title: Cell-free DNA in human follicular fluid as a biomarker of embryo quality.
abstract: OBJECTIVE
  Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for in vitro fertilization (IVF) outcomes?
  
  
  CONCLUSIONS
  CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes.
  
  
  BACKGROUND
  CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated.
  
  
  METHODS
  One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (n = 26) or ICSI (n = 17). CfDNA level was quantified in each individual follicular fluid sample.
  
  
  METHODS
  At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers.
  
  
  RESULTS
  Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean ± SD, 1.62 ± 2.08 ng/µl). CfDNA level was significantly higher in small follicles (8-12 mm in diameter) than in large ones (>18 mm) (mean ± SD, 2.54 ± 0.78 ng/µl versus 0.71 ± 0.44 ng/µl, respectively, P = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (r = -0.34; P = 0.003). A weak significant negative correlation between DNA integrity and 17β-estradiol level in follicular fluid samples at oocyte collection day was observed (r = -0.26; P = 0.008). CfDNA level in follicular fluid samples corresponding to top quality embryos was significantly lower than in follicular fluid samples related to poor quality embryos (P = 0.022). Similarly, cfDNA level was also significantly lower in follicular fluid samples related to embryos with low fragmentation rate (≤25%) than with high fragmentation rate (>25%) (P = 0.02).
  
  
  CONCLUSIONS
  A larger study should be conducted in order to establish the predictive value of cfDNA level for embryo quality and to investigate whether follicular fluid cfDNA levels are correlated with embryo implantation rates and pregnancy outcomes. Moreover, the role of follicular fluid cfDNA on embryo quality should be studied to determine whether high cfDNA concentration in follicular fluid is only a consequence or also a cause of follicular dysfunction.
  
  
  CONCLUSIONS
  CfDNA evaluation in individual follicular fluid samples might represent an innovative biomarker of embryo quality to use as a supplemental tool to predict embryo quality during IVF.
  
  
  BACKGROUND
  This study was partially supported by the University Hospital of Montpellier and Ferring Pharmaceuticals. The authors of the study have no competing interests to report.
authors.affiliation: CHU Montpellier, Institute for Research in Biotherapy, Hôpital Saint-Eloi, INSERM U1040, Montpellier, France Montpellier 1 University, UFR of Medicine, Montpellier, France ART-PGD Department, Arnaud de Villeneuve, CHU Montpellier, Montpellier, France.
authors.forename: E
authors.initials: E
authors.lastname: Scalici
authors.affiliation: CHU Montpellier, Institute for Research in Biotherapy, Hôpital Saint-Eloi, INSERM U1040, Montpellier, France.
authors.forename: S
authors.initials: S
authors.lastname: Traver
authors.affiliation: UMR 1049, DIM, CHRU, Montpellier, France.
authors.forename: N
authors.initials: N
authors.lastname: Molinari
authors.affiliation: Montpellier 1 University, UFR of Medicine, Montpellier, France ART-PGD Department, Arnaud de Villeneuve, CHU Montpellier, Montpellier, France.
authors.forename: T
authors.initials: T
authors.lastname: Mullet
authors.affiliation: ART-PGD Department, Arnaud de Villeneuve, CHU Montpellier, Montpellier, France.
authors.forename: M
authors.initials: M
authors.lastname: Monforte
authors.affiliation: ART-PGD Department, Arnaud de Villeneuve, CHU Montpellier, Montpellier, France.
authors.forename: E
authors.initials: E
authors.lastname: Vintejoux
authors.affiliation: CHU Montpellier, Institute for Research in Biotherapy, Hôpital Saint-Eloi, INSERM U1040, Montpellier, France Montpellier 1 University, UFR of Medicine, Montpellier, France ART-PGD Department, Arnaud de Villeneuve, CHU Montpellier, Montpellier, France s-hamamah@chu-montpellier.fr.
authors.forename: S
authors.initials: S
authors.lastname: Hamamah
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D010064:Embryo Implantation; D005260:Female; D005307:Fertilization in Vitro / Q000379:methods; D015571:Follicular Fluid / Q000378:metabolism*; D006801:Humans; D008297:Male; D054315:Oocyte Retrieval; D006080:Ovarian Follicle / Q000166:cytology / Q000502:physiology; D011247:Pregnancy; D011256:Pregnancy Outcome; D011446:Prospective Studies
chemical_list: D015415:Biomarkers; D004247:DNA
keywords: IVF outcomes; cell-free DNA; embryo quality; follicle size; human follicular fluid