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Superovulation-induced intrauterine growth retardation in mice
Superovulation-induced in mice MARK
I.
JOSEPH
EVANS, D.
LINDSAY
M.D.
SCHULMAN, GOLDEN,
ANIL
B.
Chicago,
Illinois,
MUKHERJEE, and
intrauterine growth retardation
Bethesda,
M.D. B.S. M.D.,
PH.D.
Maryland
ExWng animal models for inducing intrauterine growth retardation (NJGR~ involve sewere maternal compromise or acute fetal insu by enhancing fetal competition and human chorionk gonadotopm mean number of fetuees was sig reduced; and brain/liver weight ratios oophorectomy before superovulation. horns of the same mother was produ smaller brains and livers and inqeased brainfkir rat&s. In the preconceptual method for achieving IUGR and may bs combir4 with oophorectomy to permit comparisons between fetuses with different gmwth chanxMs&s withln the same mother. (AM. J. OBSTET. GYNECOL. 141:433, 1981.)
INTRAUTERINE GROWTH retardation(IUGR) is associated with significant increases in perinatal morbidity and deaths.lm3 Although studies comparing characteristics of IUGR and normal gestations have been numerous, clinical investigations to elucidate the etiology and pathophysiology of IUGR have intrinsic limitations. Consequently, many investigators have attempted to develop suitable animal models. A widely used system for inducing IUGR in the rat was developed by Wigglesworth.*, 5 One uterine artery is surgically ligated at approximately day 17 of gestation. Although fetal growth retardation occurs in the ligated uterine horn subsequent to the suddenly re-
Fwm the Department of O&et&s and Gynecology, University of Chicago-Pritzker School of Medicine, Chicago, and the Section on Human Biachemical and Developmemtol Genetics, awl Pregnancy Research Branch, Nation& Institute Child Health and Humun Development, National Institutes of Health, Bethesda
af
Received
for publication
Revised
May
Accepted May
December
9, 1980.
8, 1981, 13, 1981.
Re@int re+wts: Joseph D. Schuhnan, M.D., Section it Human Biochemical and Developmental Genetics, Nat&w1 Institutzz af Child Heal& and Human Development, Bethesda, Mqkznd 20205.
duced supply of nutrients to the fetuses,K operation during late pregnancy and the gross abrupt disruption of uterine blood flow are not typical characteristics of naturally occurring IUGR. Several other animal models of I UGR have also been deveIoped, including .etibolization of the placental bkd, stress from hypothermia or hyperthermia, chronic hypoxia, induced msernal renal insufficiency, deprivation of food, and the addition of nutritional or chemical agents (such as cadmium) to the diet.lT-13 Unfortunately, these methods may cause acute fetal insult or substantially alter normal maternal metabolism. Most investigators have considered inadequate placental exchange to be a major cause of IUCR. An animal IUGR model that brought about progressively increasing demands on placental exchange without the need for sudden intrapregnancy intervention OP maternal metabolic alteration would be desirable. Hormonally iridiuced superovulation substanti&y increases the normal number of embryos in the mouse, and thus should enhance competition for maternal nutrients and oxygen. This report describes the fetal effects of superovulaton in the mouse. Superovulation appears to represent a simple and useful method for inducing IUGR in this species. Furthermore, superovulation, combined with a partial unilateral oophorec433
434
Evans
October 15. 19x1 Am. J. Obstet. Gynewl.
et al.
Table I. Effect of superovulation
on fetal characteristics
I
(nonsurgical
Control (n = 19)
Fetuses per mother Body weight (gm) Brain weight (gm) Liver weight (gm) Brain/liver ratio Placental weight (gm) Maternal glucose (mg/dl serum)
6.3 1.26 0.076 0.074 1.04 0.11 114
I
in 0.5
60.2
Superovulated (?I = 100) 20.0 0.97 0.067 0.045 1.58 0.07 129
zt 0.11 t 0.009
IT 0.011 2 0.019 t 0.02 2 10.2
(n = 3) Fetal glucose (mg/dl serum)
group)
” ” f k r + f
I
Superovulation versus control*
p p p p p p
4.7 0.16 0.011 0.014 0.40 0.02 8.1
< 0.01 < 0.05 < 0.05 < 0.01 < 0.001 < 0.05 NSt
(n = 5) 60.9 -+ 23.2
rt 13.7
NS
All values are expressed as mean f SD. *Student’s t test for independent samples. TNot significant.
