unit 5. application of biomarkers to disease

chapter 25. Disorders of reproduction Anne Sweeney and Deborah del Junco

Summary Introduction

This chapter focuses on biomarkers The utilization of biomarkers in Reproductive epidemiology of reproductive health and disease reproductive and perinatal health studies often consist of a triad, that have been developed in the research has greatly enhanced including the , father and past 15 years. Due to the gender- our understanding of these critical conceptus, which constitutes the unit and age-dependency of most areas of public health. There has of both observation and analysis. of the advances in measuring been increasing emphasis on these The ability to obtain biomarkers for reproductive health status and time periods in early development all three of these subjects varies outcomes, these biomarkers have as vulnerable windows over the life greatly, compounded by the differing been categorized with respect to the course, during which humans are time intervals of concern for each unique member of the reproductive most highly susceptible to the effects subject in terms of biomarkers of triad of interest (i.e. mother, of exposure to toxic agents in the exposure, susceptibility and effect. father, conceptus). Biomarkers of environment. The periconceptional, Other challenges include the and male , female prenatal, perinatal and peripubertal interrelatedness of reproductive reproductive function, fetal and time periods are considered to outcomes across the spectrum infant development, and male be the most susceptible intervals of time-dependent endpoints of reproductive function are discussed. for adverse health events (1–7). interest and the accuracy and The strengths and limitations of However, methodologic issues reliability of the markers available developing and implementing unique to this area of research for evaluation. Examination of biomarkers in reproductive health render the identification of effects that occur at later time studies over the past decade are appropriate biomarkers a daunting points in (e.g. recognized explored. challenge. spontaneous or preterm Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 453 delivery) are restricted to those to these topics (12–20). Likewise, association studies of reproductive conceptions that have survived readers are referred to Chapter health outcomes (26–33). long enough to be identified for 7 and the wealth of resources Figure 25.1 provides an evaluation. This reinforces the urgent described in Perera and Herbstman illustration of the spectrum of need to develop methodologies, (14), Burke et al. (21), Seminara et reproductive outcomes (although including biomarkers, that enable al. (22), Field & Sansone (23) and not exhaustive) that are available us to examine the earliest outcomes Ho & Tang (24) for more in-depth for investigation. This figure along this spectrum (8). information on developments in attempts to present these topics in genomic, transcriptomic, proteomic, a chronological fashion, from the Context and public health metabolomic and epigenomic earliest sentinel of potential adverse significance technologies to examine disease reproductive function among males susceptibility and etiopathogenetic and , to early or delayed In developing biomarkers that pathways in reproductive onset of puberty, and extending to would be appropriate for use in epidemiology research. An childhood in their offspring large-scale epidemiological studies overview (25) describes how the that may be linked to prenatal of reproductive outcomes, one combination of bioengineering exposures. Again, the earliest events must consider not only sensitivity, and bioinformatics has evolved to along this chronological spectrum specificity, predictive value, within- help reveal integrated, dynamic represent the target areas of greatest subject reliability (low coefficients molecular networks underlying focus, as these outcomes enable of variation) and cost, but also complex functions in biological the examination of a representative acceptability and ease of use by systems like cohort at risk, and may serve as study participants (9). Several and early development. Also early sentinels of exposure to studies have reported an increase beyond the scope of this chapter, toxic agents in the environment, a in participation rates when study but covered well in several recent major and unresolved public health subjects are taught how to collect publications, are genome-wide concern. and ship biological specimens from the privacy of their own homes, as Figure 25.1. Selection of reproductive outcomes available for study opposed to having the samples collected in clinics or field offices Age at pubertal onset (10). Decreased (males, females) In this chapter, the earlier review by Lemasters and Schulte (11) is Menstrual cycle function updated, focusing on biomarkers / abnormalities of reproductive health and disease that have been developed in the Time-to- 15 years since that publication. Due to the gender- and age- Early pregnancy loss dependency of most of these Recognized spontaneous advances in measuring reproductive health status and outcomes, the biomarkers have been categorized Neonatal/infant mortality with respect to the unique member Congenital anomalies of the reproductive triad of interest Intrauterine growth restriction (i.e. mother, father, conceptus). Low birth weight Detailed discussions of advances in molecular biomarker technologies to measure exposure to environmental Sudden Infant Death Syndrome and infectious agents in reproductive Neuropsychological/cognitive disorders epidemiology studies (beyond the scope of this chapter) are covered Developmental disorders in Chapters 9–13 of this text and Childhood in comprehensive reviews devoted

454 Biomarkers of female age and certain psychological a correlation of r = 0.65 (P < 0.01) and male puberty disorders (40). Moreover, many between serum and urinary leptin young individuals are reluctant to levels (46). Of note, urinary leptin There has been persistent, perform this self-assessment, even levels corresponded to serum levels increasing concern over the in the privacy of their own homes; and patterns by gender during past several years regarding the one study had a 61% response rate puberty. observation that children in the USA when participants were asked to The gonadotropins, luteinizing are entering puberty at younger ages. complete the procedure at home (LH) and follicle While advancing age at puberty and mail in their information (41). stimulating hormone (FSH), and the may reflect inadequate nutritional or There are several biomarkers sex steroid , and socioeconomic conditions, younger currently undergoing evaluation for , also increase in early ages may be indicative of other use in ascertaining pubertal status. puberty, and can be