Skip to main content
SpringerLink
Log in

Controlled Ovarian Stimulation Protocols Alter Endometrial Histomorphology and Gene Expression Profiles

  • Original Article
  • Published:
Reproductive Sciences Aims and scope Submit manuscript

Abstract

Although it is well appreciated that ovarian stimulation protocols for in vitro fertilization (IVF) alter endometrial receptivity, the precise cellular mechanisms are not known. To gain insights into potential mechanisms by which different ovarian stimulation protocols alter the endometrium, we compared histologic and gene expression profiles of endometrium from women undergoing conventional ovarian stimulation for IVF (C-IVF) with those undergoing minimal stimulation with clomiphene citrate (MS-IVF). Sixteen women undergoing MS-IVF (n = 8) or C-IVF (n = 8) were recruited for endometrial biopsy at the time of oocyte retrieval. Endometrial glands were large, tortuous, and secretory with C-IVF but small and undifferentiated with MS-IVF. Whereas RNA sequencing did not reveal changes in estrogen receptor or its co-regulators or classic proliferation associated genes in MS-IVF, together with immunohistochemistry, Wnt signaling was disrupted in endometrium from MS-IVF cycles with significant upregulation of Wnt inhibitors. Secreted frizzled-related protein 1 (sFRP1) was increased fourfold (p < 0.01), and sFRP4 was upregulated sixfold (p < 0.01) relative to C-IVF. Further these proteins were localized to subepithelial endometrial stroma. These data indicate that MS-IVF protocols with CC do not seem to impact endometrial estrogen signaling as much as would be expected from the reported antiestrogenic properties of CC. Rather, the findings of this study highlight Wnt signaling as a major factor for endometrial development during IVF cycles.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Practice Committee of the American Society for Reproductive Medicine. Electronic address Aao. Comparison of pregnancy rates for poor responders using IVF with mild ovarian stimulation versus conventional IVF: a guideline. Fertil Steril 2018;109(6):993–999.

  2. Heijnen EMEW, Eijkemans MJC, De Klerk C, et al. A mild treatment strategy for in-vitro fertilisation: a randomised non-inferiority trial. Lancet. 2007;369(9563):743–9.

    Article  Google Scholar 

  3. Karimzadeh MA, Ahmadi S, Oskouian H, Rahmani E. Comparison of mild stimulation and conventional stimulation in ART outcome. Arch Gynecol Obstet. 2010;281(4):741–6.

    Article  Google Scholar 

  4. Reed B, Babayev S, Bukulmez O. Shifting paradigms in diminished ovarian reserve and advanced reproductive age in assisted reproduction: customization instead of conformity. Semin Reprod Med. 2015;33(03):169–78.

    Article  Google Scholar 

  5. Dehbashi S, Parsanezhad ME, Alborzi S, Zarei A. Effect of clomiphene citrate on endometrium thickness and echogenic patterns. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics. 2003;80(1):49–53.

    Article  CAS  Google Scholar 

  6. Haritha S, Rajagopalan G. Follicular growth, endometrial thickness, and serum estradiol levels in spontaneous and clomiphene citrate-induced cycles. International journal of gynaecology and obstetrics: the official organ of the International Federation of Gynaecology and Obstetrics. 2003;81(3):287–92.

    Article  CAS  Google Scholar 

  7. Nakamura Y, Ono M, Yoshida Y, Sugino N, Ueda K, Kato H. Effects of clomiphene citrate on the endometrial thickness and echogenic pattern of the endometrium. Fertil Steril. 1997;67(2):256–60.

    Article  CAS  Google Scholar 

  8. Reed BG, Wu JL, Nemer LB, Carr BR, Bukulmez O. Use of clomiphene citrate in minimal stimulation in vitro fertilization negatively impacts endometrial thickness: an argument for a freeze-all approach. JBRA Assist Reprod. 2018;22(4):355–62.

    PubMed  PubMed Central  Google Scholar 

  9. Badawy A, Abdel Aal I, Abulatta M. Clomiphene citrate or anastrozole for ovulation induction in women with polycystic ovary syndrome? A prospective controlled trial. Fertil Steril. 2009;92(3):860–3.

    Article  CAS  Google Scholar 

  10. Barroso G, Menocal G, Felix H, Rojas-Ruiz JC, Arslan M, Oehninger S. Comparison of the efficacy of the aromatase inhibitor letrozole and clomiphene citrate as adjuvants to recombinant follicle-stimulating hormone in controlled ovarian hyperstimulation: a prospective, randomized, blinded clinical trial. Fertil Steril. 2006;86(5):1428–31.

    Article  CAS  Google Scholar 

  11. Gerli S, Gholami H, Manna C, Di Frega AS, Vitiello C, Unfer V. Use of ethinyl estradiol to reverse the antiestrogenic effects of clomiphene citrate in patients undergoing intrauterine insemination: a comparative, randomized study. Fertil Steril. 2000;73(1):85–9.

    Article  CAS  Google Scholar 

  12. Amita M, Takahashi T, Tsutsumi S, Ohta T, Takata K, Henmi N, et al. Molecular mechanism of the inhibition of estradiol-induced endometrial epithelial cell proliferation by clomiphene citrate. Endocrinology. 2010;151(1):394–405.