T&le II. Comparisons
between
fetuses in different
uterine
Control* (n = 26)
No. of fetuses per side Body weight (gm) Brain weight (gm) Liver weight (gm) Brain/liver ratio Placental weight (pm) Fetal glucose (mg/dl serum)
4.5 ” 0.063 0.047
t 0.014 z 0.023
1.52 f 0.50 0.83
PUO*
+: 0.16
51.9 f 13.8
1.14 ” 0.28 0.078 2 0.013 0.070 k 0.027
1.19 * 0.33
0.92 65.2
Dtfferencest (control uersm PUO)
(n = 5)
1.0 f 0.0
1.3
0.99 -+ 0.27
horns after PUO
2 0.17 +. 33.8
3.5 0.15 0.015 0.023 0.33 0.09 13.3
2 2.2
p < 0.05
f 0.09
NS p < 0.05 p < 0.05
f r r rt f
0.012 0.017 0.09 0.01 16.4
P ;;i05 NS
*Mean ? SD. tMean f SD of difference, Student’s t test for paired samples $Not significant. tomy prior the number permitting degrees of
to pregnancy, produces an asymmetry in of fetuses in the two uterine horns, thereby comparisons of fetuses exposed to different crowding within the same mother.
fiia#da and matedal Virgin female randomly bred National Institutes of Health (N-NIH[S]) mice, average age 8 to 10 weeks, were housed under standard laboratory conditions and given food and water ad libitum. In the major study group, the animals did not undergo operation. One week before mating, a smaller group of mice underwent partial unilateral left oophorectomies (-50%) by means of a flank incision and with the use of 0.5 ml of sodium thiopental (Pentothal) anesthesia. The major study group was subdivided into two categories: superovulation and spontaneous ovulation. Superovulated mice were given 5 IU pregnant mare serum (PMS) subcutaneously followed in 48 hours by 5 IU human chorionic gonadotropin (hCG) subcutaneously, and were mated 12 hours later. Control mice were mated without prior administration of hormones. All of the partially oophorectomized mice were also superovulated with PMS and hCG.
For mating, each female was caged with one male overnight. Copulation was confirmed by vaginal~piug the following morning, and the females were then segregated. Animals were killed by cesareansection on day 19 of gestation (term = 20 days). For each fetus, serum glucose and wet weights of the body, placenta, brain, and liver at birth were recorded. Statistical analyses were performed with paired or nonpaired t tests, as indicated, or Pearson product moment correlation analysis. Results SuperovuW~I mice veraus eoss&oIa. Superovulation produced significant alterations in the number of fetuses, body, brain, and liver weights, brain/liver weight ratios, and placental weights compared to controls (Table I). Superovulation resulted in a reduction in mean fetal body weight of 29%, mean brain weight of 12%, mean liver weight of 39%, and mean,placental weight of 34%. The average brain/liver ratio was 1.04 in controls and increased to 1.58 primarily because af the marked reduction in the size of the liver. Mean fetal serum glucose and maternal glucose levels at delivery were similar in the two groups.
Volume 141 Number 1
Superovulation-induced
In mice, most of the uterine blood supply flows from the lateral aspect to midline. In control animals, there was a positive correlation between fetal body weight and proximity to the lateral blood supply (r = 0.77 p < 0.01). With superovulation, however, this relationship was no longer demonstrable (r = 0.10, p = NS). Effect of unilateral oophorectomy on intrauterine comparisons after superovulation. Partial unilateral oophorectomy (PUO) and subsequent superovulation were performed in a small group of animals to assess the feasibility of comparing fetal growth differences within the same pregnant female (analogous to the model of Wigglesworth). PUO resulted in an average of approximately four fetuses on the control side to one fetus on the PUO side. Fetuses on the two sides manifested significant differences in mean brain weight and liver weight. A significantly increased brain/liver weight ratio on the more crowded side was demonstrable (Table II).
