Cumulus cell pappalysin-1, luteinizing hormone/choriogonadotropin receptor, amphiregulin and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 mRNA levels associate with oocyte developmental competence and embryo outcomes

  • Richard J. Kordus
  • Akhtar Hossain
  • Michael C. Corso
  • Hrishikesh Chakraborty
  • Gail F. Whitman-Elia
  • Holly A. LaVoieEmail author
Assisted Reproduction Technologies



To determine whether a selected set of mRNA biomarkers expressed in individual cumulus granulosa cell (CC) masses show association with oocyte developmental competence, embryo ploidy status, and embryo outcomes.


This prospective observational cohort pilot study assessed levels of mRNA biomarkers in 163 individual CC samples from 15 women stimulated in antagonist cycles. Nineteen mRNA biomarker levels were measured by real-time PCR and related to the development of their corresponding individually cultured oocytes and subsequent embryos, embryo ploidy status, and live birth outcomes.


PAPPA mRNA levels were significantly higher in CC from oocytes that led to euploid embryos resulting in live births and aneuploid embryos compared to immature oocytes by ANOVA. LHCGR mRNA levels were significantly higher in CC of oocytes resulting in embryos associated with live birth compared to immature oocytes and oocytes resulting in arrested embryos by ANOVA. Using a general linearized mixed model to assess ploidy status, CC HSD3B mRNA levels in oocytes producing euploid embryos were significantly lower than other oocyte outcomes, collectively. When transferred euploid embryos outcomes were analyzed by ANOVA, AREG mRNA levels were significantly lower and PAPPA mRNA levels significantly higher in CC from oocytes that produced live births compared to transferred embryos that did not form a pregnancy.


Collectively, PAPPA, LHCGR, and AREG mRNA levels in CC may be able to identify oocytes with the best odds of resulting in a live birth, and HSD3B1 mRNA levels may be able to identify oocytes capable of producing euploid embryos.


Cumulus cells Real-time PCR mRNA levels Oocyte developmental competence Euploid embryo 



This study was supported by an ASPIRE-I grant from the University of South Carolina. MCC was supported by the University of South Carolina School of Medicine Research Program for Medical Students.

Compliance with ethical standards

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and in its later amendments or comparable ethical standards. This study was approved by the University of South Carolina Institution Review Board (IRB registration number: 00000240).

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10815_2019_1489_Fig6_ESM.png (733 kb)
Supplemental Fig. 1

Biomarkers for CC mRNA expression not associated with mature oocyte competence and embryo outcomes. To determine the differences in CC mRNA between groups where oocytes had different developmental and embryo outcomes target mRNA levels were compared using repeated measures ANOVA followed by Tukey’s post hoc test (adjusted P-values) for pairwise comparisons. Groups represent CC mRNA from oocytes with the following descriptions: Aneuploid = mature oocytes resulting in aneuploid embryos; Arrested = mature oocytes resulting in embryos that did not reach the blastocyst stage; Failed Fert = mature oocytes that did not fertilize; Immature = immature oocytes that were not fertilized; Live Birth = oocytes that resulted in transferred euploid embryos that resulted in live births; No Pregnancy = oocytes that resulted in transferred euploid embryos that did not result in a pregnancy. No differences were seen in these biomarkers (P > 0.05). Data are presented as stated in fig. 3 legend (PNG 732 kb)

10815_2019_1489_MOESM1_ESM.tif (911 kb)
High resolution image (TIF 911 kb)
10815_2019_1489_Fig7_ESM.png (262 kb)
Supplemental Fig. 2

