title: Zona pellucida gene mRNA expression in human oocytes is related to oocyte maturity, zona inner layer retardance and fertilization competence.
abstract: Do the mRNA expression levels of zona pellucida (ZP) genes, ZP1, 2, 3 and 4 in oocyte and cumulus cells (CC) reveal relevant information on the oocyte?
  
  
  
  The ZP mRNA expression in human oocytes is related to oocyte maturity, zona inner layer (IL) retardance and fertilization capacity.
  
  
  
  ZP structure and birefringence provide useful information on oocyte cytoplasmic maturation, developmental competence for embryonic growth, blastocyst formation and pregnancy. In order to understand the molecular basis of morphological changes in the ZP, in the current study, the polarized light microscopy (PLM) approach was combined with analysis of the expression of the genes encoding ZP1, 2, 3 and 4, both in the oocytes and in the surrounding CC.
  
  
  
  This is a retrospective study comprising 98 supernumerary human cumulus oocyte complexes (COC) [80 Metaphase II (MII), 10 Metaphase I (MI) and 8 germinal vesicle (GV)] obtained from 39 patients (median age 33.4 years, range 22-42) after controlled ovarian stimulation.
  
  
  
  Single oocytes and their corresponding CC were analysed. Oocytes were examined using PLM, and quantitative RT-PCR was performed for ZP1, 2, 3 and 4 in these individual oocytes and their CC. Ephrin-B2 (EFNB2) mRNA was measured in CC as a control. Presence of ZP3 protein in CC and oocytes was investigated using immunocytochemistry. Data were analysed using one-parametric and multivariate analysis and were corrected for the potential impact of patient and cycle characteristics.
  
  
  
  Oocytes contained ZP1/2/3 and 4 mRNA while in CC only ZP3 was quantifiable. Also ZP3 protein was detected in human CC. When comparing mature (MII) and immature oocytes (MI/GV) or their corresponding CC, ZP1/2 and 4 expression was lower in mature oocytes compared to the expression in immature oocytes (all P < 0.05) and ZP3 expression was lower in the CC of mature oocytes compared to the expression in CC of immature oocytes (P < 0.05). This coincided with a significantly smaller IL-ZP area and thickness in mature oocytes than in immature oocytes (all P < 0.05). In mature oocytes, IL-ZP retardance was significantly correlated with the expression of all four ZP mRNAs (all P < 0.05). The oocyte ZP3 expression was the main predictor of the fertilization capacity, next to IL-retardance and IL-thickness. Using stepwise regression analysis, IL-thickness combined with EFNB2 expression in CC and the patient's ovarian response resulted in a noninvasive oocyte fertilization prediction model.
  
  
  
  Not applicable.
  
  
  
  This is a retrospective study and the relation of oocyte mRNA levels to fertilization capacity is indirect as oocyte gene expression analysis required lysis of the oocyte.
  
  
  
  Overall relations between PLM observations, mRNA expression changes and intrinsic oocyte competence were successfully documented. As such PLM and CC gene expression are confirmed as valuable noninvasive techniques to evaluate oocyte competence.
  
  
  
  This study was funded by University of Torino, Italy, WFWG UZ-Brussel and Agentschap voor Innovatie door Wetenschap en Technologie IWT 110680, Belgium. All authors declare that their participation in the study did not involve actual or potential conflicts of interests.
authors.affiliation: Chair Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, University Department of Surgical Sciences, S. Anna Hospital, Via Ventimiglia 3, 10126 Torino, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Canosa
authors.affiliation: Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium.
authors.forename: T
authors.initials: T
authors.lastname: Adriaenssens
authors.affiliation: Department of Clinical Biology, Scientific Institute of Public Health, 1050 Brussels, Belgium.
authors.forename: W
authors.initials: W
authors.lastname: Coucke
authors.affiliation: Medical Statistics Unit, Department of Public Health and Paediatrics, University of Torino, Via Santena 5b, 10126 Torino, Italy.
authors.forename: P
authors.initials: P
authors.lastname: Dalmasso
authors.affiliation: Chair Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, University Department of Surgical Sciences, S. Anna Hospital, Via Ventimiglia 3, 10126 Torino, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Revelli
authors.affiliation: Chair Gynecology and Obstetrics 1, Physiopathology of Reproduction and IVF Unit, University Department of Surgical Sciences, S. Anna Hospital, Via Ventimiglia 3, 10126 Torino, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Benedetto
authors.affiliation: Follicle Biology Laboratory, Vrije Universiteit Brussel (VUB), Laarbeeklaan 101, 1090 Brussels, Belgium.
authors.forename: J
authors.initials: J
authors.lastname: Smitz
mesh_terms: D000328:Adult; D002454:Cell Differentiation; D054885:Cumulus Cells / Q000166:cytology / Q000378:metabolism*; D036388:Ephrin-B2 / Q000235:genetics / Q000378:metabolism; D005260:Female; D005306:Fertilization / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D008677:Metaphase; D009865:Oocytes / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D010062:Ovulation Induction; D011247:Pregnancy; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D012189:Retrospective Studies; D015398:Signal Transduction; D015044:Zona Pellucida / Q000378:metabolism*; D000072216:Zona Pellucida Glycoproteins / Q000235:genetics* / Q000378:metabolism
chemical_list: C000601239:EFNB2 protein, human; D036388:Ephrin-B2; D012333:RNA, Messenger; C000606514:ZP1 protein, human; C000606515:ZP2 protein, human; C000606517:ZP3 protein, human; C082833:ZP4 protein, human; D000072216:Zona Pellucida Glycoproteins
keywords: ZP1; ZP2; ZP3; ZP4; cumulus cells; polarized light microscopy; zona pellucida