title: Association of high fetuin-B concentrations in serum with fertilization rate in IVF: a cross-sectional pilot study.
abstract: OBJECTIVE
  Is serum fetuin-B associated with the fertilization rate in in vitro fertilization (IVF)?
  
  
  CONCLUSIONS
  Serum fetuin-B increased during IVF cycles when oocytes could be fertilized while remained unchanged in fertilization failure.
  
  
  BACKGROUND
  Fetuin-B deficiency in mice causes premature zona pellucida hardening mediated by the zona protease ovastacin. Thus fetuin-B deficiency renders females infertile.
  
  
  METHODS
  We determined the human serum fetuin-B reference range, studying longitudinally, over the course of one month, five male and seven female volunteers without hormone treatment and four female volunteers on varying hormonal contraception. We sampled blood and determined serum fetuin-B, luteinizing hormone (LH), estradiol (E2) and progesterone (P4). In addition, we determined serum fetuin-B and estradiol in eight women undergoing intracytoplasmatic sperm injection (ICSI, nine ICSI cycles) and 19 women undergoing IVF (21 IVF cycles) after ovarian stimulation with recombinant human follicular stimulating hormone (rFSH) and/or a combined medication of FSH and LH. At least three blood samples were analyzed in each cycle. We compared serum fetuin-B and follicular fluid fetuin-B in nine patients by measuring follicular fetuin-B in pooled follicular fluid, and in fluid obtained from individual follicles. Samples were drawn from January 2012 to March 2014.
  
  
  METHODS
  All volunteers and patients gave informed consent. Fetuin-B was measured employing a commercial sandwich enzyme-linked immunosorbent assay. Serum fetuin-B was determined as duplicates in 5 male (34 ± 14.6 years) and 11 female volunteers (29.4 ± 4.1 years) as well as in female volunteers on hormonal contraception (30.0 ± 6.5 years). The duplicate standard deviation was 4.0 ± 2.3%. The contraceptive drugs were mono or combined preparations containing 0-0.03 mg ethinyl estradiol, and 0.15-3.0 mg of various progestins. In addition, serum fetuin-B was determined as triplicates in 27 female patients undergoing conventional IVF (19) or ICSI (8). The triplicate standard deviation was 3.3 ± 1.8%. IVF was declared as 'successful', if at least one oocyte was fertilized, and 'unsuccessful', if no oocyte could be fertilized. Patient age was 34.4 ± 4.4 years in successful IVF, and 35.4 ± 3.3 years in unsuccessful IVF. Serum and follicular fluid of patients undergoing controlled ovarian hyperstimulation were analyzed. Serum was drawn at the day of follicle aspiration.
  
  
  RESULTS
  Serum fetuin-B and follicular fluid fetuin-B were not significantly different in six out of nine patients suggesting, in principle, free exchange of fetuin-B between serum and follicular fluid. Thus serum fetuin-B may be used as a proxy of follicular fluid fetuin-B. Serum fetuin-B increased during successful IVF cycles (n = 15, P < 0.0001), but did not change in unsuccessful IVF cycles (n = 6, P = 0.118) despite increased estradiol levels (P = 0.0019 and P = 0.0254, respectively).
  
  
  CONCLUSIONS
  The female volunteers self-reported their respective hormone medication. Medication was verified by serum estradiol, LH and progesterone measurements. For oocyte harvesting, the vaginal wall was punctured once only to minimize co-morbidity. Low grade cross-contamination of individual follicular fluid aspirates and contamination of the follicular fluid with small amounts of blood were inevitable. Samples were routinely checked for the presence of hemoglobin that would suggest blood contamination. Only samples containing <250 erythrocyte equivalents/µl were used for analysis.
  
  
  CONCLUSIONS
  Serum fetuin-B may be used as a marker to predict the fertilization success in IVF. Fetuin-B levels attained during IVF stimulation may help to make an informed decision whether oocytes should be fertilized by IVF or by ICSI to overcome the zona pellucida as a barrier.
  
  
  BACKGROUND
  The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. J.F., E.D., J.N., B.R. and W.J.-D. declare that they are named inventors on the RWTH Aachen University patent application EP 13157317.2, 'Use of fetuin-B for culture of oocytes', applied for by RWTH Aachen University.
authors.affiliation: Helmholtz Institute for Biomedical Engineering, Biointerface Laboratory, 52074 Aachen, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Floehr
authors.affiliation: Helmholtz Institute for Biomedical Engineering, Biointerface Laboratory, 52074 Aachen, Germany.
authors.forename: Eileen
authors.initials: E
authors.lastname: Dietzel
authors.affiliation: Clinic for Gynecological Endocrinology and Reproductive Medicine, RWTH Aachen University Clinics, 52074 Aachen, Germany.
authors.forename: Josef
authors.initials: J
authors.lastname: Neulen
authors.affiliation: Clinic for Gynecological Endocrinology and Reproductive Medicine, RWTH Aachen University Clinics, 52074 Aachen, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Rösing
authors.affiliation: Clinic for Gynecological Endocrinology and Reproductive Medicine, RWTH Aachen University Clinics, 52074 Aachen, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Weissenborn
authors.affiliation: Helmholtz Institute for Biomedical Engineering, Biointerface Laboratory, 52074 Aachen, Germany willi.jahnen@rwth-aachen.de.
authors.forename: Willi
authors.initials: W
authors.lastname: Jahnen-Dechent
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood; D003430:Cross-Sectional Studies; D004958:Estradiol / Q000097:blood; D005260:Female; D005306:Fertilization; D005307:Fertilization in Vitro; D060752:Fetuin-B / Q000378:metabolism*; D015571:Follicular Fluid / Q000378:metabolism; D006801:Humans; D007986:Luteinizing Hormone / Q000097:blood; D008297:Male; D010865:Pilot Projects; D015044:Zona Pellucida / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D015415:Biomarkers; C504687:FETUB protein, human; D060752:Fetuin-B; D004958:Estradiol; D007986:Luteinizing Hormone
keywords: IVF fertilization rate; early embryo development; embryo hatching; fetuin-B; follicular fluid; infertility; sperm attachment; zona pellucida hardening