GLMM model distinguishing CC from oocytes giving rise to live births and oocytes with outcomes not resulting in live birth, collectively. To evaluate the potential of CC biomarker mRNA levels to identify oocytes capable of producing euploid embryos resulting in a live birth versus groups not producing live births, the best fitting model included 110 CC samples from 14 patients and included biomarkers: CYP11A1, CYP19A1, IGFBP5, PAPPA, PGRMC1, and STARD1. Increased PAPPA mRNA expression (P < 0.05) significantly increased the odds of an oocytes producing an embryo resulting in a live birth (OR = 4.591, 95% CI: 1.098, to 19.201). All previously stated categories were included in this model except oocytes yielding euploid embryos that were not transferred. Groups represent CC mRNA from oocytes with the following descriptions: Live Birth = mature oocytes that resulted in euploid embryos that lead to live births; Non-Viable = immature oocytes, mature oocytes resulting in aneuploid blastocysts, oocytes producing embryos that did not reach the blastocyst stage, and mature oocytes that failed to fertilize, collectively. Data are presented as in fig. 3 legend (PNG 262 kb)

10815_2019_1489_MOESM2_ESM.tif (428 kb)
High resolution image (TIF 428 kb)
10815_2019_1489_MOESM3_ESM.docx (33 kb)
ESM 1 (DOCX 32 kb)