measured in adverse scenarios, including obesity Most have limited feasibility for use urine (47,48). A small longitudinal and exposure to endocrine active in large, population-based studies, study recently evaluated the compounds in the environment (34). as the components of interest have relationship between urinary leptin Most expert panelists assembled short serum half-lives and would and gonadotropin levels in 13 boys by the US Environmental Protection require the collection of serial blood and seven girls over a six-month Agency (EPA), the National Institute samples. These include leptin, period as they were expected of Environmental Health Sciences an adipocyte hormone involved to approach puberty (49). Three (NIEHS) and Serono Inc. to evaluate in energy that also consecutive first morning urine secular trends in the timing of interacts with the reproductive axis, samples were collected each month. puberty concluded that there is and Müllerian inhibiting substance These results indicated significant sufficient evidence of earlier (MIS), a glycoprotein hormone correlations between urinary leptin development onset and produced by the Sertoli cells of the and LH levels (r = 0.43, P < 0.001) in girls (35). On the other hand, male during fetal development that and FSH levels (r = 0.32, P < 0.001). almost all the panelists agreed causes regression and atrophy Moreover, urinary leptin levels were that there is insufficient evidence of the Müllerian ducts (42–44). higher among the girls, and were regarding trends in male pubertal Leptin is a critical regulator of body increased among both girls and development. fat stores, which may underlie boys nearing puberty compared The Tanner scales have been its role as a possible biomarker with those remaining prepubertal widely used by clinicians for several of approaching puberty. Given over the course of the study. years to examine onset of puberty in the well-known changes in body Both MIS and inhibins, peptides girls and boys. The scales assess fat mass and percent body fat that suppress FSH levels, have stages of , associated with puberty, it is been characterized as potential pubic hair growth, genitalia hypothesized that leptin may serve biomarkers of pubertal onset in changes, and age at menarche as a biomarker of peripuberty males. MIS is detected at high (36). A method of self-assessment and pubertal advancements (45). levels during late infancy in males, using photographs and written In girls, serum leptin levels rise then declines gradually until the descriptions of the various stages markedly as they approach puberty, presence of primary of development was developed and this increase is correlated with are detected, which appear to and evaluated by researchers in body fat mass. The levels continue inhibit MIS (50). In contrast, MIS different populations (37–39). A to increase throughout puberty, is only synthesized postnatally by recent review of biological markers whereas among boys, there is an granulosa cells in pubertal girls, for assessing puberty status, initial increase during peripuberty, and is measured in serum (51,52). however, indicated that many study followed by a return to prepubertal Serum concentrations are similar in participants are reluctant to undergo levels as they advance through both sexes after puberty (53). Inhibin this examination by a clinician, and puberty (43). The ability to measure B is a gonadal polypeptide hormone several studies have indicated a leptin in urine would greatly that regulates, via a negative range of correlations between self- enhance the utility of this biomarker feedback loop, the synthesis and reported and physician Tanner in studies of peripubertal events. A secretion of FSH (54). Similar to scores, depending upon many recent cross-sectional study of 188 MIS, among males there is a peak factors including race/ethnicity, children, aged 5–19 years, reported concentration of serum inihibin B in Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 455 infancy, followed by a rapid decline the association between endocrine- have been tested for validity and until onset of puberty, when another active chemicals in the environment reliability, a biomarker for HSDD in rise in serum levels is noted. Inhibin and altered timing of pubertal women remains elusive. Although B is also associated with FSH, LH, onset concluded that the evidence there is a growing body of evidence testosterone and testicular volume in available appears suggestive (62). supporting the role of testosterone varying patterns throughout puberty Future epidemiologic research and in women, the (54,55). In girls, serum inhibin B is to elucidate gene-environment association between decreased positively correlated with age and interactions and the molecular serum androgen levels and women FSH levels during childhood, and pathways mediating the influence reporting low libido remains unclear an increase during early breast of environmental exposures on (73,74). The development of assays development stages (56). However, pubertal development is eagerly to measure testosterone levels because these biomarkers currently awaited. in saliva samples would greatly require drawing blood samples facilitate the investigation into the for measurement and biological Biomarkers of female potential relationship between variability (e.g. diurnal fluctuations) reproductive function exposure to endocrine active is unknown, research is needed to compounds in the environment and develop valid and sensitive urinary Female libido decreased libido (75,76). biomarkers that will also be feasible in terms of cost and acceptance by Changes in usual patterns of sexual Menstrual cycle study participants in longitudinal desire can also serve as an early characteristics studies (57). sentinel for exposures that may adversely affect reproductive health. Alterations in menstrual cycle Molecular epidemiology Most of the concerns regarding a characteristics may also serve studies associating relationship between exposure to as early sentinels of exposure to environmental exposures endocrine active compounds and potentially harmful environmental with puberty onset decreased libido have focused contaminants. Furthermore, on males, although some have changes in menstrual cycle The few epidemiologic studies questioned whether this may also parameters have been associated that have associated pubertal be a problem among women (63). with adverse reproductive development with exposure to The complex interactions between outcomes, including infertility and endocrine disrupting chemicals (e.g. sex steroid hormones and the spontaneous abortion (77–80). One polychlorinated and polybrominated hypothalamic-pituitary-gonadal axis small study (n = 14) determined that biphenyls (PCBs and PBBs) and at varying times over the menstrual urinary FSH was significantly