Developing a deeper insight into reproductive biomarkers.

Any biological index with the potential to be measured that indicates a defined biological endpoint, such as a disease or developmental stage, is known as a biomarker. Biomarkers may include a wide range of measurable, significant targets, including cellular, biochemical, immunological, genetic, physiological, and molecular changes. Biomarkers help us acquire knowledge about the effects and nature of an exposure and the vulnerability of organisms towards the noxious impacts of that exposure. Large-scale research has been conducted into the development of biomarkers to predict patients' responses to drugs and to diagnose clinical conditions, extending to all therapeutic areas, including reproductive medicine. Biomarkers can be used to identify subgroups for whom treatment is likely to be successful, to improve the assessment of exposures, and to predict the results, consequences, and classification of subgroups of potentially diverse disease etiologies [ 1 ]. It has been reported that 1.5 million women are infertile [ 2 ]; likewise, another study documented a prevalence of infertility of 9% among males [ 3 ]. Biomarkers can serve as a measurable indicator of proliferation and differentiation indices [ 4 ], the formation of DNA adducts or damage [ 5 ], apoptotic endpoints [ 6 ], chromosomal abnormalities [ 7 ], measurements of enzyme activity [ 8 ], micronucleus formation [ 9 ], changes in gene expression profiles [ 10 ], the expression of specific genes [ 11 ], and the presence of a parent compound or metabolite [ 12 ]. Biomarkers associated with reproductive impairment are principal early-warning signals of ecosystem impacts, and they require complete validation and characterization in an ecosystem [ 13 ]. Potential biomarkers of reproductive impairment are in various stages of development. Biomarkers serve several distinct purposes in reproductive medicine, including: (1) biomarkers of a disease or developmental stage: some biomarkers serve as indicators of specific phases in normal and abnormal developmental processes; (2) biomarkers of effect: physical or chemical environmental exposures can generate an assembly of localized or systemic effects and can be measured at the molecular, cellular, or clinical level; (3) biomarkers of exposure: biomarkers can be used to identify potential toxic exposures because of significant changes in biological function or appearance that signify exposure to a specific stimulus of a biological, physical, or chemical nature; (4) biomarkers of susceptibility: genetic biomarkers may also be used to identify predisposition to develop specific conditions [ 14 ]. Improving exposure assessments refers to differentiating subfertile patients from infertile patients and identifying the severity and extent of subfertility. A marker can also identify when a couple is best served by in vitro fertilization. Monitoring susceptibility to the effects of treatment and identifying subgroups may help individualize treatment of patients who are in dire need of a higher dose of medication or alteration of standard laboratory conditions such as oxygen tension or media. The accuracy of the prognosis may be improved by early detection of the number of embryos or which embryo is to be used in in vitro fertilization and the prediction of abortion, obstetric complications (preterm labor or preeclampsia), and ectopic pregnancy. The classification of patients into subtypes with different etiologies helps differentiate between implantation failure and unexplained infertility. Most reproductive biomarkers that have been developed so far deal with diagnostics, such as the diagnosis of polycystic ovarian disease, endometriosis, infertility, and the viability and location of early pregnancy. Multivariate biomarkers have also been developed; for example, the first diagnostic test based on a multivariate biomarker was OVA1, developed by Vermillion intended to be used for debulking surgery of patients suffering from ovarian adnexal masses. Recently, another multivariate biomarker to diagnose endometriosis has also been developed. To date, about 20 biomarkers have been identified in the serum for the early diagnosis of ectopic pregnancy [ 15 ]. Biomarkers approved by the Food and Drug Administration are shown in Table 1 .