  1. 1.
    Uyar A, Torrealday S, Seli E. Cumulus and granulosa cell markers of oocyte and embryo quality. Fertil Steril. 2013;99(4):979–97. S0015-0282(13)00185-4. Scholar
  2. 2.
    Chen M, Wei S, Hu J, Quan S. Can comprehensive chromosome screening technology improve IVF/ICSI outcomes? A meta-analysis. PLoS One. 2015;10(10):e0140779.;PONE-D-15-32011.Google Scholar
  3. 3.
    Scott RT Jr, Upham KM, Forman EJ, Hong KH, Scott KL, Taylor D, et al. Blastocyst biopsy with comprehensive chromosome screening and fresh embryo transfer significantly increases in vitro fertilization implantation and delivery rates: a randomized controlled trial. Fertil Steril. 2013;100(3):697–703. S0015–0282(13)00549–9. Scholar
  4. 4.
    Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet. 2012;5(1):24.: 1755-8166-5-24. Scholar
  5. 5.
    Chang J, Boulet SL, Jeng G, Flowers L, Kissin DM. Outcomes of in vitro fertilization with preimplantation genetic diagnosis: an analysis of the United States Assisted Reproductive Technology Surveillance Data, 2011–2012. Fertil Steril. 2016;105(2):394–400. S0015-0282(15)02029-4. Scholar
  6. 6.
    Kushnir VA, Darmon SK, Albertini DF, Barad DH, Gleicher N. Effectiveness of in vitro fertilization with preimplantation genetic screening: a reanalysis of United States assisted reproductive technology data 2011-2012. Fertil Steril. 2016;106(1):75–9. S0015-0282(16)00140-0. Scholar
  7. 7.
    Zhang S, Luo K, Cheng D, Tan Y, Lu C, He H, et al. Number of biopsied trophectoderm cells is likely to affect the implantation potential of blastocysts with poor trophectoderm quality. Fertil Steril. 2016;105(5):1222–7. S0015-0282(16)00046-7. Scholar
  8. 8.
    Nel-Themaat L, Nagy ZP. A review of the promises and pitfalls of oocyte and embryo metabolomics. Placenta. 2011;32(Suppl 3):S257–S63. S0143-4004(11)00208-6. Scholar
  9. 9.
    Uyar A, Seli E. Metabolomic assessment of embryo viability. Semin Reprod Med. 2014;32(2):141–52. Scholar
  10. 10.
    Kordus RJ, LaVoie HA. Granulosa cell biomarkers to predict pregnancy in ART: pieces to solve the puzzle. Reproduction. 2017;153(2):R69–83. :REP-16-0500. Scholar
  11. 11.
    Tanghe S, Van SA, Nauwynck H, Coryn M, de KA. Minireview: functions of the cumulus oophorus during oocyte maturation, ovulation, and fertilization. Mol Reprod Dev. 2002;61(3):414–24.;10.1002/mrd.10102.Google Scholar
  12. 12.
    Ekart J, McNatty K, Hutton J, Pitman J. Ranking and selection of MII oocytes in human ICSI cycles using gene expression levels from associated cumulus cells. Hum Reprod. 2013;28(11):2930–42: det357. Scholar
  13. 13.
    Iager AE, Kocabas AM, Otu HH, Ruppel P, Langerveld A, Schnarr P, et al. Identification of a novel gene set in human cumulus cells predictive of an oocyte’s pregnancy potential. Fertil Steril. 2013;99(3):745–52. S0015-0282(12)02385-0. Scholar
  14. 14.
    Wathlet S, Adriaenssens T, Segers I, Verheyen G, Van d V, Coucke W, et al. Cumulus cell gene expression predicts better cleavage-stage embryo or blastocyst development and pregnancy for ICSI patients. Hum Reprod. 2011;26(5):1035–51. der036. Scholar
  15. 15.
    Fragouli E, Wells D, Iager AE, Kayisli UA, Patrizio P. Alteration of gene expression in human cumulus cells as a potential indicator of oocyte aneuploidy. Hum Reprod. 2012;27(8):2559–68. :des170. Scholar
  16. 16.
    Green KA, Franasiak JM, Werner MD, Tao X, Landis JN, Scott RT Jr, et al. Cumulus cell transcriptome profiling is not predictive of live birth after in vitro fertilization: a paired analysis of euploid sibling blastocysts. Fertil Steril. 2018;109(3):460–6. S0015-0282(17)32045-9. Scholar
  17. 17.
    Parks JC, Patton AL, McCallie BR, Griffin DK, Schoolcraft WB, Katz-Jaffe MG. Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth. Reprod BioMed Online. 2016;32(5):518–26. :S1472-6483(16)00052-3. Scholar
  18. 18.
