Biobanking of different body fluids within the frame of IVF—a standard operating procedure to improve reproductive biology research

Purpose The aim of the present study was to develop a standard operating procedure (SOP) for the collection, transport, and storage of human cumulus cells, follicular fluid, blood serum, seminal plasma, embryo culture supernatant, and embryo culture supernatant control obtained within the IVF process under approved protocols and written informed consent from participating patients. The SOP was developed at the Kinderwunsch Institut Schenk, Dobl, Austria, together with Biobank Graz of the Medical University of Graz, Austria. Methods The SOP provides comprehensive details of laboratory procedures and sampling of the different fluids within the IVF process. Furthermore, information on sample coding, references of involved laboratory techniques (e.g., oocyte retrieval with a Steiner-TAN needle), ethical approvals, and biobanking procedures are presented. Results The result of the present study is a standard operating procedure. Conclusions The SOP ensures a professional way for collection and scientific use of IVF samples by the Kinderwunsch Institut Schenk, Dobl, Austria, and Biobank Graz of the Medical University of Graz, Austria. It can be used as a template for other institutions to unify specimen collection procedures in the field of reproductive health research.


Introduction
Infertility is a global phenomenon, affecting an estimated 48.5 million of reproductive-aged couples worldwide in 2010. The overall burden of infertility has remained similar in estimated levels and trends according to a WHO study published in 2012 [1]. This persisting problem of infertility explains the ever increasing number of people receiving care in terms of in vitro fertilization (IVF), the assisted reproductive technique (ART), which is now existing for almost 40 years [2]. What is known so far is that success in the IVF treatment depends on a complex interplay of reproductive medicine and clinical embryology. Changes in stimulation protocols were thought to optimize the therapeutic outcome; however, clinical pregnancy rates or baby take home rates did not increase as expected [3]. Improving the process of IVF by different research approaches is tightly linked to state-of-the-art methods of sampling, transporting, and storing of biological materials.
In terms of storage, biobanks have become indispensable institutions for archiving biological materials. In their professional capacity, they do not only provide a vast amount of different sample types, but are also in the ascendant to act as research partners for upcoming scientific questions. Biobanking studies in the field of reproductive medicine are still a niche in IVF research, with only a few publications so far [4,5]. This unused scientific potential is possibly caused Capsule The SOP provides comprehensive information on collection, transport and storage of different body fluids within the IVF process to assure comparability and sample reliability for state of the art molecular analysis in future studies in the field of reproductive health research. by additional ethical hurdles concerning handling of embryonic specimens, cells, fluids, and tissues in terms of processing and storage (i.e., cord blood, oocytes, etc.).
The mechanisms of collecting the samples are crucial for the overall usefulness of the specimens for scientific research and clinical purpose. Thus, standard operating procedures (SOP) are required to standardize the different collections and to facilitate comparability of international institutions of reproductive medicine.
Here, we describe an SOP for collecting, processing, handling, and storing samples derived from patients undergoing IVF treatment comprising cumulus cells (CC), follicular fluid (FF), blood serum (SR), seminal plasma (SE), embryo culture supernatant (SU), and respective culture control media (SUC). These fluids are promising candidates to investigate and correlate a variety of different parameters within the process of IVF.
A major advantage of the present SOP is the fact that follicular fluid is collected with the Steiner-TAN-Needle, which allows the collection of follicular fluid per oocyte instead of pooling all the follicular fluids [6]. This technique allows linking of one particular oocyte to the respective IVF outcome. Together with modern IVF techniques like time-lapse culture systems [7] and preimplantation genetic screening (PGS) [8], this SOP provides an efficient and powerful method, addressing a broad range of applications for different scientific questions and IVF studies.
In the recent years, follicular fluid (FF) has become a major target for oocyte quality in human IVF. To limit embryo Boverproduction,^there is a trend to start the selection process with the oocytes rather than with the already-fertilized oocytes [9]. The follicular fluid comprises a variety of different proteins including anti-apoptotic proteins and metalloproteinaseand IGF-related proteins as well as other growth factors like angiotensinogen (AGT), growth hormone receptor (GHR), or hepatocyte growth factor-like protein (MST1) [10], thus serving as potential target for scientific questions. Besides blood serum, seminal, and embryo culture supernatant, cumulus cells have evolved to become interesting targets as predictors of blastocyst formation and pregnancy success overall [11].

