- Email: [email protected]
Progesterone administration induces preimplantation embryonic loss in mice
Progesterone administration induces preimplantation embryonic loss in mice Challa Harini, Ph.D.,a Sri Bhashyam Sainath, M.Sc.,a and Pamanji Sreenivasula Reddy, Ph.D.a,b a
Department of Biotechnology, Sri Venkateswara University, Tirupati, India; and b Department of Zoology, Sri Venkateswara University, Tirupati, India
Objective: To evaluate the effect of progesterone during early pregnancy on pregnancy maintenance in mice. Design: Experimental study or laboratory investigation. Setting: An established molecular reproduction laboratory, Department of Biotechnology, S.V. University. Animal(s): Healthy virgin female Swiss albino mice of 8 weeks of age. Intervention(s): After confirmation of the heat period, adult female mice were mated with mature healthy males to achieve pregnancy. Inseminated females received intraperitoneal injections of progesterone at doses of 0, 1, 3.5, 7, 15, 25, or 50 mg/kg body weight on the first, third, and seventh day of pregnancy. Main Outcome Measure(s): Preimplantation embryonic loss. Result(s): Pregnancy failure was evidenced by reduction in the number of embryos in females injected with 7 (25.12 %), 15 (38.44 %), 25 (100%), and 50 (100%) mg progesterone/kg body weight. In females with a successful pregnancy, the numbers of corpora lutea and postimplantation loss per dam were comparable across all groups. No increase in the incidence of malformed fetuses was found in any progesterone-treated groups. Conclusion(s): Administration of supranormal levels of progesterone during early pregnancy caused a reduction in the number of implantations and an increase in preimplantation loss in mice. (Fertil Steril 2009;91:2137–41. 2009 by American Society for Reproductive Medicine.) Key Words: Early pregnancy, pregnancy failure, preimplantation loss
Hormone interplay is a key process in the maintenance of pregnancy in mammals. Pregnancy is a dynamic process with immense anatomic and physiologic changes that occur from fertilization to parturition. Depending on the stage of embryo and fetal development, any agent administered at this stage interferes with the secretion of estrogens and progesterone, and results in termination of gestation (1–3). Exposure to diethylstilbesterol (a drug that was extensively used to support pregnancy) resulted in reduced fertility, reproductive tract anomalies, and increased incidence of vaginal adenocarcinoma in women (4). Several xenoestrogens have also been reported to affect implantation, gestation, and fetal growth in mice and rats (5–7). In view of this, an elaborate program was initiated in our laboratory to evaluate the effect of synthetic hormones on reproduction in rats and mice. Earlier studies from our laboratory suggests that in utero exposure to hydroxyprogesterone decreased the activity levels of testicular steroidogenic enzymes (8) and suppressed spermatogenesis (9) in rats. The circulatory levels of testosterone, follicle stimulating hormone, and luteinizing hormone in adult male rats exposed to hydroxyprogesterone during embryonic development Received March 8, 2008; revised May 29, 2008; accepted June 12, 2008; published online August 9, 2008. C.H. has nothing to disclose. S.B.S. has nothing to disclose. P.S.R. has nothing to disclose. Reprint requests: Pamanji Sreenivasula Reddy, Ph.D., Department of Biotechnology, Sri Venkateswara University, University Campus, Tirupati, Andihra Pradesh 517 502, India (FAX: 877-224-9611; E-mail: [email protected]).