    Assidi M, Dufort I, Ali A, Hamel M, Algriany O, Dielemann S, et al. Identification of potential markers of oocyte competence expressed in bovine cumulus cells matured with follicle-stimulating hormone and/or phorbol myristate acetate in vitro. Biol Reprod. 2008;79(2):209–22. Scholar
  19. 19.
    Blaha M, Nemcova L, Kepkova KV, Vodicka P, Prochazka R. Gene expression analysis of pig cumulus-oocyte complexes stimulated in vitro with follicle stimulating hormone or epidermal growth factor-like peptides. Reprod Biol Endocrinol. 2015;13:113. Scholar
  20. 20.
    Christenson LK, Gunewardena S, Hong X, Spitschak M, Baufeld A, Vanselow J. Research resource: preovulatory LH surge effects on follicular theca and granulosa transcriptomes. Mol Endocrinol. 2013;27(7):1153–71. me.2013-1093. Scholar
  21. 21.
    Glister C, Satchell L, Knight PG. Changes in expression of bone morphogenetic proteins (BMPs), their receptors and inhibin co-receptor betaglycan during bovine antral follicle development: inhibin can antagonize the suppressive effect of BMPs on thecal androgen production. Reproduction. 2010;140(5):699–712. Scholar
  22. 22.
    Nivet AL, Vigneault C, Blondin P, Sirard MA. Changes in granulosa cells’ gene expression associated with increased oocyte competence in bovine. Reproduction. 2013;145(6):555–65. Scholar
  23. 23.
    Saini N, Singh MK, Shah SM, Singh KP, Kaushik R, Manik RS, et al. Developmental competence of different quality bovine oocytes retrieved through ovum pick-up following in vitro maturation and fertilization. Animal. 2015;9(12):1979–85. Scholar
  24. 24.
    Vigone G, Merico V, Prigione A, Mulas F, Sacchi L, Gabetta M, et al. Transcriptome based identification of mouse cumulus cell markers that predict the developmental competence of their enclosed antral oocytes. BMC Genomics. 2013;14:380. Scholar
  25. 25.
    Allegra A, Raimondo S, Volpes A, Fanale D, Marino A, Cicero G, et al. The gene expression profile of cumulus cells reveals altered pathways in patients with endometriosis. J Assist Reprod Genet. 2014;31(10):1277–85. doi. Scholar
  26. 26.
    Barcelos ID, Donabella FC, Ribas CP, Meola J, Ferriani RA, de Paz CC, et al. Down-regulation of the CYP19A1 gene in cumulus cells of infertile women with endometriosis. Reprod BioMed Online. 2015;30(5):532–41. S1472-6483(15)00058-9. Scholar
  27. 27.
    Greenseid K, Jindal S, Hurwitz J, Santoro N, Pal L. Differential granulosa cell gene expression in young women with diminished ovarian reserve. Reprod Sci. 2011;18(9):892–9. 18/9/892 [pii]. Scholar
  28. 28.
    May-Panloup P, Ferre-L'Hotellier V, Moriniere C, Marcaillou C, Lemerle S, Malinge MC, et al. Molecular characterization of corona radiata cells from patients with diminished ovarian reserve using microarray and microfluidic-based gene expression profiling. Hum Reprod. 2012;27(3):829–43. der431. Scholar
  29. 29.
    Gardner DK, Lane M, Stevens J, Schlenker T, Schoolcraft WB. Blastocyst score affects implantation and pregnancy outcome: towards a single blastocyst transfer. Fertil Steril. 2000;73(6):1155–8. doi:S0015028200005185. Scholar
  30. 30.
    Nelson-Degrave VL, Wickenheisser JK, Hendricks KL, Asano T, Fujishiro M, Legro RS, et al. Alterations in mitogen-activated protein kinase kinase and extracellular regulated kinase signaling in theca cells contribute to excessive androgen production in polycystic ovary syndrome. Mol Endocrinol. 2005;19(2):379–90. :me.2004-0178. Scholar
  31. 31.
    Bates D, Machler M, Bolker B, Walker S. Fitting linear mixed-effects models using lme4. J Stat Softw. 2015;67(1):1–48. Scholar
  32. 32.
    Hothorn T, Bretz F, Westfall P. Simultaneous inference in general parametric models. Biom J. 2008;50(3):346–63. doi. Scholar
  33. 33.
    Laursen LS, Overgaard MT, Soe R, Boldt HB, Sottrup-Jensen L, Giudice LC, et al. Pregnancy-associated plasma protein-A (PAPP-A) cleaves insulin-like growth factor binding protein (IGFBP)-5 independent of IGF: implications for the mechanism of IGFBP-4 proteolysis by PAPP-A. FEBS Lett. 2001;504(1–2):36–40 doi:S0014-5793(01)02760-0 [pii].Google Scholar
  34. 34.
