Adverse effects of a superovulatory regimen of equine chorionic gonadotropin and gonadotropin-releasing hormone on embryo implantation in mice

Document Type : Original Article

Authors

1 College of Animal Science and Technology, Hebei Technology Innovation Center of Cattle and Sheep Embryo, Hebei Agricultural University, Baoding, China

2 College of Veterinary Medicine, Hebei Agricultural University, Baoding, China

10.30466/vrf.2025.2054637.4679
Abstract
Although equine chorionic gonadotropin (eCG) combined with gonadotropin-releasing hormone (GnRH) is known to improve reproductive rates by increasing the numbers of ovulating oocytes, the actions of these hormones on embryo implantation have not been elucidated. We herein eliminated the effects of embryos by performing embryo transfer and elucidated the effects on embryo implantation after hormonal treatment. The recipient mice were allocated to four groups, including control, eCG, GnRH, and eCG + GnRH groups. Well-developed blastocysts were transferred into recipients, and implantation sites were counted five days after embryo transfer. Our results revealed that embryo implantation rates in the eCG and eCG + GnRH groups were significantly lower than GnRH and control groups; eCG and eCG + GnRH groups didn’t differ, and the implantation rate was the lowest in the eCG + GnRH group. When we evaluated estrogen and progesterone concentrations in four groups on embryonic day 0.5 to embryonic day 3.5, we noted that serum estrogen levels in eCG + GnRH group were significantly higher than the other groups, while progesterone levels were significantly lower than the control group, and that the progesterone/estrogen ratio was significantly reduced relative to the control group. Finally, the expression of the endometrial receptivity-related Lif gene was significantly diminished in the eCG + GnRH and eCG groups compared to the other groups. These results suggest that eCG combined with GnRH as a super-ovulatory regimen reduced mouse embryo implantation rates via an estrogen-mediated impairment of endometrial receptivity.

