**5. Conclusions**

*Innovations in Assisted Reproduction Technology*

human placental trophoblast cell line [98].

of single protein of interest.

**4. Future development of ASA contraceptives**

efforts are needed to make ASA contraceptive become reality.

peptide [90].

The use of synthetic PCSK4 inhibitors could also serve as an option for contraception. A synthetic inhibitor of PCSK has been developed based on the knowledge, that prodomain removal is essential for activation of the enzyme. Binding of this domain to PCSK4 active enzyme could hypothetically inactivate the enzyme. A peptide, mimicking prodomain sequence near its primary activation site, was engineered and an *in-vitro* assay using a recombinant PCSK4 showed that PCSK4 mediated proteolysis was efficiently blocked by synthetic prodomain rPC4101–116

Other potent synthetic PCSK4-inhibitors: tetrapeptide chloromethyl ketone and the Dec-RVKR/K-cmk (Decanoyl-RVKR/K-chloromethyl ketone) were reported to inhibit PCSK4 more potently than synthetic prodomain rPC4101–116 peptide [90]. Another substance, synthetic enediyne amino acid containing peptides, was developed and reported to inhibit PCSK4 activity *in-vitro* [97]. Further, dimeric form of CRES was reported to moderately block the PCSK4 activity to human proIGF-2 in

Only a few of aforementioned ASA underwent animal models *in-vivo* study. Among are EQT, FA-1, YLP12, *Eppin*, PCI, HSA, AKAP 4 and PCSK4. None of these molecules however, shows full inhibition of fertilisation in animal models, whereas YLP12 contraceptive studies reported various results from partial to lack of inhibition of fertility to animal models [37, 53, 63–65, 82] [53, 55, 70]. Therefore, further

Future development of ASA contraceptives might include the studies of underexplored proteins such as those involve in the energy production in the mitochondria of sperm. Proteomic approach assistance, in this regard, have enabled the discovery of large number of novel proteins [59] and allows further investigations

On the other hand, as the usage of single molecule have been impractical, alternative of future ASA development might include the study of efficacy of multivalent vaccines, in order to boost the final effective contraception effect as well as reducing potential toxic effect of high dose administration of a single substance. Study of multivalent vaccine targeting proteins of acrosomal sperm has been actually initiated. In the study, immunised monkeys recognised the five antigens used: ESP, SLLP-1, SAMP 32, SP-10 and SAMP 14, with the highest IgG average absorbance values were to ESP, SAMP 32 and SP-10 but at IgG lower values for SLLP-1 and SAMP 14 [99]. Further, capacitated sperm treated with sera from immunised monkeys showed fusion inhibition but only in two of five individuals [99]. This was a sound study that used proteins which were pre-tested to not cross react to each other. But the study used molecules that never had undergone *in-vivo* study so individual effect of the vaccine component was unknown. In addition, a study of FA-1 and YLP12 mixed vaccine in mice further support the more potential of multivalent vaccine in inducing contraceptive effects compared to monovalent

The *in-vivo* study of anti-PSCK4 administration demonstrated in our laboratory indicates that it is possible to efficaciously administer contraceptive agents in the form of antiserum rather than injecting them to an individual for a few times as immunisation [82]. In the future, this approach might be more practical if the molecule could reach therapeutic concentration in the seminal plasma once the semen ejaculated thus well mixed with sperm along their journey to egg, mimicking *in-vitro* incubation of sperm with its inhibitors prior to a fertilisation challenge with eggs.

**228**

vaccine [54].

Existing contraceptive methods have been widely practiced by couples globally. However, some side effects limit the broader utilisation. Although ASA is a promising method of contraception it is not yet practical. More studies should be done in order to enable ASA to replace common contraceptive methods.