Comment IUGR continues to be associated with significant perinatal morbidity and mortality.‘* * Animal models have allowed close experimental scrutiny of many factors related to IUGR; however, all such models have had intrinsic limitations because of various differences from human IUGR. The present report indicates that superovulation is a simple, reliable, nonsurgical method for achieving IUGR in mice. The effects on fetal growth are presumably related to the gradually increasing fetal competition for limited uterine flow and, hence, nutrient and/or oxygen supply. Changes in the brain/liver ratio
IUGR
435
(i.e., brain sparing) are often characteristic of IIJGR in man and are present in the simple superovulation model in mice described here. The usefulness’of this model in studies of IUGR, perhaps including that associated with multiple human gestation, is apparent. The value of this model may be further enhanced in certain experimental situations by the addition of PLrO, a simple surgical procedure, prior to superovulation to allow comparisons between fetuses within the same pregnant animal. Such comparisons have proved to be highly advantageous in Wigglesworth’s IUGR model in the rat. Ideally, superovulation should restore a nearly normal number of fetuses on the PUO side and produce excessive contralateral crowding. Under the conditions that we used in these initial studies, superovulation resulted in a reduced number of gestations on the operated side and modestly increased the number of fetuses on the control side in comparison to those animals without prior operation (see Tables I and II). The important observation was that a significant asymmetry (approximately 4: 1) in fetal distribution and characteristics occurred. The total number of fetuses might possibly have been greater if the postoperative recovery time had been longer prior to superovulation. Although the absolute numbers of fetuses in the PUO group were small, the data in Table II support the validity of this refinement of the induced ovulation technique for selected investigations of IUGR. Finally, these observations reinforce the hypothesis that, even within the same mother, larger numbers of. fetuses per uterine horn are associated with changes typical of IUGR.
REFERENCES
1. Low, J. A., Galbrairh, R. S., Muir, D., Killen, H., Karchmar, J., and Campbell, D.: Intrauterine growth retardation: A preliminary report of long-term morbidity, AM. J. OBSTET.
GYNECOL.
103:534,
1978.
2. Daikoker, N. H., Johnson, J. W. C., Graf, C., Kearney, K., Tyson, I. E., and King, T. M.: Patterns of intrauterine growth Retardation, &&et. Gynecol. 50:211, 1979. 3. Vohr, B. R., Oh, W., Rosenfield, A. G., and Cowett, R. M.: The preterm sma&-for-gestation&age infant: A two-year follow-up study, AM. J. OBSTET. GYNECOL. 133:425,1979. 4. Wigglesworth, J. S.: Experimental growth retardation in the foetal rat, J. Pathol. 88: 1, 1964. 5. Wigglesworth,-J. S.: Fetal growth retardation, Am. J. Pathol. 77:347, 1974. 6. Nitzan, M., Odoff, S., and Schulman, J. D.: Placental transfer of analogues of glucose and amino acids in fetal growth retardation, Pediatr. Res. 13: 100, 1979. 7. Greasy. R. K., Barrett, C. T., DeSwiet, M., Kananpaa, K. U., and Rudolph, A. M.: Experimental intrauterine growth retardation in the sheep, AM. J. OBSTET. GYNECOL. 113:566, 1972.
8. Hensleigh, P. A., and Johnson, D. C.: Heat stress effects during pregnancy. I. Retardation of fetal rat growth, Fertil. Steril. 24:522, 1971. 9. Van Geijn, H. P., Kaylon, W. M., Nicola, K. K., and Zuspan, F. P.: Induction of severe intrauterine growth retardation in the Sprague-Dawley rat, AM. J. ORSTET. GYNECOL. 137:43,
1980.
10. Nitzan, M., Orlo:Ff, S., Chrzanowski, B. L., and Schulman, .I. D.: Intrauterine mowth retardation in renal insufficiency: An experimental model in the rat, AM. J. OBSTET. GYNECCIL. 133:40. 1979. 11. Anderson, L. L. Embryonic and placental development during prolonged inanition in the pig, Am. J. Physiol. 429: 1687, 1975. 12. Webster, W. S.: Cadmium induced fetal growth retardation in the mouse, Arch. Environ. Heakh 33t36, 1978. 13. Scott, J. R.: Fetal growth retardation associated with maternal administration of immunosuppressive drugs, AM. J. OBSTET. GYNECOL. 112:668, 1977.