    Firth SM, Baxter RC. Cellular actions of the insulin-like growth factor binding proteins. Endocr Rev. 2002;23(6):824–54. doi. Scholar
  35. 35.
    Wang HS, Chard T. IGFs and IGF-binding proteins in the regulation of human ovarian and endometrial function. J Endocrinol. 1999;161(1):1–13. no DOI available. Scholar
  36. 36.
    Maman E, Yung Y, Kedem A, Yerushalmi GM, Konopnicki S, Cohen B, et al. High expression of luteinizing hormone receptors messenger RNA by human cumulus granulosa cells is in correlation with decreased fertilization. Fertil Steril. 2012;97(3):592–8. :S0015-0282(11)02911-6. Scholar
  37. 37.
    Papamentzelopoulou M, Mavrogianni D, Partsinevelos GA, Marinopoulos S, Dinopoulou V, Theofanakis C, et al. LH receptor gene expression in cumulus cells in women entering an ART program. J Assist Reprod Genet. 2012;29(5):409–16. Scholar
  38. 38.
    Hamel M, Dufort I, Robert C, Gravel C, Leveille MC, Leader A, et al. Identification of differentially expressed markers in human follicular cells associated with competent oocytes. Hum Reprod. 2008;23(5):1118–27. den048. Scholar
  39. 39.
    Borgbo T, Povlsen BB, Andersen CY, Borup R, Humaidan P, Grondahl ML. Comparison of gene expression profiles in granulosa and cumulus cells after ovulation induction with either human chorionic gonadotropin or a gonadotropin-releasing hormone agonist trigger. Fertil Steril. 2013;100(4):994–1001. :S0015-0282(13)00648-1. Scholar
  40. 40.
    Hayashi KG, Ushizawa K, Hosoe M, Takahashi T. Differential genome-wide gene expression profiling of bovine largest and second-largest follicles: identification of genes associated with growth of dominant follicles. Reprod Biol Endocrinol. 2010;8:11. 1477-7827-8-11. Scholar
  41. 41.
    Kristensen SG, Mamsen LS, Jeppesen JV, Botkjaer JA, Pors SE, Borgbo T, et al. Hallmarks of human small antral follicle development: implications for regulation of ovarian steroidogenesis and selection of the dominant follicle. Front Endocrinol. 2017;8:376. Scholar
  42. 42.
    Sisco B, Hagemann LJ, Shelling AN, Pfeffer PL. Isolation of genes differentially expressed in dominant and subordinate bovine follicles. Endocrinology. 2003;144(9):3904–13. doi. Scholar
  43. 43.
    Huang Y, Zhao Y, Yu Y, Li R, Lin S, Zhang C, et al. Altered amphiregulin expression induced by diverse luteinizing hormone receptor reactivity in granulosa cells affects IVF outcomes. Reprod BioMed Online. 2015;30(6):593–601. :S1472-6483(15)00109-1. Scholar
  44. 44.
    Feuerstein P, Cadoret V, Dalbies-Tran R, Guerif F, Bidault R, Royere D. Gene expression in human cumulus cells: one approach to oocyte competence. Hum Reprod. 2007;22(12):3069–77. dem336. Scholar
  45. 45.
    Adriaenssens T, Wathlet S, Segers I, Verheyen G, De VA, Van der Elst J, et al. Cumulus cell gene expression is associated with oocyte developmental quality and influenced by patient and treatment characteristics. Hum Reprod. 2010;25(5):1259–70. deq049. Scholar
  46. 46.
    Assou S, Haouzi D, Dechaud H, Gala A, Ferrieres A, Hamamah S. Comparative gene expression profiling in human cumulus cells according to ovarian gonadotropin treatments. Biomed Res Int. 2013;2013:354582:1–13. Scholar
  47. 47.
    Grondahl ML, Borup R, Lee YB, Myrhoj V, Meinertz H, Sorensen S. Differences in gene expression of granulosa cells from women undergoing controlled ovarian hyperstimulation with either recombinant follicle-stimulating hormone or highly purified human menopausal gonadotropin. Fertil Steril. 2009;91(5):. S0015-0282(08)00499-8):1820–30. Scholar
  48. 48.
    Hurwitz JM, Jindal S, Greenseid K, Berger D, Brooks A, Santoro N, et al. Reproductive aging is associated with altered gene expression in human luteinized granulosa cells. Reprod Sci. 2010;17(1):56–67. 1933719109348028. Scholar
  49. 49.
    Al-Edani T, Assou S, Ferrieres A, Bringer DS, Gala A, Lecellier CH, et al. Female aging alters expression of human cumulus cells genes that are essential for oocyte quality. Biomed Res Int. 2014;2014:964614:1–10. Scholar