lower phthalate esters) have been the cycle also add to the challenges in the periovulatory period in cycles topic of several recent reviews in measuring these hormones as that did not result in conception (58–60). Conflicting findings and biomarkers of decreased libido (64). compared with those that did, uncertainties regarding critical There have been several recent rendering urinary FSH a potentially windows of susceptibility and the studies of diminished sexual desire useful predictor of cycle . possibility of exposure to complex in females, but the majority have Numerous studies have been mixtures of chemicals that may examined this condition among conducted using questionnaires to have antagonistic effects highlight postmenopausal women (65–68). obtain information on cycle length, the need for further research with A related outcome, hypoactive days and severity of menstrual flow, objective biomarkers. A recent sexual desire disorder (HSDD), and ; however, these epidemiologic investigation of is defined as low sexual desire methods often do not yield valid pubertal stages in nine-year-old accompanied by personal distress information. One study described inner-city girls in New York City caused by this decrease in sexual the use of urinary biomarkers to was unique in associating delayed desire (69,70). The prevalence of determine sex steroid hormone breast development with high levels low sexual desire among younger levels throughout the menstrual of the hormonally active agents and middle-aged women that is cycle among healthy premenopausal phytoestrogens and isoflavones not ascribed to menopausal effects women (n = 403) (81). The women measured in urine (61). An expert ranges from 24–31% (71,72). While were required to collect and freeze panel recently convened to review questionnaires to ascertain HSDD daily morning urine samples that

456 were analysed for pregnanediol- follicular and segments, Infertility/early pregnancy 3-glucuronide, sulfate and and occurrence and timing of loss estrone glucuronide (combined and were determined using referred to as E1C) by - urinary estrogen and The only approach that allows linked immunoassay. Using metabolite levels that were analysed researchers to distinguish between computer-generated algorithms to in daily morning urine samples. women having infertility and those define menstrual cycle events, the These efforts confirmed earlier experiencing subclinical or early researchers were able to utilize the reports that menstrual cycle pregnancy loss is the prospective hormone data to describe menstrual characteristics vary by age, race/ pregnancy study design. Though cycle length, the length of both ethnicity and lifestyle factors (e.g. beyond the scope of this chapter the follicular and luteal phases, as alcohol consumption) (84–86). because of limited application in well as occurrence and timing of Taken together, these studies large-scale epidemiologic research, ovulation. provide evidence that urinary diagnostic advances in assisted Using the same sampling frame biomarkers can be used in large, reproductive technology (ART) as the study above, the Kaiser population-based studies of have shed light on this distinction, Permanente Medical Care Program menstrual cycle function, as well as and readers are referred to several in California evaluated variability in selected and pregnancy recent and thorough reviews (92– in estrogen and progesterone outcomes. However, it must be 94). As illustrated in Figure 25.2, metabolites according to these noted that the majority of these there is increased emphasis on the menstrual cycle characteristics, protocols required daily specimen periconceptional environment that as well as demographic variables collection over varying lengths may impact fertilization as well as and reproductive history (82). With of time. While compliance rates implantation. Scientists have made an average length of participation with urine specimen collection in great strides in the ability to detect of 141 days, urine samples were prospective pregnancy studies is early once implantation collected on over 95% of the study generally quite high (8), it must also takes place. The measurement days in this sample of mostly be noted that these studies typically of urinary human chorionic white, highly-educated women. involve women (or couples) who are gonadotropin (hCG) as a marker They reported urinary estrogen planning to conceive. There may of early pregnancy loss has been metabolite levels 10–13% higher in therefore be considerable variability used since the late 1980s (95–97). the baseline interval (days 1–5) and with compliance, depending upon The algorithm for determining the during the among the goal of the study and the level of hCG that exceeds normal women who experienced shorter pregnancy intentions of the sample. background levels and indicates menstrual cycles. There was also Additional biomarkers that that conception has occurred was an association between increased would be feasible for use in large, developed by examining hCG levels urinary progesterone metabolites population-based studies focusing throughout the menstrual cycle in and a longer luteal phase. on the detection of ovulation include women who had been surgically Associations between estrogen cervical mucus monitoring, which sterilized (96). Concerns were and progesterone metabolites and has been available and used for later raised suggesting that urinary race/ethnicity, prior reproductive several years, and salivary sex hCG alone may not be a sensitive experiences, age, BMI and steroid hormones levels. However, indicator of early pregnancy among educational level were also noted. studies examining the validity and those conceptions terminating A large study was conducted feasibility of analysing salivary in subclinical losses (98–100). in 1989–1991 to determine the samples for various biomarkers of However, when patterns of serum relationship between occupational reproductive function have included and urinary hCG levels were exposures among women employed relatively small sample sizes, and compared between successful in the semiconductor industry and reported considerable intra- as well pregnancies and those terminating fertility and early pregnancy losses as interindividual variability for the in early pregnancy loss and clinical (83). Investigators evaluated the sex steroid hormones (87–91). spontaneous abortion, there were influence of demographic and no differences, thus validating the lifestyle factors on menstrual solitary measure of urinary hCG as cycle characteristics among 309 a biomarker of pregnancies around women from this cohort. Duration of the time of implantation (101). Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 457 Figure 25.2. The sensitive periods in human development (230). Copyright Elsevier (1998).