I.
JOSEPH
EVANS, D.
LINDSAY
M.D.
SCHULMAN, GOLDEN,
ANIL
B.
Chicago,
Illinois,
MUKHERJEE, and
intrauterine growth retardation
Bethesda,
M.D. B.S. M.D.,
PH.D.
Maryland
ExWng animal models for inducing intrauterine growth retardation (NJGR~ involve sewere maternal compromise or acute fetal insu by enhancing fetal competition and human chorionk gonadotopm mean number of fetuees was sig reduced; and brain/liver weight ratios oophorectomy before superovulation. horns of the same mother was produ smaller brains and livers and inqeased brainfkir rat&s. In the preconceptual method for achieving IUGR and may bs combir4 with oophorectomy to permit comparisons between fetuses with different gmwth chanxMs&s withln the same mother. (AM. J. OBSTET. GYNECOL. 141:433, 1981.)
INTRAUTERINE GROWTH retardation(IUGR) is associated with significant increases in perinatal morbidity and deaths.lm3 Although studies comparing characteristics of IUGR and normal gestations have been numerous, clinical investigations to elucidate the etiology and pathophysiology of IUGR have intrinsic limitations. Consequently, many investigators have attempted to develop suitable animal models. A widely used system for inducing IUGR in the rat was developed by Wigglesworth.*, 5 One uterine artery is surgically ligated at approximately day 17 of gestation. Although fetal growth retardation occurs in the ligated uterine horn subsequent to the suddenly re-
Fwm the Department of O&et&s and Gynecology, University of Chicago-Pritzker School of Medicine, Chicago, and the Section on Human Biachemical and Developmemtol Genetics, awl Pregnancy Research Branch, Nation& Institute Child Health and Humun Development, National Institutes of Health, Bethesda
af
Received
for publication
Revised
May
Accepted May
December
9, 1980.
8, 1981, 13, 1981.
Re@int re+wts: Joseph D. Schuhnan, M.D., Section it Human Biochemical and Developmental Genetics, Nat&w1 Institutzz af Child Heal& and Human Development, Bethesda, Mqkznd 20205.
duced supply of nutrients to the fetuses,K operation during late pregnancy and the gross abrupt disruption of uterine blood flow are not typical characteristics of naturally occurring IUGR. Several other animal models of I UGR have also been deveIoped, including .etibolization of the placental bkd, stress from hypothermia or hyperthermia, chronic hypoxia, induced msernal renal insufficiency, deprivation of food, and the addition of nutritional or chemical agents (such as cadmium) to the diet.lT-13 Unfortunately, these methods may cause acute fetal insult or substantially alter normal maternal metabolism. Most investigators have considered inadequate placental exchange to be a major cause of IUCR. An animal IUGR model that brought about progressively increasing demands on placental exchange without the need for sudden intrapregnancy intervention OP maternal metabolic alteration would be desirable. Hormonally iridiuced superovulation substanti&y increases the normal number of embryos in the mouse, and thus should enhance competition for maternal nutrients and oxygen. This report describes the fetal effects of superovulaton in the mouse. Superovulation appears to represent a simple and useful method for inducing IUGR in this species. Furthermore, superovulation, combined with a partial unilateral oophorec433
434
Evans
October 15. 19x1 Am. J. Obstet. Gynewl.
et al.
Table I. Effect of superovulation
on fetal characteristics
I
(nonsurgical
Control (n = 19)
Fetuses per mother Body weight (gm) Brain weight (gm) Liver weight (gm) Brain/liver ratio Placental weight (gm) Maternal glucose (mg/dl serum)
6.3 1.26 0.076 0.074 1.04 0.11 114
I
in 0.5
60.2
Superovulated (?I = 100) 20.0 0.97 0.067 0.045 1.58 0.07 129
zt 0.11 t 0.009
IT 0.011 2 0.019 t 0.02 2 10.2
(n = 3) Fetal glucose (mg/dl serum)
group)
” ” f k r + f
I
Superovulation versus control*
p p p p p p
4.7 0.16 0.011 0.014 0.40 0.02 8.1
< 0.01 < 0.05 < 0.05 < 0.01 < 0.001 < 0.05 NSt
(n = 5) 60.9 -+ 23.2
rt 13.7
NS
All values are expressed as mean f SD. *Student’s t test for independent samples. TNot significant.
