RBMOnline - Vol 15 No 4. 2007 451-456 Reproductive BioMedicine Online; www.rbmonline.com/Article//2988 on web 17 August 2007 Outlook Emerging technologies for the molecular study of infertility, and potential clinical applications Dr Ashok Agarwal is a Professor in the Lerner College of Medicine at Case Western Reserve University and the Director of Reproductive Research Center, and the Clinical Andrology Laboratory at The Cleveland Clinic, Cleveland, Ohio, United States. He has published over 300 scientific articles, reviews and book chapters in different areas of andrology and reproductive biology. His long-term research interests include studies on the role of oxidative stress, DNA integrity, and apoptosis in the pathophysiology of male and female reproduction. Dr Ashok Agarwal Alex C Varghese1,2, Eric Goldberg3, Asok K Bhattacharyya1,2, Ashok Agarwal3,4 1DeCode Life Foundation; 2 Department of Biochemistry, University of Calcutta, Kolkata, India; 3Reproductive Research Centre, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, Ohio, USA 4Correspondence: Tel: +1 216 4449485; Fax: +1 216 4456049; e-mail: [email protected] Abstract The techniques currently used to treat infertility cases are quite limited in their capabilities, due to an incomplete understanding of the molecular activities of germ cells. Fortunately, several technologies are presently being researched that should aid in the understanding of the various molecular causes of germ cell pathologies. This review discusses microarray technology, proteomics, metabolic profiling, the PolScope, atomic force microscopy and microfluidics. These technologies have all seen success in preliminary studies, and promise directly or indirectly to improve the low success rates of IVF and other related therapies. However, their widespread use in laboratories and clinics may not be seen until preliminary studies confirming their safety and effectiveness are published, and until standardized protocols for their utilization are established. Keywords: infertility, IVF, microbiology, research, technology Introduction It is estimated that worldwide, 15% of couples of reproductive this relates to the molecular pathways in the testis remains age encounter fertility problems (Nishimune and Tanaka, rudimentary. To gain a more detailed understanding of the 2006). This has been attributed to a variety of factors, including molecular basis of male infertility, it would be advantageous environmental pollutants (He et al., 2006), and the increasing age to study the differences in gene expression between fertile and at which couples decide to have children (Manipalviratn et al., infertile males. If the differences in mRNA profiles, otherwise 2006). There are currently a number of techniques available to known as transcriptomes, are uncovered, there will be greater clinicians in diagnosing and managing infertility, but there are insight into identifying potential biochemical markers for still limitations in the field. Regarding IVF, multiple pregnancies infertility, as well as discovering clues to its indirect causes continue to be a problem (Manipalviratn, et al., 2006), and or direct triggers. Fortunately, the microarray is capable of success rates are greatly limited by the current embryo culture analysing the transcriptome of cells and tissues (He et al., methods (Suh et al., 2003). Fortunately, several technologies are 2006). presently being researched that should aid in the understanding of the various molecular causes of germ cell pathologies, thereby To learn more about the molecular events of spermatogenesis, improving the clinical management of infertility cases. researchers have used the microarray to compare the gene expression of spermatozoa from fertile and infertile men, and Microarray to compare the transcriptomes of germ cells at various stages in spermatogenesis. A number of novel genes associated with Despite detailed knowledge of testicular structure and male fertility have even been identified using the microarray. accompanying cell biology, the current understanding of how As more research is conducted using this technology, it can be 451 © 2007 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB3 8DB, UK Article - Potential of modern technologies in infertility treatment - AC Varghese et al. anticipated that several molecular pathways associated with informed choices regarding the selection of ovarian stimulation sperm physiology and pathology will be uncovered (He et al., protocols in female infertility cases. Such information could 2006). also lead to the selection of higher quality oocytes for IVF, and improve the culture and oocyte manipulation techniques The microarray also has potential for use in clinical diagnosis that are used (Assou et al., 2006). However, one must always of male infertility. One large step in this direction was the keep in mind that even if a specific gene transcript is observed, creation of a spermatogenesis-related gene expression profile not much information can be realistically known regarding the for human spermatozoa. This was paired with a demonstration metabolic events within the cell. If microarray is to be utilized that one could legitimately assess the overall health and quality for studies of cellular activity, it is imperative that proteomics of a sperm cell by analysing its gene expression signature using technology is also incorporated into the studies. the microarray (Wang et al., 2004). To use this non-invasive technology to assess a male’s fertility Proteomics technology status, a semen sample is first collected, followed by isolation of spermatozoa from the ejaculate, and then isolation of mRNA One complement to the field of gene expression analysis is from the spermatozoa. While functionally mature spermatozoa proteomics (Figure 1). This is the study of protein abundance in do not undergo transcription, they have been shown to contain cells or tissues, and involves the discovery of protein functions mRNA, which was synthesized during spermatogenesis, at the biochemical level (Rocken et al., 2004). providing insight of the specific spermatogenic events of an individual. The microarray is used to create an mRNA profile Fundamental proteomic techniques include protein separation that can be compared with a physiologically normal gene and the identification of proteins in biological samples. These expression profile for sperm. This would help to identify if can then be coupled to computational algorithms that allow the cause of infertility for a male is of spermatogenic origin. the extraction of relevant information from the totality of Utilizing the microarray to analyse a male’s fertility status is data. Fortunately, several advances in proteomics, including sure to reveal much more information when compared with the the association of mass spectrometry with techniques of techniques currently employed in clinics (Moldenhauer et al., electrospray ionization (ESI) and matrix-associated laser 2003). desorption/ionization (MALDI), have greatly facilitated the ability to sequence and characterize peptides and proteins There would be many advantages to creating microarray-based (Shankar et al., 2005). Additionally, proteomics researchers transcription profiles of oocytes at various stages of growth are currently developing a method to compare protein profiles and maturation. It would result in a better understanding of of cells and tissues in varied biological states. With such a the genes expressed during oocyte development, as well as development, it will be easier to learn more about the molecular those expressed at the various checkpoints that regulate the activity and behaviour of cells (Rocken et al., 2004). process. As of now, it is known that in a fully mature oocyte arrested in metaphase II (MII), the mRNA profile is essentially One such development, surface-enhanced laser desorption/ a collection of the mRNA transcripts created during its growth ionization time-of-flight mass spectrometry (SELDI-TOF MS), phase. In addition, disruption of transcription within an oocyte involves affinity-based mass spectrometry whereby proteins or modification of its current transcriptome could negatively are absorbed to a chemically modified surface (e.g. cationic or effect its growth and development, as well as that of the embryo anionic) or to a biochemical molecular surface (e.g. receptors (Wood et al., 2007). or ligands). These different chip surfaces allow the various classes of proteins (hydrophobic, hydrophilic, acidic or basic) Microarray analysis has indicated that the transcriptomes of MII to be captured for analysis (Merchant and Weinberger, 2000). oocytes of high morphological quality from normal women are SELDI-TOF MS has been shown to capture, detect and analyse distinctly different from the morphologically normal oocytes proteins directly from crude biological fluids. Alternatively, of women with polycystic ovaries (PCO). These molecular quadruple TOF (Q-TOF) identifies proteins by searching defects could help to reveal why women with PCO tend to have appropriate sequence databases. The high accuracy of the reduced fertility (Wood et al., 2007). It has also been shown Q-TOF technology makes the hybridization of MALDI and with microarrays that oocyte quality can be estimated based on Q-TOF a superior option for de-novo protein sequencing (Gygi the expression levels of at least 160 different genes (Zhang et and Aebersold, 2000). al., 2005). Many of these have been shown to be involved in pathways