In mice fetuin-B is essential for fertilization. Fetuin-B deficiency (Fetub-/-) leads to female infertility due to premature zona pellucida hardening. The zona pellucida (ZP) is a glycoprotein matrix surrounding the oocyte. The penetration of the ZP by sperm is a critical factor for fertilization; fetuin-B maintains the fertility of oocytes by inhibiting premature ZP. The fetuin-B gene is well conserved between mammals with 61% sequence homology in mice and humans. To study the role of fetuin-B in human female reproduction serum and follicular fluid fetuin-B were determined in healthy volunteers and in patients consulting the fertility clinic. The results suggest an estrogen-mediated regulation of hepatic fetuin-B expression. Furthermore, serum fetuin-B increased in women with successful in vitro fertilization (IVF) while serum fetuin-B remained unchanged in women with fertilization failure. Using fetuin-B as an additional marker in assisted reproductive technology may increase the fertilization rate. Biochemical studies showed that fetuin-B is a potent ovastacin inhibitor. Ovastacin induces ZP hardening by cleavage of the zona pellucida protein 2 (ZP2). Using conventional breeding, fetuin-B/ovastacin double deficient (Fetub-/-, Astl-/-) mice were generated. Fertility of female Fetub-/-, Astl-/- mice showed in vivo that fertility of Fetub-/- females was restored by additional ovastacin deficiency. Fetub-/-, Astl-/- females produced healthy offspring, confirming the proteinase ovastacin as the target of the inhibitor fetuin-B. Following natural mating, the litter size of Fetub-/-, Astl-/- females were highly variable from unusually low to normal litter sizes. Because the IVF rates were unusually high in these oocytes a defect in fertilization was definitely not the primary reason for small litter sizes suggesting that embryo abortion occurred following successful fertilization. This was attributed to a complete loss of fertilization-induced ZP hardening, and thus a lack of pre-implantation embryo stability. Nevertheless, the fact that the infertility of Fetub-/- mice was fully restored in Fetub-/-, Astl-/- mice underscored the important role of fetuin-B in fertilization, which might be exploited as a potential contraceptive target. Corroborating this view, antisense oligonucleotide-mediated down-regulation of fetuin-B expression reversibly prevented pregnancy. Due to the high homology between fetuin-B and ovastacin in mice and humans it is hypothesized that pharmacological fetuin-B down-regulation may be also contraceptive in women