A prospective, longitudinal The development of fertility time, or the onset of the LH surge study of early pregnancy loss monitors and sensitive urinary hCG and the subsequent two days. The (conceptions ending within five assays that can easily be used by control group does not receive any weeks of ovulation) among women in the home to detect early fertility monitor feedback concerning indigenous women of rural Bolivia pregnancy has greatly enhanced fertile windows. This same fertility correlated salivary progesterone the ability to examine fecundability, monitor is also currently being used measurements with urinary hCG infertility, time-to-pregnancy and in the Longitudinal Investigation of measurements (102). Among the early pregnancy loss (103,104). Fertility and the Environment (LIFE) 191 study women who were eligible The Oxford Conception Study is a study, a prospective examination (taking no active steps to either randomized clinical trial to determine of the impact of environmental prevent or achieve conception) if knowledge of the timing of peak exposures and lifestyle factors on and visited a clinic every other day fertility increases conception rates fecundability and fertility, which will (to collect samples and record the among couples trying to achieve be described later. first day of menstrual bleeding), pregnancy (103). The study is using There are additional advantages eight early pregnancy losses were the Clearblue® Easy fertility monitor, to using the fertility monitors. detected and 32 pregnancies were which measures levels of urinary Women are requested to collect first sustained past the five-week cut estrone-3-glucuronide (E3G) and morning urine samples beginning on point. Overcoming some of the LH. The monitor screen displays day six of their cycle, and continue limitations of salivary progesterone bars to indicate high and peak (which for 10 or 20 days depending upon measurements described above, displays an symbol) fertile days in their cycle length; thus the need the investigators were able to detect the cycle. There are two intervention for daily urine sample collection is ovulation as a sudden, steep rise arms to the study: one third of women reduced somewhat. Moreover, the in salivary progesterone, and they receive feedback only on the early monitors store data on the estrogen reported a significant association fertile time, defined as the first rise in and progesterone metabolite between elevated follicular phase E3G until the LH surge is detected, levels, which may be downloaded (pre-ovulatory) progesterone and and another one third receive using a data card and processed subsequent early pregnancy loss. information only about the late fertile for data analysis. These hormone

458 data are also used to generate compared with 25 non-pregnant in population-based prospective graphs for study participants, which women (6.58, 5.71, and 3.44 RTI, pregnancy studies. provide a clear illustration of their respectively (P < 0.001). Moreover, fertile windows during each cycle, there was a significant correlation Biomarkers of fetal and reinforce the importance of between serum and cervical mucus and infant development engaging in on RTI values, r = 0.611 (P < 0.0005). those days to increase likelihood of Additional research on EPF Adverse fetal conception. activity in cervical mucus around or infant outcomes The utilization of fertility the time of ovulation is needed monitors and home pregnancy to determine the feasibility of this The major focus of this review test kits as described above has biomarker in identifying fertilization is the identification of new greatly enhanced the information and preimplantation events in biomarkers of reproductive health that can be obtained in prospective fecundability and fertility studies. that can be employed in large- pregnancy studies. However, the The challenges inherent in scale epidemiologic studies of home pregnancy tests cannot establishing and maintaining reproduction. Although the many detect fertilization, and only begin prospective pregnancy study promising biomarkers developed to measure hCG around the cohorts underscore the importance recently for use in clinical time of implantation. Thus, the of developing biomarkers that are and reproductive endocrinology interval between fertilization and sensitive, specific and cost-effective are beyond the scope of this implantation remains a ‘black box’ in but also acceptable and relatively chapter, readers are referred to the investigation of factors affecting easy to use. The LIFE study, in-depth discussions of advances fertility. There have been some designed to examine the relationship in the detection of pregnancy attempts to measure a substance between environmental and complications (e.g. pre- known as early pregnancy factor lifestyle factors and fecundability and intrauterine growth retardation) (EPF) (105–107). EPF is believed and fertility, is funded by the US and other adverse prenatal events to be a substance secreted by National Institute for Child Health provided elsewhere (109–112). the in response to a trigger and Human Development and is Several resources are also available from the zygote (107). This ‘ovum currently enrolling couples in Texas for updates on screening and factor’ is secreted at the time that and Michigan (http://www.lifestudy. diagnostic tests for aneuploidy, the ovum is fertilized, and can be us). This study methodology is including Down syndrome (113– measured in the maternal serum the only approach that allows for 117), other congenital anomalies within 2–6 days post ovulation ascertainment and examination (118), fetal lung maturation (119) (106). Despite the fact that several of the early critical events in the and haematologic disorders earlier studies demonstrated the reproductive process in humans and complications (120) during ability of EPF to detect fertilization (i.e. to distinguish between failures gestation. Epidemiologic studies in humans before implantation, this of fertilization versus subclinical of perinatal outcomes and long- biomarker has not been evaluated pregnancy losses), and hopefully term health in children conceived for use in prospective studies of soon, implantation failures versus by ARTs have been an important fecundability and fertility. Again, these