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was also significantly altered (10). The gestational exposure to hydroxyprogesterone also suppressed reproductive potential in adult male rats (11). The present study was undertaken to examine the effects of graded doses of progesterone on dams and development of their offspring when administered to pregnant mice during very early pregnancy. The mouse, as one of the most common laboratory animals, is widely used in basic biologic research, and could provide useful information that is relevant to human biology. MATERIALS AND METHODS Animals and Maintenance Swiss albino mice, purchased from the Indian Institute of Sciences, Bangalore, were used for the present study. Virgin female mice of 8 weeks of age (body weight 25 2 g) at late proestrus cycle were mated with fertile males (one female with one male mouse). Female mice showing sperm in the vaginal smear on the next day, designated as day 1 of pregnancy, were selected for the experiment. Different stages of the estrus cycle and confirmation of pregnancy were determined according to Deb et al. (12). The pregnant mice were isolated and distributed on a random basis into seven groups and housed individually. The animals were maintained in an air-conditioned animal house facility (temperature: 25 1 C; light:dark, 12-hour:12-hour; relative humidity 55 5%) at the Department of Biotechnology, Sri Venkateswara University, Tirupati, India. The mice were housed in sterilized polypropylene cages lined with paddy husk and reared on a pellet diet (HLL Animal Feed,
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Bangalore, India) and water ad libitum. All animal procedures were approved by the institutional review board and Institutional Animal Care and Ethics Committee at S.V. University. Test Chemical and Dosing The pregnant mice in group 1 that served as controls were treated the same as the experimental group but received injections of caster oil and benzyl benzoate (1:1.7) in a 20-mL volume. The pregnant mice in groups 2 to 7 were injected with progesterone at a dose of 1, 3.5, 7, 15, 25, and 50 mg/kg body weight, respectively, on the first, third, and seventh day of pregnancy. Progesterone was purchased from Sigma Aldrich Company (St. Louis, MO) and dissolved in caster oil and benzyl benzoate (1:1.7). The volume of each dose was adjusted to 20 mL/mice. Progesterone injections were given to women at a dose of 25 to 100 mg twice weekly, or maximum daily, from day 15 to weeks 8 to 16 of pregnancy (Monthly Index of Medical Specialties, ISSN: 0970-1036, Vol. 26: p. 158). Observations Maternal body weight gain, food consumption, and clinical signs of toxicity were recorded daily. Eight mice from each group were autopsied following cervical dislocation on day 6 of gestation. The peritoneal cavity and uterus were opened and the number of implantation sites and resorption sites were recorded (13). The ovaries were isolated and the number of corpora lutea was determined. The remaining animals (n ¼ 8) were sacrificed on day 16 of gestation and the numbers of live and dead fetuses in the uterus were determined. It should be noted that no attempt was made to further characterize the dead or resorbed fetuses. All live fetuses removed from the uterus were sexed, weighed, and inspected for external malformations. The fetuses were also examined for skeletal malformations using Alizarin red-S according to the protocol described earlier (14). Data were analyzed to determine the preimplantation loss (difference between the number of corpora lutea and the number of implantation sites, expressed as per number of corpora lutea), and postimplantation loss (difference between the number of implantation sites and the number of live fetuses, expressed as per number of implantation sites). Data Analysis Statistical analysis of the offspring data was performed using the litter as a unit. The data were evaluated by analysis of variance and Dunnet’s multiple comparison test. RESULTS Table 1 shows the findings in pregnant mice administered with progesterone on the first, third, and seventh day of pregnancy. Neither deaths nor clinical signs of toxicity occurred in the pregnant mice in any of the groups. The net body weight gain and food consumption of the progesterone-in2138
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jected mice were also not significantly different than that in the control mice (data not shown). In the control group, all inseminated females become pregnant and the number of implantations were 11.94 1.11 (Table 1; Fig. 1A). Pregnancy failure, as evidenced by decrease in number of implantations, was found in the 7- and 15-mg/kg group (Fig. 1B). The number of implantations decreased significantly in the 7- and 15-mg/kg groups (25.12% and 38.44%, respectively) when compared with the control group. No implantations were found in the sperm-positive mice injected with 25 mg (Fig. 1C) and the 50-mg progesterone/kg group. The numbers of corpora lutea per mouse in the progesterone-treated groups were comparable to the control group. The number of implanted embryos per mouse significantly decreased in the progesterone-injected groups (7 mg/kg body weight onward) in a dose-dependent manner. A significant increase in the incidence of preimplantation loss was also observed in the 7and 15-mg progesterone/kg body weight injected groups (25.56 and 38.07, respectively), whereas there was no significant difference in the incidence of postimplantation loss between the progesterone-injected groups and the control group. The sex ratio of live fetuses was comparable across all the groups. The body weights of fetuses were not significantly different in experimental groups when compared with those in the control group. The results of external and skeletal examinations in fetuses of mice administered progesterone during pregnancy is shown in Table 2. No significant external (cleft palate, polydactyly, syndactyly, macrophthalmia, microphthalmia, blunttipped tail, kinky tail, short tail, drooping wrist, subdermal hemorrhagic patches, anogenital distance) and skeletal (fusion of sternebrae, fusion of ribs, ossification of nasal, frontal, parietal, interparietal, caudal, carpal, metacarpal, tarsal, and claws) anomalies were observed in the fetuses of progesterone-injected mice.
