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The individual research group or independent investigator often requires access to samples from a unique well characterized subject population. Cohorts of such samples from a well-defined comparative population are rare and limited access can impede progress. This bottleneck can be removed by accessing the samples provided by biorepositories such as the NIH/NICHD Cooperative Reproductive Medicine Network (RMN) Biorepository (detailed in the accompanying manuscript in this issue. In those cases where the individual research group or independent investigator already has access to a unique population, comparisons between well-defined groups are often sought to contextualize the data. In both cases seamless integration of data resources associated with the samples is required to ensure optimal comparisons. At the most basic level this requires standardization of sample collection and storage, as well as a de-identified data base containing demographic, clinical, and laboratory values. To facilitate such interoperability, the reagents and protocols that have been adopted by the RMN Biorepository for the collection and storage of serum, blood, saliva and sperm are described.
The Reproductive Medicine Network (RMN) is comprised of a nationwide series of clinical sites and a data coordination center (DCC). It is charged with designing, implementing, and publishing the results of high quality clinical research in reproductive medicine. Various specimens have been and continue to be collected as part of the ongoing research activities of this network. Additional aliquots of samples are often retained for additional analyses and new tests as they are introduced. Specimen collection is conducted under the auspices of each local Institutional Review Board (IRB), with attainment of participants’ consent detailing whether use will be allowed for future projects that are either related to the initial project and/or other fields of medicine as outlined in the accompanying manuscript Casson et al. 2011. Because of the ongoing rapid advance of genome technologies such consent must be mindful of whether genetic information could be collected and used.
Samples derived from randomized controlled trials in well-defined populations, represent a valuable resource for future research endeavors, both inside and outside the RMN. With the increased availability and capabilities of the rapidly evolving genome-wide-technologies, the biology of the system underlying the trial results now becomes accessible. Progress is often first revealed with the introduction of a suite of biomarkers [Sistare et al. 2010]. These advances are poised to markedly change clinical practice as we know it today while moving towards personalized systems medicine.
Sample acquisition protocols along with repositories continue to be described [Ayers et al. 2007; Ennis et al. 2010; Gohagan et al. 2000; Ugolini et al. 2008]. One is referred to best practice resources of the International Society for Biological and Environmental Repositories [ISBER 2008] and the National Cancer Institute Best Practices for Biospecimen Resources [National Cancer Institute 2007]. Within this framework, the RMN established a biorepository that provides access to a source of clinically well-characterized human reproductive samples. These samples will be collected as part of three studies, which will be complemented by biorepository specimen storage as summarized in Table 1. All will have blood specimens collected including card blood spots, whole blood, serum, and the residual blood clot. Additionally, the Assessment of Multiple Intrauterine Gestations From Ovarian Stimulation (AMIGOS) trial will collect semen for biorepository storage. Anonymized specifics of the study populations and clinical characteristics can be obtained from the DCC. It is projected that in total this will include at least 10,000 serum samples, 15,000 samples of whole blood, 7,500 FTA blood spot cards, 1,000 Oragene® recovered saliva samples, and 7,500 sperm samples. A description of the methods by which the samples are being collected and stored is outlined below in this Applications Note.
Prior to beginning any collection of human specimens for research and/or when beginning any novel research protocol, approval must be obtained from the local IRB committee [Baranzini et al. 2010; Roach et al. 2010]. Informed consent by participants must then be obtained before collecting any biological samples for research purposes and personnel must be appropriately trained in sample collection and personal biosafety. As discussed in the Casson et al.  paper, it cannot be overemphasized that the informed consent must be robust as to withstand continued scrutiny of the IRB regarding the use of these samples as our ability to extract other information continues to grow. In addition, the shipment of biological specimens must be certified and certification programs are available through Saf-T-Pak (http://www.saftpak.com). Compliance is federally mandated.
The protocols were adopted from standard clinical practice to ensure ease of implementation and compliance across collection sites. All procedures are carried out at room temperature unless otherwise noted. While the site-specific protocol may vary slightly, maintenance of cellular integrity is maintained and thus non-biological variance is assumed to be minimal. Similarly, although not definitive, there is no reason to expect that this methodology will alter the epigenetic signature in an unusual manner compared to standard clinical practice. Although the primary objective of the RMN’s sample collection is to utilize whole blood for DNA extraction, samples retrieved and stored in this manner could also provide a source of proteins, RNA, and metabolites. Routine methods of cellular recovery include venipuncture, heel stick, and buccal swab. Venipuncture is typically used for adult collections compared to buccal swabs for infants and children. While heel sticks are an integral part of standard newborn screening programs, their primary use is dedicated to providing early assessment and a baseline of newborn health status immediately following birth. Logistically, buccal swab collection may be preferred as the samples are directly purposed. Accordingly, the DNA prepared from these samples should be suitable for most analyses including, SNP, genotyping, and methylation analysis as well as direct sequencing which is becoming economically feasible. FTA cards were adopted as a medium since they offered long-term stable cost effective room temperature storage in minimal space. They have even been used previously to store cervical cells [Gustavsson et al. 2009] and provide a means to immediately freeze the sample in biological time, thus preserving integrity.
