU No. of metaphases examined No. of metaphases with 2n Hyperploldy (>40) Hypoploidy (<40) Tnploidy * ab. P < 0.05.
3.0 IU
10.0 IU
61
47
69
58 0 0 3~,*
37 2 2 6
55 0 3 11b
1. Marrs RE March CM, Mishell DR Jr: A comparison of clinical and laboratory methods in monitoring human menopausal gonadotropin therapy. Fertil Steril 1980;34:542 2. Marrs RE Vargyas JV, March CM: Correlation of ultrasonic and endocrinologic measurements m human menopausal gonadotropin therapy. Am J Obstet Gynecol 1983;145:417 3. Vargyas JM, Morente C, Shangold G, Marrs RP: The effect of different methods of ovarian stimulation for human in vitro fertilization and embryo replacement. Fertll Steril 1984;42:745
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PMSG EFFECTS ON MOUSE EMBRYOS
4. Jones GS: Update on in vitro femlization. Endocrine Rev 1984;5:62 5. Beaumont HM, Smith AF: Embryonic mortahty during the pre- and postimplantation periods of pregnancy in mature mice after superovulation. J Reprod Fertil 1975,3:437 6. Maudlin I, Fraser IR: The effect of PMSG dose on the incldence of chromosomal anomalies m mouse embryos fertilized in vitro. J Reprod Fertil 1977;50:275 7. Fraser LR, Zanellotti HM, Paton GR, Drury LM: Increased incidence of triploidy in embryos derived from mouse eggs fertilized in vitro. Nature 1976;260:39 8. Takagi N, Sasaki M: D1gynic triploldy after superovulation in mice. Nature 1976;264:278 9. Fujimoto S, Pahlavan N, Dukelow WR: Chromosome abnormalities in rabbit preimplantation blastocysts induced by superovulation. J Reprod Fertil 1974;40:177 !0, Walton EA, Evans G, Armstrong DT: Ovulation response and fertilization failure in immature rats induced to superovulate. J Reprod Fert 1983;67:91 11, Gray MH, Chnsman CL: The effect of pregnant mare serum
12.
13.
14.
15.
16. t7. 18.
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gonadotropin on mouse oocytes as monitored at metaphase II. Theriogenology 1979:13:165 Boue JG, Boue A: Increased frequency of chromosomal anomalies in abortions after induced ovulation. Lancet 1973; 1:679 Latt SA: Sister chromatid exchanges, indices of human chromosome damage and repair: Detection by fluorescence and induction by mitomycin C: Proc Natl Acad Sci USA 1974:71:3162 Perry R Evans HG: Cytological detection of mutagen-carcinogen exposure by sister chromatid exchange. Nature 1975;258:121 Carrano AV, Thompson LH, Lindl PA. Minkler JL: Sister chromatid exchange as an indicator of mutagenesis. Nature 1978:2781:551 Saito H, Berger T. Mishell DR Jr, Marrs RP: The effect of serum fractions on embryo growth. Fertil Steril 1984;41:761 Tarkowski AK: An air-drying method for chromosome preparation from mouse eggs. Cytogenetics 1966:5:394 Perry R Wolff S: New Method for differential staining of sister chromatids. Nature 1974:258:156