DISCUSSION In the present study, mice injected with graded doses of progesterone during the early stages of pregnancy showed a reduction in the rate of successful pregnancy in female mice that had a positive mating. In females with embryos, however, progesterone had no adverse effect on the numbers of corpora lutea. It is unlikely that progesterone adversely affects maternal gamete production. However, a decreased number of fetuses was found at 7-mg progesterone/kg body weight and above in a dose-dependent manner in mice. The possibility still remains that progesterone during very early stages of pregnancy may be interfering with implantations. Further studies are required to characterize this effect more precisely. Exposure of female rodents to diethylstilbesterol, a very potent synthetic estrogen that was used extensively to support pregnancy, caused multiple reproductive disorders in experimental animals including early embryonic loss (15, 16). The possible mechanisms by which an exogenous chemical Vol. 91, No. 5, Supplement, May 2009
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TABLE 1 Effect of injection of graded doses of progesterone in first, third, and seventh day of pregnancy on reproductive findings in mice. Dose (mg/kg body weight) Parameters No. of pregnant mice No. of corpora lutea/mousea No. of implantations/ mousea No. of preimplantation loss/mouse % of preimplantation loss/mouseb No. of embryos resorbed No. of fetuses/mousea
0 (control) 6
1 6
3.5 6
7 6
15
25
50
6
6
6
12.07 1.21 11.97 1.12 11.98 1.05 12.01 1.09 11.87 1.13 11.78 1.20 11.94 1.17 (0.82 %) (0.74%) (0.49%) (1.65%) (2.4%) (1.07%) 11.94 1.11 11.72 1.13 11.08 1.21 8.94 0.75 7.35 0.67 — — (1.84) (7.202) (25.12) (38.44) 0.13 0.25 0.9 3.07 4.52 — — 1.07
2.08
7.512
0
0
0
25.56 0
38.07 0
11.43 0.48 11.31 0.34 10.66 0.42 8.83 0.31 7.16 0.31 (0.78%) (6.7%) (22.74%) (37.35%) No. of postimplantation 0.51 0.41 0.42 0.11 0.19 loss/mouse % of postimplantation 4.27 3.50 3.79 1.23 2.58 loss/mousec Body weight of 2.31 0.21 2.29 0.24 2.31 0.22 2.30 0.18 2.28 0.20 fetuses (g) (0.865%) (0) (0.43%) (1.29%)
Note: ns ¼ not significant. a Values are given as mean SD. b (No. of preimplantation loss/corpora lutea) 100. c (No. postimplantation loss/no. implantations) 100. Harini. Effect of progesterone on implantations. Fertil Steril 2009.
—
—
—
—
—
—
—
—
—
—
—
—
F value
P
0.042
ns
23.807
< .0001
105.63
0.0229
< .0001
ns
2139
FIGURE 1 (A) Uterus of control showing normal implantations on 18th day of pregnancy. At autopsy diestrus. (B) Uterus showing implantations of pregnant mice treated with progesterone (15 mg/kg body weight) on first, third, and seventh day of pregnancy. At autopsy diestrus. (C) Uterus showing no implantations of pregnant mice treated with progesterone (25 mg/kg body weight) on the first, third, and seventh day of pregnancy. At autopsy diestrus.
Harini. Effect of progesterone on implantations. Fertil Steril 2009.
interferes in early pregnancy loss includes toxic effects on the gametes, alteration in the genital tracts transport of early conceptus, impairment of endometrial receptivity to the implanting blastocyst, and postimplantation abnormalities of development, hormone transport, or nutrition (17). In the present study, the time points selected for progesterone administration, days 1, 3, and 7 of pregnancy, correspond to the period that begins after fertilization and involves the before and after implantation stages. The uterus is in a receptive stage during days 2 to 3 of pregnancy in mice, and the timing of blastocyst implantation occurs normally at night (2200–2300 h) of day 4 (18, 19). Ovarian steroid hormones are necessary to prepare the endometrium for the process of
implantation. In mice, both ovarian progesterone and estrogen are required for implantation. The ovary secretes a small amount of estrogen in addition to progesterone on the morning of day 4 of pregnancy in mice. This preimplantation estrogen secretion is an absolute requirement for blastocyst activation, preparation of the uterus, and initiation of the implantation process. Any chemical insult before completion of the implantation process should result in preimplantation embryo loss. In the present study, inseminated females injected with progesterone on days 1, 3, and 7 revealed increases in the preimplantation loss but not postimplantation. Thus, it is likely that progesterone may affect adversely during the
TABLE 2 Effect of injection of graded doses of progesterone in first, third, and seventh day of pregnancy on external and skeletal changes in fetuses of rats on 18th day of pregnancy. Dose (mg/kg body weight) 0 (Control) 1 3.5 7 15 25 50
No. of fetuses examined
No. of fetuses with external anomalies
No. of fetuses with skeletal anomalies
65 (6) 63 (6) 64 (6) 53 (6) 43 (6) — —
0 0 0 0 1 — —
0 0 0 0 2 — —
Note: Values in the parentheses are number of mice in each group. Harini. Effect of progesterone on implantations. Fertil Steril 2009.