other two outcomes. The source of prospective and high the need to draw blood samples LIFE study recruitment efforts quality data. Outcomes following the during each menstrual cycle to have determined that willingness to more recently developed ARTs (e.g. detect EPF, renders this less than comply with protocol requirements, intracytoplasmic sperm injection) optimal in large-scale population- including collection and testing of are currently being compared based studies, but a recent study biological samples and completion with outcomes following the more reported the presence of EPF in the of diaries during the interval of established technologies, such as in cervical mucus of pregnant women attempting to conceive, as well as vitro fertilization (121–123). (108). Mean EPF activity, measured changes in pregnancy intentions due There have been recent in rosette inhibition titres (RIT), to varied life events while enrolled in advances in the utilization of was significantly higher among 53 the study, illustrate the need both dried blood spots (DBS) that hold pregnant women during their first for the development of sensitive great promise for large-scale trimester of gestation, and seven and acceptable biomarkers, as epidemiologic studies aiming to women in the second trimester, well as very large sampling frames identify valuable biomarkers of Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 459 susceptibility, exposure and effect. variability in whole blood matrices; is as yet no compelling evidence Blood spots, obtained via heel and issues related to stability, that any other hormonally active stick, are collected within 24–48 recoverability, half-life and storage agents increase the risk of TDS. hours after birth on nearly all over time. Great care must be taken An enormous amount of valuable newborns in the USA. The spots in the collection, drying, storage and information on the reproductive and are stored on special filter paper transport of the spots if they are to developmental effects of the atomic Guthrie cards and used by states in be of future use in research studies bombing of Hiroshima and Nagasaki screening programmes to diagnose (131). There is also the critical need has been compiled and summarized a variety of disorders among to develop policies regarding the (136). The unique and precedent- newborns. The number of different ethics and human subjects research setting contributions to the field of disorders assessed in the screening challenges presented by these reproductive epidemiology, as a programmes is determined by specimens (132,133). result of the investigators’ careful each state’s department of health. and extensive use of biomarkers There has been a surge of interest Molecular epidemiology throughout the extended course recently in the analyses of DBS to studies associating of this research, are remarkable. investigate diverse disorders in large environmental exposures with Recent studies reporting increased population-based studies, including fetal and infant development risks of preterm birth, low birth environmental exposures and their weight, and small for gestational age relationship to congenital anomalies An extensive review of the among the births to female survivors and developmental disorders, the epidemiologic literature (including of childhood cancer further illustrate prevalence of infectious diseases original studies, expert panel the advantages of accurate, among newborns, and genetic reports, meta-analyses, and carefully documented biomarkers disorders and potential biomarkers pooled analyses) published in revealing the reproductive health of susceptibility (124–129). between 1970 and 2006 examined effects of exposures at time points A recent meeting discussed the adverse reproductive and early in the mother’s development issues and approaches to using DBS developmental outcomes in relation (137,138). in studies investigating environmental to preconceptual and prenatal exposures and infant health exposures to environmental Gene–environment outcomes (130). Preliminary work compounds (18). The pregnancy interaction studies in has been conducted to determine outcomes examined included fetal pregnancy outcomes the feasibility of analysing DBS loss, intrauterine growth retardation, for several exposures of concern, preterm birth, birth weight, Molecular epidemiology studies including persistent bioaccumulative congenital anomalies and childhood examining the influence of gene- toxics (PBTs is the term used by the cancers among others. The environment interactions on US EPA, whereas the environmental exposures included reproductive health have focused Environment Programme uses the metals, pesticides and hormonally most often on the relatively common term persistent organic pollutants, active agents (e.g. methyl mercury birth defects, such as neural tube or POPs), metals, infectious and PCBs). The question of whether defects, oral clefts, hypospadias agents, immune factors and genetic in utero exposure of male offspring and gastroschisis (139–147). disorders. The scientists concluded to hormonally active agents in the Several these studies have linked that DBS represent a very valuable environment (e.g. the plasticisers, either folate metabolism genes with source of biomarkers of exposure, phthalates) (134) increases their maternal nutritional factors (139– effect and susceptibility, but there risk of testicular dysgenesis 141) or metabolic/detoxification are limitations that must be resolved syndrome (TDS) (i.e. impaired pathway genes with maternal before they can be used to the , hypospadias, smoking (143,144,146). Maternal maximum potential. The limitations cryptorchidism and testicular exposure has been assessed by include inadequate sample volume, cancer) in a manner similar to in questionnaire or interview (without as many laboratory techniques utero exposure to diethylstilbestrol biomarkers) and genotyping has to measure environmental (DES), was evaluated in a recent been performed on DNA from the contaminants require relatively large meta-analysis (135). Although mother (146), infant (142,145,147), or volumes; development of reference the meta-analysis confirmed the rarely, the entire triad including the values for elements with large association of DES with TDS, there father (143,144).