Keywords

Subjects

1.     Behringer R, Gertsenstein M, Nagy KV, et al. Administration of gonadotropins for superovulation in mice.Cold Spring Harb Protoc 2018; 2018(1): 092403. doi: 10.1101/pdb.prot092403.
2.     Crispo M, Meikle MN, Schlapp G, et al. Ovarian superstimulatory response and embryo development using a new recombinant glycoprotein with eCG-like activity in mice. Theriogenology 2021; 164: 31-35.
3.     Baojiang W, Hong-yan X,Liping C, et al. Effect of PMSG/hCG superovulation on mouse embryonic development. J Integr Agr 2013; 12(06): 1066-1072.
4.     Brasil OO, Moreira NH, Santos Júnior G, et al. Super-ovulatory and embryo yielding in sheep using increased exposure time to progesterone associated with a GnRH agonist. Small Rumin Res 2016; 136: 54-58.
5.     Humaidan P, Papanikolaou EG, Tarlatzis BC, et al. GnRHa to trigger final oocyte maturation: a time to reconsider. Hum Reprod 2009; 24(10): 2389-2394.
6.     Atsuko K, Minako T, Mami M, et al. Establishment of superovulation procedure in Japanese field vole, Microtus montebelli. Theriogenology 2016; 86(3): 899-905.
7.     Kageyama A, Tanaka M, Morita M, et al. Effect of GnRH agonist and letrozole treatment in women with recurrent implantation failure.Fertil Steril 2019; 112(1):98-104.
8.     Segal T, Amini P, Wang J, et al. Superovulation with human chorionic gonadotropin (hCG) trigger and gonadotropin releasing hormone agonist (GnRHa) trigger differentially alter essential angiogenic factors in the endometrium in a mouse ART model. Biol Reprod 2020; 102(5): 1122-1133.
9.     Miller I, Chuderland D, Ron-El R, et al. GnRH agonist triggering modulates PEDF to VEGF ratio inversely to hCG in granulosa cells. J Clin Endocrinol Metab 2015; 100(11): E1428-E1436.
10. Techakumphu M, Phutikanit N, Suadsong S, et al. The effect of GnRH supplement of FSH and PMSG treatments for prepubertal swamp buffalo calves (Bubalus bubalis). J Vet Med Sci 2000; 62(3): 269-272.
11. Walker SK, Smith DH, Seamark RF.Timing of multiple ovulations in the ewe after treatmentwith FSH or PMSG with and without GnRH. J Reprod Fertil 1986; 77(1): 135-142.
12. Libby V, Wilson R, Kresak A, et al. Superovulation with gonadotropin-releasing hormone agonist or chorionic gonadotropin for ovulation trigger differentially affects leukocyte populations in the peri-implantation mouse uterus. F S Sci 2021; 2(2): 198-206.
13. Richardson A, Brearley S, Ahitan S, et al. A clinically useful simplified blastocyst grading system. Reprod Biomed Online 2015; 31(4): 523-553.
14. Pierce CA, Block RA., Aguinis H. Cautionary note on reporting eta-squared values from multifactor ANOVA designs. Educ Psychol Meas 2004; 64(6): 916-924.
15. Zhang J, Wu X, Li X. GnRH administration after estrus induction protocol decreases the pregnancy rate of recipient ewes following transfer of frozen-thawed embryos. Small Rumin Res 2022; 217: 106849. doi: 10.1016/j.smallrumres.2022.106849.
16. Ertzeid G, Storeng R. Adverse effects of gonadotrophin treatment on pre- and postimplantation development in mice. J Reprod Fertil 1992; 96(2): 649-655.
17. Kelley RL, Kind KL, Lane M, et al. Recombinant human follicle-stimulating hormone alters maternal ovarian hormone concentrations and the uterus and perturbs fetal development in mice. Am J Physiol Endocrinol Metab 2006; 291(4): E761-E770.
18. Ezoe K, Murata N, Yabuuchi A, et al. Evaluation of uterine receptivity after gonadotropin releasing hormone agonist administration as an oocyte maturation trigger: a rodent model. Sci Rep 2019; 9: 12519. doi: 10.1038/s41598-019-48918-3.
19. Lin LZ, NI HM, Liu YH, et al. Effect of anti-PMSG on distribution of estrogen receptor alpha and progesterone receptor in mouse ovary, oviduct and uterus. Zygote 2015; 23(5): 695-703.
20. Simón C, Garcia Velasco JJ, Valbuena D, et al. Increasing uterine receptivity by decreasing estradiol levels during the preimplantation period in high responders with the use of a follicle-stimulating hormone step-down regimen. Fertil Steril 1998; 70(2): 234-239.
21. Aliabadi E, Makoolati Z, Talaei-Khozani T, et al. Histochemical study of the rat uterine glycoconjugate alteration following treatment with exogenous gonadotropic hormones during the implantation period. Biomed Res Int 2020; 2020: 3967427. doi: 10.1155/2020/3967427.