  50. 50.
    Rabinowitz M, Ryan A, Gemelos G, Hill M, Baner J, Cinnioglu C, et al. Origins and rates of aneuploidy in human blastomeres. Fertil Steril. 2012;97(2):395–401. S0015-0282(11)02810-X. Scholar
  51. 51.
    Assidi M, Montag M, Van d V, Sirard MA. Biomarkers of human oocyte developmental competence expressed in cumulus cells before ICSI: a preliminary study. J Assist Reprod Genet. 2011;28(2):173–88. Scholar
  52. 52.
    Tsutsumi R, Hiroi H, Momoeda M, Hosokawa Y, Nakazawa F, Koizumi M, et al. Inhibitory effects of cholesterol sulfate on progesterone production in human granulosa-like tumor cell line, KGN. Endocr J. 2008;55(3):575–81. Scholar
  53. 53.
    Gonzalez-Fernandez R, Pena O, Hernandez J, Martin-Vasallo P, Palumbo A, Avila J. Patients with endometriosis and patients with poor ovarian reserve have abnormal follicle-stimulating hormone receptor signaling pathways. Fertil Steril. 2011;95(7):2373–8. Scholar
  54. 54.
    Wang HX, Tong D, El-Gehani F, Tekpetey FR, Kidder GM. Connexin expression and gap junctional coupling in human cumulus cells: contribution to embryo quality. J Cell Mol Med. 2009;13(5):972–84. Scholar
  55. 55.
    Walker G, MacLeod K, Williams AR, Cameron DA, Smyth JF, Langdon SP. Insulin-like growth factor binding proteins IGFBP3, IGFBP4, and IGFBP5 predict endocrine responsiveness in patients with ovarian cancer. Clin Cancer Res. 2007;13(5):1438–44. Scholar
  56. 56.
    He YY, Huang JL, Sik RH, Liu J, Waalkes MP, Chignell CF. Expression profiling of human keratinocyte response to ultraviolet A: implications in apoptosis. J Invest Dermatol. 2004;122(2):533–43. Scholar
  57. 57.
    Seder CW, Hartojo W, Lin L, Silvers AL, Wang Z, Thomas DG, et al. Upregulated INHBA expression may promote cell proliferation and is associated with poor survival in lung adenocarcinoma. Neoplasia. 2009;11(4):388–96. Scholar
  58. 58.
    Yung Y, Maman E, Ophir L, Rubinstein N, Barzilay E, Yerushalmi GM, et al. Progesterone antagonist, RU486, represses LHCGR expression and LH/hCG signaling in cultured luteinized human mural granulosa cells. Gynecol Endocrinol. 2014;30(1):42–7. Scholar
  59. 59.
    Wagner PK, Otomo A, Christians JK. Regulation of pregnancy-associated plasma protein A2 (PAPPA2) in a human placental trophoblast cell line (BeWo). Reprod Biol Endocrinol. 2011;9:48. Scholar
  60. 60.
    Guzman L, Adriaenssens T, Ortega-Hrepich C, Albuz FK, Mateizel I, Devroey P, et al. Human antral follicles <6 mm: a comparison between in vivo maturation and in vitro maturation in non-hCG primed cycles using cumulus cell gene expression. Mol Hum Reprod. 2013;19(1):7–16. Scholar
  61. 61.
    Zachariades E, Foster H, Goumenou A, Thomas P, Rand-Weaver M, Karteris E. Expression of membrane and nuclear progesterone receptors in two human placental choriocarcinoma cell lines (JEG-3 and BeWo): Effects of syncytialization. Int J Mol Med. 2011;27(6):767–74. Scholar
  62. 62.
    Chen Q, Sun X, Chen J, Cheng L, Wang J, Wang Y, et al. Direct rosiglitazone action on steroidogenesis and proinflammatory factor production in human granulosa-lutein cells. Reprod Biol Endocrinol. 2009;7:147. Scholar
  63. 63.
    Nelson-Degrave VL, Wickenheisser JK, Cockrell JE, Wood JR, Legro RS, Strauss JF III, et al. Valproate potentiates androgen biosynthesis in human ovarian theca cells. Endocrinology. 2004;145(2):799–808. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Cell Biology and AnatomyUniversity of South Carolina School of MedicineColumbiaUSA
  2. 2.Fertility Center of the Carolinas, Department of Obstetrics and GynecologyPrisma Health – UpstateGreenvilleUSA
  3. 3.Department of Epidemiology and BiostatisticsUniversity of South CarolinaColumbiaUSA
  4. 4.Duke Clinical Research InstituteDuke UniversityDurhamUSA
  5. 5.Advanced Fertility and Reproductive Endocrinology Institute, LLCColumbiaUSA
  6. 6.Piedmont Reproductive Endocrinology GroupColumbiaUSA

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