460 Researchers have emphasized consent for sensitive, invasive or review of employment records, the utility of examining Mendelian complicated sample collection or by environmental monitoring randomization, or the random procedures); exposure assessment techniques. None of the studies had transmission of genes that occurs (that may include idiosyncratic measured exposure to pesticides between parent and offspring, in sample collection, processing using biological samples (e.g. urine or drawing inferences from studies and storage requirements and blood). This is understandable given that examine the developmental expensive analytical techniques); that over time, study participants health effects of in utero exposures and data analysis strategies that may have been exposed to several in combination with candidate must take into account important different compounds (either one susceptibility genes (148). Studies interrelationships (e.g. confounding at a time or in complex mixtures), that genotype each member of and effect-measure modification) that many pesticides have short the triad separately may confer across large numbers of measured half-lives in the biological samples an important advantage to the independent and dependent commonly used for analysis, and interpretation of study results. variables. that the limits of detection for many Specifically, if the genetic variant of the standard chemical analyses under study either influences Childhood cancer are relatively high. exposure to the etiologic factor of interest or modifies the exposure– The extent to which the etiology Transgenerational health disease relationship, then the of childhood cancer involves in effects and epigenetic expected relative risk for the utero or preconceptional parental mechanisms adverse pregnancy outcome exposure to environmental agents would be farthest from the null remains a major public health Several recent studies have when presence of the risk gene is concern and an important research advanced yet another challenging measured in ’ DNA, closer question for both reproductive and but essential area of research to to the null when the presence of the cancer epidemiologists. Biomarkers determine adverse reproductive risk gene is measured in fathers’ of childhood cancer and a review of effects of environmental DNA, and somewhere in-between the epidemiologic investigations that exposures—the conduct of second- when presence of the risk gene is have incorporated molecular markers generation studies in humans. measured in infants’ DNA. of exposure and susceptibility are The critical importance of this Research interest in the discussed in detail in Chapter 26. effort is illustrated by the studies interactions between metabolic/ A recent comprehensive review of the offspring of women who detoxification genes and maternal/ of pesticides and childhood took DES during pregnancy to in utero exposure to environmental cancer (153) is discussed only prevent spontaneous abortions. agents has begun to extend briefly here to emphasize the Earlier animal studies suggested across the spectrum of pregnancy critical need for improvements that the carcinogenic effects of outcomes including preterm delivery in exposure assessment (e.g. prenatal DES exposure may be (149), infant birth weight (150), objective biomarkers of exposure transgenerational, reporting an pervasive developmental disorders to environmental chemicals) and increase in reproductive tract (e.g. autism) (151) and childhood- for the detection of specific gene– tumours among the offspring of mice onset attention deficit disorder (152). environment interactions that are with prenatal exposure to DES (154– These studies further illustrate likely contributors to the complex 156). After noting the occurrence of the methodologic challenges etiology of childhood cancer. Among hypospadias among two boys born often confronted by molecular the 77 studies included in the to mothers who had been exposed epidemiologists: constraints on the review (153), parental exposure to to DES prenatally, researchers in study design (e.g. statistical power pesticides (including herbicides and the Netherlands conducted a cohort issues and the need to rely on insecticides) was measured indirectly study among women experiencing case-control or case-only designs); (e.g. by proximity of residential fertility problems to examine this source populations of varying race, address to chemical production association (157). Among 205 ethnicity and genetic backgrounds plants or by classification of usual women who reported having been (e.g. case and control groups may occupation on birth certificate) by exposed to DES in utero, four gave not be comparable); participant responses to interviews or self- birth to sons with hypospadias, recruitment (e.g. requiring administered questionnaires, compared with eight cases reported Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 461 among the 8729 sons born to adults), epigenetic mechanisms are highly uncertain at this time, non-exposed women (prevalence regulate gene expression through and gold standard methodologies ratio = 21.3; 95% CI = 6.5–70.1). DNA methylation, histone have yet to emerge (24). In humans, This finding generated several modification of chromatin structure, molecular epidemiology studies able studies in more representative and autoregulatory DNA binding to examine biomarkers of epigenetic populations, all indicating an . mechanisms and reveal the ways increase in hypospadias among Although epigenetic in which such mechanisms mediate sons of prenatally exposed DES mechanisms can cause phenotypic the relation between exposure mothers. However, the magnitude discordance between monozygotic to environmental chemicals and was much lower, with prevalence , epigenetic influences can also reproductive outcomes may lag ratios of 5.0 (95% CI = 1.2–16.8) be inherited (e.g. an imprinted gene only a few years behind the ground- among a French population and 1.7 in which the only allele expressed breaking studies in animal models. (95% CI = 0.4–6.8) among women in the offspring is the one inherited However, it may take many years in the USA, and a case–control from either the mother or the father, before molecular epidemiologists study of both maternal and paternal never both). Epigenetic alterations can measure epigenetic markers and in utero DES exposures yielded can occur at the level of transcription, their ultimate