Many environmental chemicals act as endocrine disruptors, such as dichlorodiphenyl-trichloroethane, dioxins, and polychlorinated biphenyls, which are anti-androgenic and estrogen-like in nature. They impede natural hormonal action and cause infertility in males. Prostate and testicular cancers, hypospadias, undescended testis, abnormal sexual development, Sertoli cell-only patterns, and altered thyroid and pituitary gland functions have been found to be caused by endocrine disruptors [ 109 ]. Many genes identified in the uterus have served as marker genes to evaluate the estrogenicity of endocrine disruptors. For example, the plasma glycoprotein clusterin gene, genes responsible for gap junction connexins, such as connexin 26 and connexin 43, and complement component 3 (C3) have been shown to be controlled by the endometrium of rats. C3 was found to be produced in the uterus of female mice. C3 is a protein that is an important contributor to immunity. In the uterus, endocrine disruptors increase the expression of the gene for ornithine decarboxylase. Therefore, these genes sensitive to estrogen have served as markers to analyze the estrogenic potential of endocrine disruptors of the uterus [ 110 111 112 ]. A study done on fish has reported that vitellogenin (VTG) is a precursor protein of egg yolk secreted by liver cells and released into the blood, from which it reaches the ovaries and promotes oocyte development; moreover, it has been proposed that VTG can also be used as a potential biomarker for estrogenic chemicals [ 113 ]. Although VTG is also present in male fish, it is not normally expressed due to the low circulating level of Estradiol (E 2 ) in the blood plasma; however, males have been shown to have ability to express VTG under the influence of estrogenic endocrine disruptors; thus, it has been proposed that the VTG gene in male fish may be used as a biomarker for analyzing the effects of endocrine disruptors [ 114 115 116 ]. A study revealed that estrogenic compounds and E 2 triggered the expression of pS2 in the cloning of the pS2 gene in the MCF-7 breast cancer cell line [ 117 ]. The production of pS2 mRNA can be induced by E 2 in some breast cancers, but not in normal breast tissue or in any other human cell lines. Therefore, the expression of pS2 mRNA in MCF-7 cells is an ideal model for estimating the impact of estrogenic compounds [ 117 118 119 ]. Mucin1 is an extended rod-like molecule protruding above the surface of epithelial cells, and it acts as a well-known marker of breast cancer [ 120 ]. The steroid receptor known as progesterone receptor binds with progesterone, and it is involved in a several physiological functions such as homeostasis, cell differentiation, and the control of embryonic development [ 121 122 123 ]. To evaluate the estrogenicity of endocrine disruptors efficiently in a cost- and time-effective manner, the expression levels of progesterone receptor genes are measured [ 116 ]. The use of calbindin-D9k (CaBP-9k) as a biomarker for endocrine disruptors has been reported. The injection of estrogenic chemicals such as nonylphenol, bisphenol A, 17β-estradiol, and 4- tert -octylphenol in immature mice led to CaBP-9k protein assembly and uterine localization, which were confirmed by western blotting and immunohistochemical staining, respectively. A time- and dose-dependent increment in the CaBP-9k protein was identified in the uterus of immature rats when treated with 4- tert -octylphenol and nonylphenol; thus, the CaBP-9k protein could serve a potent biomarker for evaluating the estrogenicity of putative estrogenic compounds [ 124 ].

It is necessary to develop clinically useful biomarkers to inform therapeutic and regulatory decision-making bodies about candidate drugs and their effects in an attempt to resolve reproductive impairments through the development of new diagnostic methods and medicines based on biomarkers. The number of candidate markers in reproductive medicine is increasing, and it is urgently necessary to comprehend the development pathway from discovery to clinical utility. Extensive testing and modification, along with validation, must be performed before a biomarker is demonstrated to have clinical utility. New partnerships and opportunities exist and should hasten the development of reproductive biomarkers. The classification of biomarkers discussed in this review is presented in Table 2 .

Neohormones are new paracrine or endocrine adaptations that have the ability to define mammalian success to complement physiological functions. Relaxin like peptide hormones are common neohormones, as they define basic reproductive physiology, such as viviparity with placentation or implantation, lactation, and adaptations required by sperm cells for successful internal fertilization, thereby acting as highly useful biomarkers to characterize and monitor reproductive diseases. H2-relaxin assists in implantation and the development of the placenta in the ovary, whereas its levels change in cases of early miscarriage. During fetal development, testicular INSL3 is important for the first phase of testicular descent, but it may be disrupted in cryptorchidism. In adults, INSL3 is considered to be an antiapoptotic factor in follicle selection (female) and germ cell survival (male), and serves as an excellent indicator of Leydig cell functional capacity, especially in aging males. Likewise, INSL5 and INSL6 have been reported to be involved in the maintenance of adequate spermatogenesis [ 16 ].

Endocannabinoids and anandamide have proven to be potential biomarkers of reproductive impairments. These are a group of bioactive lipids that act as crucial signals in human reproduction. Fluctuations in the balance between the degradation or decay and synthesis of endocannabinoids lead to local changes in the human male and female reproductive tracts, which in turn adjust and control several pathophysiological processes, including sperm and oocyte maturation [ 17 ]. A study revealed that endocannabinoids measured in the saliva could be used as a biomarker of obesity [ 18 ].