    Article  CAS  Google Scholar 

  13. Ferraretti AP, La Marca A, Fauser BCJM, et al. ESHRE consensus on the definition of 'poor response' to ovarian stimulation for in vitro fertilization: the Bologna criteria. Hum Reprod. 2011;26(7):1616–24.

    Article  CAS  Google Scholar 

  14. Noyes RW, Hertig AT, Rock J. Dating the endometrial biopsy. Fertil Steril. 1950;1(1):3–25.

    Article  Google Scholar 

  15. Hart SN, Therneau TM, Zhang Y, Poland GA, Kocher JP. Calculating sample size estimates for RNA sequencing data. J Comput Biol. 2013;20(12):970–8.

    Article  CAS  Google Scholar 

  16. Aronesty E. Comparison of sequencing utility programs. The Open Bioinformatics Journal. 2013;7(1):1–8.

    Article  Google Scholar 

  17. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013;14(4):R36–6.

    Article  Google Scholar 

  18. Liao Y, Smyth GK, Shi W. FeatureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923–30.

    Article  CAS  Google Scholar 

  19. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2009;26(1):139–40.

    Article  Google Scholar 

  20. Kamath MS, Maheshwari A, Bhattacharya S, Lor KY, Gibreel A. Oral medications including clomiphene citrate or aromatase inhibitors with gonadotropins for controlled ovarian stimulation in women undergoing in vitro fertilisation. Cochrane Database Syst Rev. 2017;11:CD008528.

    PubMed  Google Scholar 

  21. Haskell SG. Selective estrogen receptor modulators. South Med J. 2003;96(5):469–76.

    Article  Google Scholar 

  22. Amita M, Takahashi T, Igarashi H, Nagase S. Clomiphene citrate down-regulates estrogen receptor-alpha through the ubiquitin-proteasome pathway in a human endometrial cancer cell line. Mol Cell Endocrinol. 2016;428:142–7.

    Article  CAS  Google Scholar 

  23. Kiewisz J, Wasniewski T, Kmiec Z. Participation of WNT and beta-catenin in physiological and pathological endometrial changes: association with angiogenesis. Biomed Res Int. 2015;2015:854056.

    Article  Google Scholar 

  24. Tranguch S, Daikoku T, Guo Y, Wang H, Dey SK. Molecular complexity in establishing uterine receptivity and implantation. Cell Mol Life Sci. 2005;62(17):1964–73.

    Article  CAS  Google Scholar 

  25. Cooke PS, Spencer TE, Bartol FF, Hayashi K. Uterine glands: development, function and experimental model systems. Mol Hum Reprod. 2013;19(9):547–58.

    Article  CAS  Google Scholar 

  26. Gustafson CT, Mamo T, Maran A, Yaszemski MJ. Molecular strategies for modulating Wnt signaling. Front Biosci (Landmark Ed). 2017;22:137–56.

    Article  CAS  Google Scholar 

  27. Rattner A, Hsieh JC, Smallwood PM, et al. A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors. Proc Natl Acad Sci U S A. 1997;94(7):2859–63.

    Article  CAS  Google Scholar 

  28. Bafico A, Gazit A, Pramila T, Finch PW, Yaniv A, Aaronson SA. Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling. J Biol Chem. 1999;274(23):16180–7.

    Article  CAS  Google Scholar 

  29. Carmon KS, Loose DS. Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells. Mol Cancer Res. 2008;6(6):1017–28.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

C. X. was partially supported by National Institutes of Health Grant UL1TR001105. Tissues from which cells were cultured were obtained from the Human Biological Tissues and Cell Core funded by NIH P01 HD087150 (RAW).

Author information

Authors and Affiliations

  1. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    John L. Wu, Patrick Keller, Samir N Babayev, Bruce R Carr, Orhan Bukulmez & R. Ann Word

  2. The Cecil H. and Ida Green Center for Reproductive Biology Sciences, Department of Obstetrics and Gynecology, The Green Center for Reproductive Biological Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Patrick Keller & R. Ann Word

  3. Eugene McDermott Center for Human Growth and Development, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Mohammed Kanchwala & Chao Xing

  4. Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Chao Xing

  5. Department of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Chao Xing

Authors
  1. John L. Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Patrick Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohammed Kanchwala
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chao Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Samir N Babayev
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bruce R Carr
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Orhan Bukulmez
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. R. Ann Word
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J. W. conducted the experiments, analysis, and wrote the manuscript. P. K. contributed to experimental design. M.K. and C.X. provided bioinformatic and statistical analysis. S.B., J.W., and O.B. identified patients, obtained informed consent, and conducted endometrial biopsies. B.C., O.B., and R.A.W. contributed to experimental design, analysis, data interpretation, and writing of the manuscript. All authors gave final approval of the article.

Corresponding author

Correspondence to R. Ann Word.

Ethics declarations

Conflict of Interests

BC received research grants from AbbVie and Medicines360. JW, PK, MK, CX, SB, OB, and RW have nothing to declare. No other potential conflicts of interest relevant to this article were reported.

Electronic Supplementary Material

ESM 1

(PDF 143 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wu, J.L., Keller, P., Kanchwala, M. et al. Controlled Ovarian Stimulation Protocols Alter Endometrial Histomorphology and Gene Expression Profiles. Reprod. Sci. 27, 895–904 (2020). https://doi.org/10.1007/s43032-019-00093-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s43032-019-00093-6

Keywords

Search

Navigation