effects on reproductive an adjusted odds ratio (OR) of 4.9 translation or post-translation, and health across multiple generations. (95% CI = 1.1–22.3) for maternal appear to mediate the development There is hope that answers will come exposures, but no increase of adverse reproductive health from the National Children’s Study among males whose fathers were effects following experimental (http://www.nationalchildrensstudy. prenatally exposed to DES (OR = exposure to several hormonally gov), which plans to examine 0.9; 95% CI = 0.1–6.7) (158–160). In active environmental toxicants, at mothers and fathers before and addition, there were case reports of least in animal models (e.g. dioxins during pregnancy, and to follow their other congenital anomalies among and PBBs) (169). In rodents, for children for decades thereafter (172). second-generation offspring of example, in utero exposure to DES-exposed women, including the endocrine-active compounds Biomarkers of male limb reduction defects, deafness bisphenol A (170) and vinclozolin reproductive function and ovarian carcinoma (161,162). (171) produced epigenetically- There have been several recent mediated changes in coat colour Male libido reports on approaches to examine and in reproductive organs including the transgenerational adverse testicular defects and prostate Biomarkers of male reproductive reproductive effects of in utero tumours, respectively. In the latter function have been reviewed (173), DES exposure in animal models. study of rats, the epigenetic changes as well as a comprehensive and in- Experiments in mice exposed to and adverse reproductive health depth look at the current array of DES within 1–5 days after birth effects appeared to be transmitted diagnostic tests available for male have supported the hypothesis through a paternal allele in three sexual dysfunction (174). Libido that epigenetic dysregulation (e.g. consecutive generations (i.e. is the biological need for sexual hypomethylation of multiple CpG transgenerational), despite the lack activity (i.e. the sex drive), and sites of the proto-oncogene c-fos) of any vinclozolin exposure beyond male sexual desire is regulated by could be a causal mechanism the first generation of pups. past sexual activity, psychosocial underlying the adverse effects of As is so often the case for factors, activation of brain and DES on uterine tissue (163). As findings from novel and intriguing spinal cord dopamine receptors, described in greater detail in Chapter animal experiments, they await and gonadal hormones. Little is 26 and recent reviews (164,165) confirmation in other experiments, known of the physiologic basis of of the ‘developmental origins’ or since replication is the hallmark libido, and assessments of libido, Barker hypothesis (166–168) (i.e. of good science. The validity , , and that environmental exposures and reliability (e.g. coefficient of detumescence would be difficult to during the earliest and most plastic variation) of the intricate molecular make in large-scale epidemiologic stages of human development may techniques to measure epigenetic studies. There are electronic devices be among the most significant mechanisms in different human adapted for home use that monitor causal factors underlying many biological matrices with varying nocturnal penile tumescence chronic diseases in children and amounts of each individual sample (the penile that occur

462 spontaneously during rapid eye semen quality in population-based male hormones and semen quality, movement stages of sleep) (174,175). epidemiologic investigations, the allowing for a more thorough Though relatively little is known identification of alternative reliable assessment of the potentially about the effect of occupational or biomarkers is a pressing need (20). complex environmental effects on environmental exposures on sexual In a recent comparison with the gold male reproduction (180–182). desire in men, it has been suggested standard measurement of sperm that lead, carbon disulfide, stilbene concentration in semen, serum Semen characteristics or cadmium exposure may have levels of inhibin B, the peptide adverse effects (176). The few hormone produced in Sertoli cells, Despite conflicting reports and studies that have associated looked promising as a potential substantial geographic variation, erectile dysfunction with exposure surrogate biomarker for large- the question of whether declines to hazardous environmental and scale epidemiologic research (178). in semen quality and sperm counts occupational chemicals, and the Although serum FSH has also been over the past several decades fewer still that have incorporated used as a surrogate biomarker for have resulted from exposure to biomarkers of exposure, have been semen quality, FSH levels may be post-industrial age environmental carefully reviewed (175). Clearly, less desirable on the biological toxicants remains a major unresolved male libido and erectile dysfunction grounds that they are affected by public health concern. While the are important reproductive health gonadotropin releasing hormone, number of reports of declining sperm outcomes requiring further and testosterone, and counts continues to grow, there is epidemiologic research and unlike inhibin B, FSH is not as yet no compelling evidence of biomarker development. produced in the testes (178). In the decreased fertility in the human future, serum biomarkers for other populations studied (173,183). The Hormones male hormones, such as activin and assessment of reproductive function follistatin, may be explored for their in males usually begins with semen Chemical analyses for male utility in epidemiologic studies of analysis (177). In addition to the hormones are usually performed male reproductive function (173). challenges of recruiting participants on serum or urine samples, and willing to submit semen samples for the latter are readily available for Molecular epidemiology large-scale epidemiologic studies, use in large-scale epidemiologic studies associating the samples must be collected in studies. Abnormal levels of male environmental exposures appropriate containers, analysed hormones in serum or urine are with male hormone levels within one hour of collection, and kept indicators of problems in the warm during transportation (177). hypothalamic-pituitary-gonadal A recent, unique molecular The systematic analysis of axis that underlie abnormalities epidemiology study