Association of high fetuin-B concentrations in serum with fertilization rate in IVF: a cross-sectional pilot study.

STUDY QUESTION: Is serum fetuin-B associated with the fertilization rate in in vitro fertilization (IVF)? SUMMARY ANSWER: Serum fetuin-B increased during IVF cycles when oocytes could be fertilized while remained unchanged in fertilization failure. WHAT IS KNOWN ALREADY: Fetuin-B deficiency in mice causes premature zona pellucida hardening mediated by the zona protease ovastacin. Thus fetuin-B deficiency renders females infertile. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHOD: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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). LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDING: 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. STUDY FUNDING/COMPETING INTERESTS: 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.

Cryopreservation of spermatozoa in alginic acid capsules.

OBJECTIVE: To develop a method of freezing small amounts of spermatozoa in polymerized alginic acid drops, which can be liquified after thawing for recovery of the spermatozoa. DESIGN: Prospective clinical study. SETTING: Medical School, RWTH Aachen, Aachen Germany. PATIENT(S): None. INTERVENTION(S): Validation of the encapsulation method with bovine sperm; cryopreservation of human spermatozoa in alginic capsules. MAIN OUTCOME MEASURE(S): We optimized the cryopreservation method by testing different parameters influencing the freezing procedure, such as concentration of alginic acid, size of drops, time of polymerization, and culture media. RESULT(S): The final protocol was as follows: encapsulation by 7.3 mg/mL alginic acid forming 10-muL drops polymerized for 30 seconds and liquefied for 2.5 minutes in sodium citrate. Cryopreservation of human spermatozoa by this protocol resulted in a decreased motility of 18.3% compared with standard protocols but a 19.9% higher vitality of the immotile spermatozoa. CONCLUSION(S): No difference in viability of spermatozoa after both sperm-freezing procedures could be observed. Further investigation will be undertaken to reduce the amount of immotile but viable sperm after microencapsulation in alginic acid.

Heparin and aspirin attenuate placental apoptosis in vitro: implications for early pregnancy failure.

OBJECTIVE: Live birth rates are increased by treatment with heparin and aspirin in cases of poor pregnancy outcome such as antiphospholipid syndrome. Both drugs may attenuate miscarriage by inhibiting aberrant coagulation or by modulating trophoblast apoptosis. Here we assessed their roles in trophoblast apoptosis in vitro. STUDY DESIGN: BeWo cells and placental villi were cultured in sera from women with successful or failing in vitro fertilization, with and without heparin or aspirin. Apoptosis was assessed by using DNA laddering, cytokeratin 18 neoepitope formation, Bcl-2, and caspase 7 expression. RESULTS: In BeWo cells, sera from in vitro fertilization failure increased trophoblast apoptosis, whereas heparin and aspirin reversed these effects. In villous trophoblast, heparin increased Bcl-2 and cytokeratin 18 protein expression. Heparin and aspirin inhibited DNA laddering. CONCLUSION: Heparin and aspirin modulate trophoblast apoptosis suggesting a direct impact on trophoblast biology, thus providing an additional mechanism to explain the clinical benefits of heparin and aspirin on recurrent pregnancy loss.