22. Evans J, Salamonsen LA, Winship A, et al. Fertile ground: human endometrial programming and lessons in health and disease. Nat Rev Endocrinol 2016; 12(11): 654-667.
23. Davidson LM, Coward K. Molecular mechanisms of membrane interaction at implantation. Birth Defects Res C Embryo Today 2016; 108(1): 19-32.
24. Lubahn DB, Moyer JS, Golding TS, et al. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci U S A 1993; 90(23): 11162-11166.
25. Tranguch S, Daikoku T, Guo Y, et al. Molecular complexity in establishing uterine receptivity and im-plantation. Cell Mol life Sci 2005; 62(17): 1964-1973.
26. Wang H, Dey SK. Roadmap to embryo implantation: clues from mouse models. Nat Rev Genet 2006; 7(3): 185-199.
27. Maclin VM, Radwanska E, Binor Z, et al. Progesterone: estradiol ratios at implantation in ongoing pregnancies, abortions, and nonconception cycles resulting from ovulation induction. Fertil Steril 1990; 54(2): 238-244.
28. Basir GS, O WS, Ng EH, et al. Morphometric analysis of peri-implantation endometrium in patients having excessively highoestradiol concentrations after ovarian stimulation. Hum Reprod 2001; 16(3): 435-440.
29. Horcajadas JA, Riesewijk A, Polman J, et al. Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles. Mol Hum Reprod 2005; 11(3): 195-200.
30. Steward RG, Zhang CE, Shah AA, et al. High peak estradiol predicts higher miscarriage and lower live birth rates in high responders triggered with a GnRH agonist in IVF/ICSI cycles. J Reprod Med 2015; 60(11-12): 463-470.
31. Xia X, Zhang Y, Cao M, et al. Adverse effect of assisted reproductive technology-related hyperoestrogensim on the secretion and absorption of uterine fluid in superovulating mice during the peri-implantation period. Front Endocrinol (Lausanne) 2023; 14: 859204. doi: 10.3389/fendo.2023.859204.
32. Junovich G, Mayer Y, Azpiroz A, et al. Ovarian stimulation affects the levels of regulatory endometrial NK cells and angiogenic cytokine VEGF. Am J Reprod Immunol 2011; 65(2): 146-153.
33. Papanikolaou EG, Bourgain C, Kolibianakis E, et al. Steroid receptor expression in late follicular phase endometrium in GnRH antagonist IVF cycles is already altered, indicatinginitiation of early luteal phase transformation in the absence of secretory changes. Hum Reprod 2005; 20(6): 1541-1547.
34. Chen JR, Cheng JG, Shatzer T, et al. Leukemia inhibitory factor can substitute for nidatory estrogen and is essential to inducing a receptive uterus for implantation but is not essential for subsequent embryogenesis. Endocrinology 2000; 141(12):4365-4372.
35. Fukui Y, Hirota Y, Saito-Fujita T, et al. Uterine epithelial lif receptors contribute to implantation chamber formation in blastocyst attachment. Endocrinology 2021; 162(11): bqab169. doi: 10.1210/endocr/bqab169.
36. Song H, Lim H, Das SK, et al. Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF- deficient mice. Mol Endocrinol 2000; 14(8): 1147-1161.
37. Stewart CL, Kaspar P, Brunet LJ, et al. Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor.Nature 1992; 359(6390): 76-79.
38. Heng-Chao R, Xiao-Ming Z, Qiong L, et al. Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin β3 and leukaemia-inhibitory factor and improves uterine receptivity in mice. Hum Reprod 2006; 21(10): 2521-2529.
39. Diao H, Xiao S, Zhao F, et al. Uterine luminal epithelium-specific proline-rich acidic protein 1 (PRAP1) as a marker for successful embryo implantation. Fertil Steril 2010; 94(7): 2808-11.e1. doi: 10.1016/ j.fertnstert.2010.06.034.
40. Xiong GF, Zhang YS, Han BC, et al. Estradiol-regulated proline-rich acid protein 1 is repressed by class I histone deacetylase and functions in peri-implantation mouse uterus. Mol Cell Endocrinol 2011; 331(1): 23-33.
41. Liu YF, Li MY, Yan YP, et al. ERα-dependent stimulation of LCN2 in uterine epithelium during mouse early pregnancy. Reproduction 2020: 159(4): 493-501.
42. Liu YF, Deng WB, Li SY, et al. Progesterone induces the expression of lipocalin-2 through Akt-c-Myc pathway during mouse decidualization. FEBS Lett 2016; 590(16): 2594-2602.
43. Deis J, Lin TY, Bushman T, et al. Lipocalin 2 deficiency alters prostaglandin biosynthesis and mTOR signaling regulation of thermogenesis and lipid metabolism in adipocytes. Cells 2022; 11(9): 1535. doi: 10.3390/ cells11091535.
Volume 17, Issue 8
August 2026
Pages 545-552

  • Receive Date 28 February 2025
  • Revise Date 16 May 2025
  • Accept Date 19 July 2025