Both serum and blood clots are also being recovered. On one hand, serum should provide an additional source of metabolite-enriched biomarkers. Its utility as a tool to predict preeclampsia, obstetrical complications, and Assisted Reproductive Technology outcomes is currently being explored [Gagnon et al. 2008; Grill et al. 2009; La Marca et al. 2010]. One must be careful to avoid hemolysis. This can compromise subsequent analyses. Serum samples appearing pink or red should be noted and if possible an additional specimen collected. On the other hand, blood clots that are typically discarded provide an additional source of DNA [Iovannisci et al. 2006; McCulloch et al. 2009]. The blood clot can be placed in reserve in case of catastrophic failure, or used in place of the blood samples.
Sperm are being collected as part of the evaluation of the reproductive function of the couple in addition to blood and sera. The manner in which the spermatozoa are being prepared and stored as described below has proven suitable for long-term storage. Maintaining the integrity and viability of the spermatozoa has enabled recovery of both biological functions well suited to fertilization (reviewed in [Pasqualotto et al. 2009]) and functional full-length RNAs [Goodrich et al. 2007; Ostermeier et al. 2005]. Spermatozoa prepared and stored as outlined below should be suitable for the current range of functional assays as well as new assays as they become available (reviewed in [Lewis et al. 2008]).
To ensure a complete study of reproductive outcomes, samples can be collected from the newborn and DNA recovered. The use of a saliva/buccal swab is now being adopted as standard practice in neonatal screening to avoid venipuncture or heel stick. Saliva can also serve as a noninvasive method of specimen collection in children and adults. It has already proven successful in various mutation screens [Doyle et al. 2004; Pawlowski et al. 2008]. In conjunction with the other biological samples obtained above, parent of origin effects can be identified.
In addition to the protocols described above, several reviews [Holland et al. 2005; Schrohl et al. 2008; Vaught 2006] are available that describe tissue isolation, preparation, and storage. As with the above, all emphasize the need for standardization to yield samples of uniform high-quality. The isolation and storage conditions of the RMN samples are summarized below in Table 2. When stored in the appropriate manner, samples can remain stable for a considerable period [Kaaks et al. 2000]. Cryovials with rubber gaskets are essential for any long–term storage to prevent sample incursion of any form to ensure integrity while maintaining fidelity.
Consideration must also be given to establishing the appropriate size of the aliquot for each sample. This primarily reflects how their downstream use is envisioned. For example, depending on the specific application, i.e., a Polymerase Chain Reaction base diagnostic–assay, each blood spot can contain sufficient material for at least 100 if not greater assays. As of today an FTA blood spot would not provide sufficient sample for an epigenome study that must rely on the use of the whole blood or other similar large sample. However, with the advances in technology of sample preparation and analysis approaching the single cell level, this may no longer present a limitation. These innovations will spur reevaluation to reduce the amount of sample made available, thereby extending the life of this resource. If viability can be assured, then prior to shipment the sample would be re-aliquoted such that each reduced aliquot would be sufficient for single use. In this manner the residual sample would be retained by the Repository for future use.
The growth and expansion of biorepositories since the late 1940s [Strong 2000] attests to the utility of these resources. Since the early 1980s various neonatal screening programs [Norgaard-Pedersen and Simonsen 1999] have created these resources providing one of the pillars of personalized medicine. For example, ELEMENT (Early Life Exposures in Mexico to Environmental Toxicants) [Pilsner et al. 2009] was one of the first studies to suggest that the maternal lead burden could modify the fetal epigenome. The spectrum of resources provided by the RMN biorepository provides a unique platform to assess developmental outcomes from conception to birth. By making the scientific community aware of the existence of the RMN repository and providing the collection and storage protocols, investigators should have sufficient information to collect samples of interest that can be directly compared to RMN cohort. In addition, the investigator may select and then request the most appropriately paired sample specimens from the RNM biorepository for inclusion in the specific study of interest.
This work was supported in part by NIH/NICHD grants U10HD055925 (HZ), U10 HD038992 (RL), U10 HD038998 (WS), U10 HD027049 (CC), U10 HD039005 (MD), U10 HD055936 (GC), U10 HD055942 (RB), and H10 HD055944 (PC). The authors would like to thank the other members of the RMN for their invaluable assistance in developing the RMN biorepository.
Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.