T&le II. Comparisons
between
fetuses in different
uterine
Control* (n = 26)
No. of fetuses per side Body weight (gm) Brain weight (gm) Liver weight (gm) Brain/liver ratio Placental weight (pm) Fetal glucose (mg/dl serum)
4.5 ” 0.063 0.047
t 0.014 z 0.023
1.52 f 0.50 0.83
PUO*
+: 0.16
51.9 f 13.8
1.14 ” 0.28 0.078 2 0.013 0.070 k 0.027
1.19 * 0.33
0.92 65.2
Dtfferencest (control uersm PUO)
(n = 5)
1.0 f 0.0
1.3
0.99 -+ 0.27
horns after PUO
2 0.17 +. 33.8
3.5 0.15 0.015 0.023 0.33 0.09 13.3
2 2.2
p < 0.05
f 0.09
NS p < 0.05 p < 0.05
f r r rt f
0.012 0.017 0.09 0.01 16.4
P ;;i05 NS
*Mean ? SD. tMean f SD of difference, Student’s t test for paired samples $Not significant. tomy prior the number permitting degrees of
to pregnancy, produces an asymmetry in of fetuses in the two uterine horns, thereby comparisons of fetuses exposed to different crowding within the same mother.
fiia#da and matedal Virgin female randomly bred National Institutes of Health (N-NIH[S]) mice, average age 8 to 10 weeks, were housed under standard laboratory conditions and given food and water ad libitum. In the major study group, the animals did not undergo operation. One week before mating, a smaller group of mice underwent partial unilateral left oophorectomies (-50%) by means of a flank incision and with the use of 0.5 ml of sodium thiopental (Pentothal) anesthesia. The major study group was subdivided into two categories: superovulation and spontaneous ovulation. Superovulated mice were given 5 IU pregnant mare serum (PMS) subcutaneously followed in 48 hours by 5 IU human chorionic gonadotropin (hCG) subcutaneously, and were mated 12 hours later. Control mice were mated without prior administration of hormones. All of the partially oophorectomized mice were also superovulated with PMS and hCG.
For mating, each female was caged with one male overnight. Copulation was confirmed by vaginal~piug the following morning, and the females were then segregated. Animals were killed by cesareansection on day 19 of gestation (term = 20 days). For each fetus, serum glucose and wet weights of the body, placenta, brain, and liver at birth were recorded. Statistical analyses were performed with paired or nonpaired t tests, as indicated, or Pearson product moment correlation analysis. Results SuperovuW~I mice veraus eoss&oIa. Superovulation produced significant alterations in the number of fetuses, body, brain, and liver weights, brain/liver weight ratios, and placental weights compared to controls (Table I). Superovulation resulted in a reduction in mean fetal body weight of 29%, mean brain weight of 12%, mean liver weight of 39%, and mean,placental weight of 34%. The average brain/liver ratio was 1.04 in controls and increased to 1.58 primarily because af the marked reduction in the size of the liver. Mean fetal serum glucose and maternal glucose levels at delivery were similar in the two groups.
Volume 141 Number 1
Superovulation-induced
In mice, most of the uterine blood supply flows from the lateral aspect to midline. In control animals, there was a positive correlation between fetal body weight and proximity to the lateral blood supply (r = 0.77 p < 0.01). With superovulation, however, this relationship was no longer demonstrable (r = 0.10, p = NS). Effect of unilateral oophorectomy on intrauterine comparisons after superovulation. Partial unilateral oophorectomy (PUO) and subsequent superovulation were performed in a small group of animals to assess the feasibility of comparing fetal growth differences within the same pregnant female (analogous to the model of Wigglesworth). PUO resulted in an average of approximately four fetuses on the control side to one fetus on the PUO side. Fetuses on the two sides manifested significant differences in mean brain weight and liver weight. A significantly increased brain/liver weight ratio on the more crowded side was demonstrable (Table II).