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preimplantation period but not after the implantation. The inhibition of implantation by chemicals and hormones is hypothesized to be because of imbalance in the estrogen and progesterone ratio in mice (20–22). The reduction in implantations in the progesterone-injected mice may be because of a similar imbalance in estrogen and progesterone ratios that are critical during normal implantation (23, 24). It was also reported recently (25) that injection of progesterone in a pregnant patient led to development of transient parkinsonism with a risk of abortion. Although the present data do not provide information about the mechanism of the preimplantation loss, we show here that maternal receptivity and/or embryos may be highly susceptible to the hormonal imbalance before completion of implantation process. The present data demonstrates that the embryos are highly susceptible to changes in hormone levels in the early stages of development. Further investigation is necessary to determine the mechanism of blockade of embryo implantation by progesterone. Extrapolation of mice data to humans is always difficult. However, it should be noted that progesterone under different trade names is routinely prescribed to protect the pregnancy in women in this part of the country; the present research cautions the importance of selection of dose and time of administration. Acknowledgments: We thank Mr. S. Umasankar for maintaining the mice colony in the department. The experiments were conducted following the rules and regulations of the Institutional Animal Care and Ethical Committee, and comply with the laws of the country. The authors gratefully acknowledge the assistance of Prof. K.V.S. Sarma for statistical analysis of data, and Dr. T. Pushpalatha for assistance with fetus collection and examination. We thank the University Grants Commission, New Delhi, for financial assistance in the form of a research grant F.No.32-502/2006 (SR) to P.S.R.
REFERENCES 1. Dickman Z, Hart JR. The significance of daily administration of progesterone and estrogen for the maintenance of pregnancy in the ovarectomized rat. Endrocrinology 1972;90:1667–9. 2. Glasser SR, Northeutt RC, Chytill F, Strott CA. The influence of an antisteroidogenic drug (Aminoglutathimide phosphate) on pregnancy maintenance. Endrocrinology 1972;22:467–72. 3. Matt DW, Borzelleca JF. Toxic effects on the female reproductive system during pregnancy, parturition and lactation. In: Raphael J, Witorsch A, eds. Reproductive toxicology. New York: Raven Press Ltd, 1995:175–93. 4. Newbold RR, Mc Lachlan JA. Transplacental hormonal carcinogenesis: diethylstilbestrol as an example. In: Huff J, Boyd JA, Barrett JC, eds. Cellular and molecular mechanisms of hormonal carcinogenesis: environmental influences. New York: Wiley-Liss, 1996:131–47. 5. Khan MA. Reproduction and growth of progeny of female mice mated after treatment with crufomate. J Environ Sci Health 1981;16:141–57. 6. Fish SA. Organophosphorus cholinesterase inhibitors and fetal development. Am J Obstet Gynecol 1966;96:48–54.
