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A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone
Michael W Pankhurst1, Ian S McLennan2
1Department of Anatomy, Otago School of Medical Sciences, New Zealand.
Abstract:
The utility of serum anti-Müllerian hormone (AMH) assays in assessment of female fertility have been investigated extensively but little is known about the biological activity of the hormone being studied. ProAMH is the proprotein precursor and is incapable of binding to the AMH-specific type II receptor. Proteolytic cleavage generates receptor-competent AMHN,C which is a non-covalent complex of the N- and C-terminal cleavage fragments. Commercially available AMH assays do not differentiate the two forms of AMH. Techniques were developed to dissociate the AMHN,C complex and abolish its two-site immunoassay immunoreactivity. This allowed specific quantification of proAMH. The surfactant sodium deoxycholate (DOC) dissociated AMHN,C without disrupting binding of proAMH to the capture-antibody with an optimal concentration of 0.1-0.2%w/v. The incorporation of a DOC incubation step into the AMH Gen II ELISA detected proAMH, with AMHN,C cross-detection conservatively estimated at 6.0% ± 2.5% (mean ± S.D.). The intra-assay and inter-assay variability were estimated at 8.0%CV and 13.0%CV respectively. The levels of proAMH and total AMH were assessed in 5 boys and 5 men and the proportion of proAMH was found to be significantly higher in boys (p = 0.005). This study will facilitate further investigation of the role of proteolytic cleavage in AMH signalling.
Insights
This study developed a method to specifically measure pro-anti-Müllerian hormone (proAMH), a precursor form of AMH. This technique revealed significantly higher proAMH levels in boys compared to men, offering new insights into AMH biology.
Area of Science:
- Reproductive Endocrinology
- Biochemistry
- Assay Development
Background:
- Serum anti-Müllerian hormone (AMH) is crucial for fertility assessment, but its biological activity and precursor forms are poorly understood.
- Commercially available AMH assays lack the ability to differentiate between the inactive proAMH precursor and the active AMHN,C complex.
- Understanding the different forms of AMH is essential for accurate interpretation of fertility assessments and AMH signaling pathways.
Purpose of the Study:
- To develop and validate a technique for the specific quantification of proAMH in serum.
- To investigate the differences in proAMH levels between prepubertal boys and adult men.
- To explore the role of proteolytic cleavage in AMH signaling and its implications for reproductive health.
Main Methods:
- Developed a modified Enzyme-Linked Immunosorbent Assay (ELISA) incorporating sodium deoxycholate (DOC) to dissociate the AMHN,C complex.
- Optimized DOC concentration (0.1-0.2% w/v) to selectively disrupt AMHN,C without affecting proAMH binding.
- Validated the assay for proAMH detection with assessed cross-reactivity, intra-assay, and inter-assay variability.
Main Results:
- The modified ELISA successfully quantified proAMH by dissociating the AMHN,C complex, with low cross-detection (6.0% ± 2.5%).
- Intra-assay and inter-assay variability were found to be 8.0%CV and 13.0%CV, respectively, indicating assay reliability.
- Serum proAMH levels were significantly higher in boys compared to men (p = 0.005), suggesting developmental changes in AMH processing.
Conclusions:
- A novel assay allows for specific measurement of proAMH, differentiating it from the active AMH form.
- The significantly higher proportion of proAMH in boys indicates developmental regulation of AMH proteolytic cleavage.
- This advancement facilitates further research into the biological significance of AMH cleavage and its impact on fertility and reproductive development.