Comment IUGR continues to be associated with significant perinatal morbidity and mortality.‘* * Animal models have allowed close experimental scrutiny of many factors related to IUGR; however, all such models have had intrinsic limitations because of various differences from human IUGR. The present report indicates that superovulation is a simple, reliable, nonsurgical method for achieving IUGR in mice. The effects on fetal growth are presumably related to the gradually increasing fetal competition for limited uterine flow and, hence, nutrient and/or oxygen supply. Changes in the brain/liver ratio
IUGR
435
(i.e., brain sparing) are often characteristic of IIJGR in man and are present in the simple superovulation model in mice described here. The usefulness’of this model in studies of IUGR, perhaps including that associated with multiple human gestation, is apparent. The value of this model may be further enhanced in certain experimental situations by the addition of PLrO, a simple surgical procedure, prior to superovulation to allow comparisons between fetuses within the same pregnant animal. Such comparisons have proved to be highly advantageous in Wigglesworth’s IUGR model in the rat. Ideally, superovulation should restore a nearly normal number of fetuses on the PUO side and produce excessive contralateral crowding. Under the conditions that we used in these initial studies, superovulation resulted in a reduced number of gestations on the operated side and modestly increased the number of fetuses on the control side in comparison to those animals without prior operation (see Tables I and II). The important observation was that a significant asymmetry (approximately 4: 1) in fetal distribution and characteristics occurred. The total number of fetuses might possibly have been greater if the postoperative recovery time had been longer prior to superovulation. Although the absolute numbers of fetuses in the PUO group were small, the data in Table II support the validity of this refinement of the induced ovulation technique for selected investigations of IUGR. Finally, these observations reinforce the hypothesis that, even within the same mother, larger numbers of. fetuses per uterine horn are associated with changes typical of IUGR.
REFERENCES
1. Low, J. A., Galbrairh, R. S., Muir, D., Killen, H., Karchmar, J., and Campbell, D.: Intrauterine growth retardation: A preliminary report of long-term morbidity, AM. J. OBSTET.
GYNECOL.
103:534,
1978.
2. Daikoker, N. H., Johnson, J. W. C., Graf, C., Kearney, K., Tyson, I. E., and King, T. M.: Patterns of intrauterine growth Retardation, &&et. Gynecol. 50:211, 1979. 3. Vohr, B. R., Oh, W., Rosenfield, A. G., and Cowett, R. M.: The preterm sma&-for-gestation&age infant: A two-year follow-up study, AM. J. OBSTET. GYNECOL. 133:425,1979. 4. Wigglesworth, J. S.: Experimental growth retardation in the foetal rat, J. Pathol. 88: 1, 1964. 5. Wigglesworth,-J. S.: Fetal growth retardation, Am. J. Pathol. 77:347, 1974. 6. Nitzan, M., Odoff, S., and Schulman, J. D.: Placental transfer of analogues of glucose and amino acids in fetal growth retardation, Pediatr. Res. 13: 100, 1979. 7. Greasy. R. K., Barrett, C. T., DeSwiet, M., Kananpaa, K. U., and Rudolph, A. M.: Experimental intrauterine growth retardation in the sheep, AM. J. OBSTET. GYNECOL. 113:566, 1972.
8. Hensleigh, P. A., and Johnson, D. C.: Heat stress effects during pregnancy. I. Retardation of fetal rat growth, Fertil. Steril. 24:522, 1971. 9. Van Geijn, H. P., Kaylon, W. M., Nicola, K. K., and Zuspan, F. P.: Induction of severe intrauterine growth retardation in the Sprague-Dawley rat, AM. J. ORSTET. GYNECOL. 137:43,
1980.
10. Nitzan, M., Orlo:Ff, S., Chrzanowski, B. L., and Schulman, .I. D.: Intrauterine mowth retardation in renal insufficiency: An experimental model in the rat, AM. J. OBSTET. GYNECCIL. 133:40. 1979. 11. Anderson, L. L. Embryonic and placental development during prolonged inanition in the pig, Am. J. Physiol. 429: 1687, 1975. 12. Webster, W. S.: Cadmium induced fetal growth retardation in the mouse, Arch. Environ. Heakh 33t36, 1978. 13. Scott, J. R.: Fetal growth retardation associated with maternal administration of immunosuppressive drugs, AM. J. OBSTET. GYNECOL. 112:668, 1977.