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7. Soratur SM, Kaliwal BB. Effect of methyl parathion on pregnancy in albino rats. Ecol Env Cons 1998;4:145–9. 8. Pushpalatha T, Ramachandra Reddy P, Sreenivasula Reddy P. Effect of prenatal exposure to hydroxyprogesterone on steroidogenic enzymes in male rats. Naturwissenschaften 2003;90:40–3. 9. Pushpalatha T, Ramachandra Reddy P, Trivikram G, Sreenivasula Reddy P. Reduced spermatogenesis in rats exposed transplacentally to hydroxyprogesterone. Cytologia 2003;68:369–73. 10. Pushpalatha T, Ramachandra Reddy P, Sreenivasula Reddy P. Impairment of male reproduction in adult rats exposed to hydroxyprogesterone caproate in utero. Naturwissenschaften 2004;91:242–4. 11. Pushpalatha T, Ramachandra Reddy P, Sreenivasula Reddy P. Gestational exposure to hydroxyprogesterone caproate suppresses reproductive potential in male rats. Naturwissenschaften 2005;92:385–8. 12. Deb K, Reese J, Paria BC. Methodologies to study implantation in mice. Methods in molecular medicine. In: Soares MJ, Hunt JS, eds. Placenta and trophoblast: methods and protocols. Vol. 121. Totowa, NJ: Humana Press Inc., 2005:9–34. 13. Hood RD. Hand book of developmental toxicology. New York: CRC Press, Inc., 1997. 14. Kawamura S, Hirohashi A, Kato T, Yasuda M. Bone-staining technique for fetal rat specimens without skinning and removing adipose tissue. Cong Anom 1990;30:93–5. 15. Mc Lachlan JA. Rodent models for perinatal exposure to diethylstilbestrol and their relation to human disease in the male. In: Herbst AL, Bern HA, eds. Developmental aspects of diethylstilbestrol (DES) in pregnancy. New York: Thime Verlag, 1981:148–57. 16. Marselos M, Tomatis L, Diethylstilbestrol II. Pharmacology, toxicology and carcinogenicity in experimental animals. Eur J Cancer 1993;29A: 149–55. 17. Wilcox A, Weinberg C, Baird D. Caffeinated beverages and decreased fertility. Lancet 1988;2:1553–5. 18. Beaudoin AR. Embryology and teratology. In: Baker HJ, Lindsey JR, Weisbroth SH, eds. The laboratory rat: research applications. New York: Academic Press, 1980:75–94. 19. Das SK, Wang XN, Paria BC, Damm D, Abraham JA, Klagsbrun M, et al. Heparin-binding EGF like growth factor gene is induced in the mouse uterus temporally by blastocysts solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF receptorin implantation. Development 1994;120:1071–83. 20. Yochim J, Zarrow MX. Action of estradiol, progesterone and relaxin in the maintenance of gestation in castrated pregnant rat. Fertil Steril 1961;12:263–76. 21. Nutting EF, Meyer RK. Implantation delay, nidation and embryonal survival in rats treated with ovarian hormones. In: Enders AC, ed. Delayed implantation. Chicago: University of Chicago Press, 1963:223–52. 22. Prasad MRN, Kalra SP, Segal SJ. Effect of clomiphone on blastocysts during delayed implantation in the rat. Fertil Steril 1965;6:101–5. 23. Morris JM, Van Wagenen G, Mc Cann T, Jacob D. Compounds interfering with ovum implantation and development II. Synthetic estrogens and antiestrogens. Fertil Steril 1967;18:18–34. 24. Morris P, Binionda Z, Gillam MP, Harkey MR, Zhou C, Henderson GL, et al. The effect of chronic cocaine exposure during pregnancy on material and infant outcomes in the rhesus monkey. Neurotoxicol Teratol 1996;18:147–54. 25. Demirkiran M, Aslan K, Bicakci S, Bozdemir H, Ozeren A. Transient parkinsonism: induced by progesterone or pregnancy? Mov Disord 2004;19:1382–4.
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Department of Biotechnology, Sri Venkateswara University, Tirupati, India; and b Department of Zoology, Sri Venkateswara University, Tirupati, India
Objective: To evaluate the effect of progesterone during early pregnancy on pregnancy maintenance in mice. Design: Experimental study or laboratory investigation. Setting: An established molecular reproduction laboratory, Department of Biotechnology, S.V. University. Animal(s): Healthy virgin female Swiss albino mice of 8 weeks of age. Intervention(s): After confirmation of the heat period, adult female mice were mated with mature healthy males to achieve pregnancy. Inseminated females received intraperitoneal injections of progesterone at doses of 0, 1, 3.5, 7, 15, 25, or 50 mg/kg body weight on the first, third, and seventh day of pregnancy. Main Outcome Measure(s): Preimplantation embryonic loss. Result(s): Pregnancy failure was evidenced by reduction in the number of embryos in females injected with 7 (25.12 %), 15 (38.44 %), 25 (100%), and 50 (100%) mg progesterone/kg body weight. In females with a successful pregnancy, the numbers of corpora lutea and postimplantation loss per dam were comparable across all groups. No increase in the incidence of malformed fetuses was found in any progesterone-treated groups. Conclusion(s): Administration of supranormal levels of progesterone during early pregnancy caused a reduction in the number of implantations and an increase in preimplantation loss in mice. (Fertil Steril 2009;91:2137–41. 2009 by American Society for Reproductive Medicine.) Key Words: Early pregnancy, pregnancy failure, preimplantation loss
Hormone interplay is a key process in the maintenance of pregnancy in mammals. Pregnancy is a dynamic process with immense anatomic and physiologic changes that occur from fertilization to parturition. Depending on the stage of embryo and fetal development, any agent administered at this stage interferes with the secretion of estrogens and progesterone, and results in termination of gestation (1–3). Exposure to diethylstilbesterol (a drug that was extensively used to support pregnancy) resulted in reduced fertility, reproductive tract anomalies, and increased incidence of vaginal adenocarcinoma in women (4). Several xenoestrogens have also been reported to affect implantation, gestation, and fetal growth in mice and rats (5–7). In view of this, an elaborate program was initiated in our laboratory to evaluate the effect of synthetic hormones on reproduction in rats and mice. Earlier studies from our laboratory suggests that in utero exposure to hydroxyprogesterone decreased the activity levels of testicular steroidogenic enzymes (8) and suppressed spermatogenesis (9) in rats. The circulatory levels of testosterone, follicle stimulating hormone, and luteinizing hormone in adult male rats exposed to hydroxyprogesterone during embryonic development Received March 8, 2008; revised May 29, 2008; accepted June 12, 2008; published online August 9, 2008. C.H. has nothing to disclose. S.B.S. has nothing to disclose. P.S.R. has nothing to disclose. Reprint requests: Pamanji Sreenivasula Reddy, Ph.D., Department of Biotechnology, Sri Venkateswara University, University Campus, Tirupati, Andihra Pradesh 517 502, India (FAX: 877-224-9611; E-mail: [email protected]).