Methods and results
The SOP was established by the Kinderwunsch Institut Schenk, Dobl, Austria, together with Biobank Graz [12] of the Medical University of Graz, Austria, to facilitate future international collaborations and to possibly improve the process of IVF by gaining more insights into molecular mechanisms and processes in early stages of embryogenesis. In the different sections of the SOP, we outline the procedure from responsibilities, via documents and references, to coding and sampling of the different fluids. Furthermore, we essentially included all of the materials and the respective companies used and list the sampling processes in detail. Below, we refer to details of the different SOP sections.
Purpose, scientific value, responsibilities, references, abbreviations, materials, and sample collection approvals Section 1 presents the purpose of the SOP, including information on collected materials and how it was developed. Sections 2 and 3 state the scientific value and the scope. Section 4 comments on the responsibilities of the involved directors/principal investigators and authorized staff members. In section 5, references and documents are highlighted which contributed to the preparation of the SOP. A list of all used abbreviations is found in section 6. Section 7 reveals a list of materials, instruments, and reagents with respective companies, for an accurate implementation of the SOP. The section is divided in general equipment, instruments, microscopes and related equipment, and culture media and embryo culture equipment as well as tubes, dishes, and tips. Section 8 shows the approval list for sampling, collecting, and storage of bio-specimens, including ethical and patient approvals.
Coding, sample preparation, and storage and use The coding system of the Kinderwunsch Institut Schenk is shown in section 9. It serves as paradigm for other institutions, including relevant information of the patient, type of sample, and collection ID. Even if other coding systems are used, it is suggested to include the essential information on the samples and make sure that the sample type can be tracked and linked to the appropriate patient record. In section 10, we provide detailed protocols of sampling of blood serum, follicular fluid, cumulus cells, seminal plasma, embryo culture supernatant, and supernatant control. The section provides information on authorities of performing the procedure, labeling of samples, and storage at the biobank. Furthermore, the specific days of sampling are provided in the SOP and in Fig. 1. Section 11 provides the sample usage with different methods of analyzing proteomics and metabolomics.
During the whole procedure of the SOP, one has to keep in mind that every sample has to be considered as potentially infectious. Thus, wearing protective latex gloves is mandatory when working with biological specimens at all time according to the Level 2 criteria of the European Biosafety Association (http://www.ebsaweb.eu/). It is not advisable to handle more than one sample at the same time to avoid any possibility of confusion and/or contamination. It is also necessary to use separate sets of sterile instruments to avoid cross-contaminations. Each institution may have their own regulations for both transporting and handling human samples under Biosafety Level 2 protocols (BSL2). However, the safety standards in the respective institution should be ensured and followed. If institutions do not have the ability to store their samples in a biobank, it is recommended to store samples at −80°C for long-term storage.

Purpose
The present standard operating procedure (SOP) describes the process of collecting, transporting, and storing human body fluid samples (blood serum, cumulus cells, follicular fluid, seminal plasma, and embryo culture supernatants) at a biobank for research purposes. The SOP was developed at the Kinderwunsch Institut Schenk, Dobl, Austria, together with Biobank Graz of the Medical University of Graz, Austria.

Scientific value
The storage of all samples enables retrospective, scientific research, leading to a better understanding of pathology and progression of diseases in the field of reproductive biology. Furthermore, it permits optimization of diagnostics and therapeutic approaches.

Scope
This procedure applies to blood serum, cumulus cells, follicular fluid, seminal plasma, embryo culture supernatant, and supernatant control samples collected for research in the field of reproductive biology.   (6)

Discussion
The Kinderwunsch Institut Schenk, Dobl, Austria, has started to collect and store samples of FF, SE, SR, SU, and SUC in 2013. To date, more than 9000 different samples have been collected and stored at Biobank Graz of the Medical University of Graz. All samples from women and men undergoing IVF treatment have been approved for sampling and storage. The high quality samples can be tracked and assured by accurately following the SOP guidelines. This SOP serves as a template for other institutions in the field of reproductive health research to unify specimen collection procedures within the process of IVF. One major advantage of the present SOP is to routinely collect samples, which would usually be designated as waste. Hence, a comprehensive dataset can be obtained prospectively for any type of retrospective study subsequently. The dataset allows linkage of specific sample types to clinical characteristics of the patients such as body mass index (BMI), smoking habit, age, and type of ovarian stimulation as well as pregnancy success and outcome.
Within the obtained dataset, follicular fluid is a parameter with increasing importance for IVF treatment. As plasma ultrafiltrate, follicular fluid comprises signaling mediators, mostly peptide hormones [13,14], responsible for communication between granulosa cells, cumulus complex, and oocyte and somatic compartment [15][16][17][18]. This signaling is mandatory for maintenance of oocyte integrity. In addition to the ultrafiltrate, proteins can pass the follicular barrier, depending on molecular weight and charge characteristics [19]. The fluid components within the follicular fluid are potential sources for biomarkers defining oocyte health. Different ways of sampling and storage of follicular fluid are critical issues between datasets and the comparability remains uncertain. The current SOP addresses these issues and provides standardized sample consistency, accuracy, and quality.
By now, the collection of samples obtained from the Kinderwunsch Institut Schenk is unique in Austria and serves as the major source for reproductive health research in Austria. To the best of our knowledge, no laboratory facility collects follicular fluid per oocyte and stores this comprehensive dataset of FF, SE, SR, SU, and SUC of patients receiving IVF treatment in a biobank. The idea of storage of all these materials refers to basic research in IVF and was invented to gain knowledge of basic procedures in order to improve clinical results and pregnancy outcome. So far, the samples collected with the present SOP have already been used for scientific purposes. Quantification of protein carbonyl groups as oxidative stress parameter has been successfully analyzed in seminal plasma, embryonal culture supernatant, and follicular fluid [20]. Since the SOP was developed quite recently, there is lots of unexploited potential for scientific questions.
The cooperation with Biobank Graz of the Medical University of Graz enables investigators from all over the world with straightforward access to stored samples. We provide this SOP for a standardized quality management to assure good scientific practice and high quality of samples within different institutions. Hence, comparability and sample reliability can be guaranteed for state of the art molecular analysis in future studies in the field of reproductive health research. By following the SOP, sample handling and storage will be standardized and research comparability in institutions implementing the same procedures can be assured. This is a big step forward towards sound and reliable research.