Development of a predictive assay to assess the presence of germ cells and their stage of maturation in the testes of infertile men. – FERTICHIP

Several technical challenges have been identified, for which we anticipate alternatives if needed.
For characterizing our 92 germ cell markers, we have developed an innovative integrative genomics approach that, in our hands, advantageously replaces the so-called differential Omics studies used for biomarker discovery worldwide. We have thus demonstrated our ability to handle technical challenges with bioinformatics. The ongoing selection of top-priority markers out of our list of 92 candidates, for antibody production, relies on our novel computing and data mining developments.

This project calls for the development of specific monoclonal antibodies against each one of the germ cell-specific biomarkers identified. For obvious financial reasons, we focus on a first short list of promising candidate biomarkers. Based on our experience and so as to keep on with our agenda, we have now decided to subcontract the production of protein for immunization prior to antibody deelopoment to CRO. The validation of specific monoclonal antibodies may be the second challenge. The choice was made of developing specific assays using the robust and tried ELISA technology for which no major difficulty is expected. Again we subcontract with a CRO for some challenging ELISAs

Our short-term objective is to produce as much as possible monoclonal antibodies against our top-priority germ cell markers and develop the corresponding specific ELISA assays.

33 peptides were designed (correponding to 10 proteins) & synthetised. The first step of the antibody production in ongoing.

During the course of the project we will focus on the validation of at least the first 3 biomarker assays. This is a prerequisite for transferring the IP to a start-up company, as recommended by Kurma Biofunds and Fadièse2, two private equity structures that are interested by our project.

The Fertichip assay represents a valuable and no invasive method to diagnose the presence of spermatozoa in the testis of azoospermic patients and should help avoid unnecessary testicular biopsy. This study should also the improvement of our knowledge on the human seminal plasma proteome of healthy fertile men and patients with distinct etiologies of azoospermia. Indeed, this project aims to develop a non-invasive simple test that will answer the following question:
• Are germ cells present or absent?
• What is the final stage of germ cell maturation?
• Is it a Sertoli cell-only syndrome?

Potential customers interested by the Fertichip assay will be either ‘infertility and family birth’ departments in public hospitals or fertility clinics in the private sector. Use of the Fertichip assay should finally help reduce the psychological cost for patients and indeed couples. Additionally, the indirect money saving for healthcare insurances should also be considered.
For clinicians in the field of ART, the Fertichip assay undoubtedly presents a technological rupture in the diagnosis of male infertility and management of infertile men. It will advantageously complement all indirect methods available so far for predicting the presence of mature germ cells in the testes of infertile patients, avoid unnecessary biopsies and select for testis biopsies only those NOA patients for which there is a high chance of spermatozoa recovery.

Rolland AD, Lavigne R, Calvel P and Pineau C. Method for predicting the presence of reproductive cells in testis. Brevet n°EP11306409. Nov 2011. in Extension (PCT) .

Rolland AD, Lavigne R, Dauly C, Calvel P, Kervarrec C, Freour T, Evrard B, Rioux-Leclercq N, Auger J, Pineau C. Identification of genital tract markers in the human seminal plasma using an integrative genomics approach. Hum Reprod. 2013; 28(1): 199-209

Although the lack of ejaculated spermatozoa makes azoospermic men naturally infertile, these men can become fathers thanks to assisted reproductive technologies (ART). When azoospermia is diagnosed, TESE (testicular sperm extraction) is proposed to the patient. In men with obstructive azoospermia (OA), this technique allows through a minimally invasive needle-biopsy to recover spermatozoa. But, in men with non-obstructive azoospermia (NOA), one or several invasive testicular biopsies are proposed to the patient for eventually recovering a few mature germ cells for entering an ICSI program (Intra-cytoplasmic sperm injection). These invasive biopsies may impair future spermatogenesis or induce testicular atrophy and only lead to sperm cell recovery in about 50% of cases.

To date, there is no powerful predictor of the probability to recover living sperm cells in NOA men. Several indicators have been proposed to be potential indirect markers for the presence of spermatozoa in the testis. These include examination parameters, measurement of circulating hormones and of factors secreted by Sertoli cells (e.g., inhibin A and B, AMH). In such context, unfortunately, surgery with testis biopsy is to date the only valuable method for answering whether or not spermatozoa can be found in the testes of NOA patients. This crucially calls for discovering and validating specific seminal plasma biomarkers that are needed by clinicians and will help predicting sperm-positive testicular biopsies.

Seminal plasma has a strong potential in providing biomarkers of reproductive disorders. We have thus conducted a large-scale proteomics study with the aim to establish a repertoire of seminal plasma proteins and have further characterized using an innovative integrative genomics approach 92 proteins specific to spermatogonia, spermatocytes and/or spermatids that could be used as predictive biomarkers of the presence of germ cells in the gonads of NOA patients. Monitoring these germ cell biomarkers directly in semen should be indeed a promising non-invasive method to predict TESE outcome and select NOA patients for a testis biopsy. This method could also be used to diagnose maturation arrests or a Sertoli cell-only syndrome (total lack of germ cells).

This project aims at:
1) Developing the Fertichip test: an ELISA-based multiplex assay to predict the presence of mature germ cells in the testes of NOA patients, document a maturation arrest of spermatogenesis or characterize a Sertoli cell-only syndrome;
2) Valorizing the technology and organizing the starting up of a Biotech company that will further develop our prototype assay and commercialize the products;
and 3) Expanding our clinician network and construct a biological sample bank to fulfill our goals.

To our knowledge there is no equivalent solution to the so-called Fertichip assay on the market. Benefits for ART clinicians to use the Fertichip assay should be multiple. It includes, facilitating the management of infertile men, avoid unnecessary biopsies, improve patient counselling, diminish psychological costs and reduce the financial cost of ART medicine. Over the past two decades, the use ART has increased dramatically worldwide and now represents a growing market. The Fertichip project is timely and answers a socioeconomic demand in both industrialized and developing countries.