0015-0282/09/$36.00 doi:10.1016/j.fertnstert.2008.06.031
was also significantly altered (10). The gestational exposure to hydroxyprogesterone also suppressed reproductive potential in adult male rats (11). The present study was undertaken to examine the effects of graded doses of progesterone on dams and development of their offspring when administered to pregnant mice during very early pregnancy. The mouse, as one of the most common laboratory animals, is widely used in basic biologic research, and could provide useful information that is relevant to human biology. MATERIALS AND METHODS Animals and Maintenance Swiss albino mice, purchased from the Indian Institute of Sciences, Bangalore, were used for the present study. Virgin female mice of 8 weeks of age (body weight 25 2 g) at late proestrus cycle were mated with fertile males (one female with one male mouse). Female mice showing sperm in the vaginal smear on the next day, designated as day 1 of pregnancy, were selected for the experiment. Different stages of the estrus cycle and confirmation of pregnancy were determined according to Deb et al. (12). The pregnant mice were isolated and distributed on a random basis into seven groups and housed individually. The animals were maintained in an air-conditioned animal house facility (temperature: 25 1 C; light:dark, 12-hour:12-hour; relative humidity 55 5%) at the Department of Biotechnology, Sri Venkateswara University, Tirupati, India. The mice were housed in sterilized polypropylene cages lined with paddy husk and reared on a pellet diet (HLL Animal Feed,
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Bangalore, India) and water ad libitum. All animal procedures were approved by the institutional review board and Institutional Animal Care and Ethics Committee at S.V. University. Test Chemical and Dosing The pregnant mice in group 1 that served as controls were treated the same as the experimental group but received injections of caster oil and benzyl benzoate (1:1.7) in a 20-mL volume. The pregnant mice in groups 2 to 7 were injected with progesterone at a dose of 1, 3.5, 7, 15, 25, and 50 mg/kg body weight, respectively, on the first, third, and seventh day of pregnancy. Progesterone was purchased from Sigma Aldrich Company (St. Louis, MO) and dissolved in caster oil and benzyl benzoate (1:1.7). The volume of each dose was adjusted to 20 mL/mice. Progesterone injections were given to women at a dose of 25 to 100 mg twice weekly, or maximum daily, from day 15 to weeks 8 to 16 of pregnancy (Monthly Index of Medical Specialties, ISSN: 0970-1036, Vol. 26: p. 158). Observations Maternal body weight gain, food consumption, and clinical signs of toxicity were recorded daily. Eight mice from each group were autopsied following cervical dislocation on day 6 of gestation. The peritoneal cavity and uterus were opened and the number of implantation sites and resorption sites were recorded (13). The ovaries were isolated and the number of corpora lutea was determined. The remaining animals (n ¼ 8) were sacrificed on day 16 of gestation and the numbers of live and dead fetuses in the uterus were determined. It should be noted that no attempt was made to further characterize the dead or resorbed fetuses. All live fetuses removed from the uterus were sexed, weighed, and inspected for external malformations. The fetuses were also examined for skeletal malformations using Alizarin red-S according to the protocol described earlier (14). Data were analyzed to determine the preimplantation loss (difference between the number of corpora lutea and the number of implantation sites, expressed as per number of corpora lutea), and postimplantation loss (difference between the number of implantation sites and the number of live fetuses, expressed as per number of implantation sites). Data Analysis Statistical analysis of the offspring data was performed using the litter as a unit. The data were evaluated by analysis of variance and Dunnet’s multiple comparison test. RESULTS Table 1 shows the findings in pregnant mice administered with progesterone on the first, third, and seventh day of pregnancy. Neither deaths nor clinical signs of toxicity occurred in the pregnant mice in any of the groups. The net body weight gain and food consumption of the progesterone-in2138
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jected mice were also not significantly different than that in the control mice (data not shown). In the control group, all inseminated females become pregnant and the number of implantations were 11.94 1.11 (Table 1; Fig. 1A). Pregnancy failure, as evidenced by decrease in number of implantations, was found in the 7- and 15-mg/kg group (Fig. 1B). The number of implantations decreased significantly in the 7- and 15-mg/kg groups (25.12% and 38.44%, respectively) when compared with the control group. No implantations were found in the sperm-positive mice injected with 25 mg (Fig. 1C) and the 50-mg progesterone/kg group. The numbers of corpora lutea per mouse in the progesterone-treated groups were comparable to the control group. The number of implanted embryos per mouse significantly decreased in the progesterone-injected groups (7 mg/kg body weight onward) in a dose-dependent manner. A significant increase in the incidence of preimplantation loss was also observed in the 7and 15-mg progesterone/kg body weight injected groups (25.56 and 38.07, respectively), whereas there was no significant difference in the incidence of postimplantation loss between the progesterone-injected groups and the control group. The sex ratio of live fetuses was comparable across all the groups. The body weights of fetuses were not significantly different in experimental groups when compared with those in the control group. The results of external and skeletal examinations in fetuses of mice administered progesterone during pregnancy is shown in Table 2. No significant external (cleft palate, polydactyly, syndactyly, macrophthalmia, microphthalmia, blunttipped tail, kinky tail, short tail, drooping wrist, subdermal hemorrhagic patches, anogenital distance) and skeletal (fusion of sternebrae, fusion of ribs, ossification of nasal, frontal, parietal, interparietal, caudal, carpal, metacarpal, tarsal, and claws) anomalies were observed in the fetuses of progesterone-injected mice.
DISCUSSION In the present study, mice injected with graded doses of progesterone during the early stages of pregnancy showed a reduction in the rate of successful pregnancy in female mice that had a positive mating. In females with embryos, however, progesterone had no adverse effect on the numbers of corpora lutea. It is unlikely that progesterone adversely affects maternal gamete production. However, a decreased number of fetuses was found at 7-mg progesterone/kg body weight and above in a dose-dependent manner in mice. The possibility still remains that progesterone during very early stages of pregnancy may be interfering with implantations. Further studies are required to characterize this effect more precisely. Exposure of female rodents to diethylstilbesterol, a very potent synthetic estrogen that was used extensively to support pregnancy, caused multiple reproductive disorders in experimental animals including early embryonic loss (15, 16). The possible mechanisms by which an exogenous chemical Vol. 91, No. 5, Supplement, May 2009
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TABLE 1 Effect of injection of graded doses of progesterone in first, third, and seventh day of pregnancy on reproductive findings in mice. Dose (mg/kg body weight) Parameters No. of pregnant mice No. of corpora lutea/mousea No. of implantations/ mousea No. of preimplantation loss/mouse % of preimplantation loss/mouseb No. of embryos resorbed No. of fetuses/mousea
0 (control) 6
1 6
3.5 6
7 6
15
25
50
6
6
6
12.07 1.21 11.97 1.12 11.98 1.05 12.01 1.09 11.87 1.13 11.78 1.20 11.94 1.17 (0.82 %) (0.74%) (0.49%) (1.65%) (2.4%) (1.07%) 11.94 1.11 11.72 1.13 11.08 1.21 8.94 0.75 7.35 0.67 — — (1.84) (7.202) (25.12) (38.44) 0.13 0.25 0.9 3.07 4.52 — — 1.07
2.08
7.512
0
0
0
25.56 0
38.07 0
11.43 0.48 11.31 0.34 10.66 0.42 8.83 0.31 7.16 0.31 (0.78%) (6.7%) (22.74%) (37.35%) No. of postimplantation 0.51 0.41 0.42 0.11 0.19 loss/mouse % of postimplantation 4.27 3.50 3.79 1.23 2.58 loss/mousec Body weight of 2.31 0.21 2.29 0.24 2.31 0.22 2.30 0.18 2.28 0.20 fetuses (g) (0.865%) (0) (0.43%) (1.29%)
Note: ns ¼ not significant. a Values are given as mean SD. b (No. of preimplantation loss/corpora lutea) 100. c (No. postimplantation loss/no. implantations) 100. Harini. Effect of progesterone on implantations. Fertil Steril 2009.
—
—
—
—
—
—
—
—
—
—
—
—
F value
P
0.042
ns
23.807
< .0001
105.63
0.0229
< .0001
ns
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FIGURE 1 (A) Uterus of control showing normal implantations on 18th day of pregnancy. At autopsy diestrus. (B) Uterus showing implantations of pregnant mice treated with progesterone (15 mg/kg body weight) on first, third, and seventh day of pregnancy. At autopsy diestrus. (C) Uterus showing no implantations of pregnant mice treated with progesterone (25 mg/kg body weight) on the first, third, and seventh day of pregnancy. At autopsy diestrus.
Harini. Effect of progesterone on implantations. Fertil Steril 2009.
interferes in early pregnancy loss includes toxic effects on the gametes, alteration in the genital tracts transport of early conceptus, impairment of endometrial receptivity to the implanting blastocyst, and postimplantation abnormalities of development, hormone transport, or nutrition (17). In the present study, the time points selected for progesterone administration, days 1, 3, and 7 of pregnancy, correspond to the period that begins after fertilization and involves the before and after implantation stages. The uterus is in a receptive stage during days 2 to 3 of pregnancy in mice, and the timing of blastocyst implantation occurs normally at night (2200–2300 h) of day 4 (18, 19). Ovarian steroid hormones are necessary to prepare the endometrium for the process of
implantation. In mice, both ovarian progesterone and estrogen are required for implantation. The ovary secretes a small amount of estrogen in addition to progesterone on the morning of day 4 of pregnancy in mice. This preimplantation estrogen secretion is an absolute requirement for blastocyst activation, preparation of the uterus, and initiation of the implantation process. Any chemical insult before completion of the implantation process should result in preimplantation embryo loss. In the present study, inseminated females injected with progesterone on days 1, 3, and 7 revealed increases in the preimplantation loss but not postimplantation. Thus, it is likely that progesterone may affect adversely during the
TABLE 2 Effect of injection of graded doses of progesterone in first, third, and seventh day of pregnancy on external and skeletal changes in fetuses of rats on 18th day of pregnancy. Dose (mg/kg body weight) 0 (Control) 1 3.5 7 15 25 50
No. of fetuses examined
No. of fetuses with external anomalies
No. of fetuses with skeletal anomalies
65 (6) 63 (6) 64 (6) 53 (6) 43 (6) — —
0 0 0 0 1 — —
0 0 0 0 2 — —
Note: Values in the parentheses are number of mice in each group. Harini. Effect of progesterone on implantations. Fertil Steril 2009.
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Effect of progesterone on implantations
Vol. 91, No. 5, Supplement, May 2009
preimplantation period but not after the implantation. The inhibition of implantation by chemicals and hormones is hypothesized to be because of imbalance in the estrogen and progesterone ratio in mice (20–22). The reduction in implantations in the progesterone-injected mice may be because of a similar imbalance in estrogen and progesterone ratios that are critical during normal implantation (23, 24). It was also reported recently (25) that injection of progesterone in a pregnant patient led to development of transient parkinsonism with a risk of abortion. Although the present data do not provide information about the mechanism of the preimplantation loss, we show here that maternal receptivity and/or embryos may be highly susceptible to the hormonal imbalance before completion of implantation process. The present data demonstrates that the embryos are highly susceptible to changes in hormone levels in the early stages of development. Further investigation is necessary to determine the mechanism of blockade of embryo implantation by progesterone. Extrapolation of mice data to humans is always difficult. However, it should be noted that progesterone under different trade names is routinely prescribed to protect the pregnancy in women in this part of the country; the present research cautions the importance of selection of dose and time of administration. Acknowledgments: We thank Mr. S. Umasankar for maintaining the mice colony in the department. The experiments were conducted following the rules and regulations of the Institutional Animal Care and Ethical Committee, and comply with the laws of the country. The authors gratefully acknowledge the assistance of Prof. K.V.S. Sarma for statistical analysis of data, and Dr. T. Pushpalatha for assistance with fetus collection and examination. We thank the University Grants Commission, New Delhi, for financial assistance in the form of a research grant F.No.32-502/2006 (SR) to P.S.R.
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