compared serum semen includes macroscopic observed in semen analysis (e.g. and inhibin B levels in male and microscopic evaluations and azoospermia and oligospermia) welders with corresponding levels chemical assays. Samples are (177). As a modestly invasive in an age-matched comparison examined for liquefaction, viscosity, procedure, requiring relatively little group. The investigators reported colour, pH (normal = 7.2–7.8), formal training, blood collection in significantly positive associations volume (normal = 2–5 millilitres), males is fairly well tolerated and after adjusting for smoking and sperm concentration (normal = 20– inexpensive. The National Institute alcohol consumption (179). Whole 50 million per millilitre), morphology for Occupational Safety and Health blood manganese concentration (e.g. size of the acrosomal cap and recommends a profile including was also positively associated with length of the tail, normal ≥ 50% of FSH, LH, testosterone and prolactin serum prolactin level. As the higher sperm have a typical acrosomal to evaluate endocrine dysfunction serum inhibin B concentrations in shape and size and a tail around 45 in the male. Although LH and FSH welders compared with the referent µm in length), motility and velocity/ can be measured in urine, prolactin group were contrary to expectation, progression (normal ≥ 50% of is currently measured only in serum the findings of this novel study observed sperm are motile and (173). await confirmation (179). Several move forward rapidly in a straight As high rates of refusal among recent epidemiologic studies have line with little lateral movement), study candidates has remained a examined the effects of exposure agglutination (clumping) and the major barrier to the assessment of to environmental agents on both presence of other cellular elements Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 463 (e.g. immature germ cells and chromatin from somatic cells (20), use light ; however, leukocytes) (20,177). and this property has led to the the Tunel technique can also be Although automated semen development of novel biomarkers performed with flow cytometry. analysis systems have been of sperm DNA integrity. Damage The SCSA method requires flow developed, visualization and to sperm from reactive oxygen cytometry (187). Challenges remain interpretation of important subtleties species (e.g. oxygen ions, free in standardization, establishing in human sperm are difficult. radicals and peroxides) has been thresholds and reference ranges, Evaluation by manual methods shown to contribute to reductions and achieving acceptable levels of remains the standard practice in male fertility (20,186). In fact, the interlaboratory reliability for these (177). Nevertheless, researchers production of free radicals due to assays (187). should be aware that evaluations oxidative stress was first reported of sperm concentration and motility in sperm cells (186). Oxidative DNA Molecular epidemiology have demonstrated acceptable damage refers to the functional or studies associating interlaboratory reliability and structural alteration of DNA that environmental exposures low coefficients of variation, but contributes to many degenerative with semen quality the assessment of other sperm diseases of aging including cancer characteristics (e.g. progression) (20). Oxidative stress to sperm DNA The literature on epidemiologic has been more difficult to integrity can arise endogenously studies relating semen quality to standardise (184). Semen contains or from exposure to environmental exposure to pesticides, and other a vast array of antigenically diverse toxicants including xenobiotics (19). endocrine disrupting chemicals proteins, including those carried on Oxidative stress leads to impaired has been thoroughly reviewed the surface of sperm, and the largely sperm motility, reduced fertilization, (188–190). The overwhelming unknown influence of specific and DNA damage. consensus is that the studies have male reproductive proteins on Although there are over 30 varied considerably in methods, human conception and pregnancy assays of oxidative stress available exposures and outcomes, and the outcomes will be an important focus for sperm assessment, the cost results have been equivocal. The for future molecular epidemiology and complexity, combined with need for further research in this area research (185). difficulties in standardization across is compelling. Several molecular Although semen has been laboratories, limit their use especially epidemiologic studies have begun used for biomonitoring of exposure for large-scale epidemiologic to incorporate biomarkers of DNA to metals and xenobiotics (20), research (186). The level of the damage to sperm (e.g. SCSA and concerns have been raised that oxidative DNA adduct 8-hydroxy- the DNA fragmentation index) as routine semen analysis may be 2'-deoxyguanosine (8-OHdG) in more sensitive measures examine an insensitive measure of many sperm is considered a sensitive the effects of exposure to potentially important reproductive health and precise biomarker of oxidative toxic environmental compounds effects resulting from environmental DNA damage (19,20). High levels (191–194). Molecular epidemiology exposures (19,20). A more of 8-OHdG are positively correlated studies have also begun to examine comprehensive approach includes with abnormal sperm morphology the influence of gene–environment assessments for cytogenetic sperm and negatively correlated with interactions on sperm DNA integrity abnormalities and DNA damage sperm concentration, number (195,196). (19,20). The fluorescent in situ and motility (19). In addition to the The evidence for male-mediated hybridization (FISH) technique 8-OHdG assay, a variety of methods reproductive and developmental has been widely used to detect to measure sperm DNA strand toxicity has been reviewed (197). aneuploidy, chromosomal breaks, breaks have developed over the past There is some evidence that and rearrangements in sperm 25 years, leading to the four major irradiation and exposure to certain cells. Although the FISH technique tests of sperm DNA fragmentation chemical compounds can be is efficient for large-scale use, an in current use today (mostly in genotoxic to sperm in experimental immense number of each subject’s ART laboratories): the Comet, animals, leading to the development sperm cells must be evaluated (up Tunel, sperm chromatin structure of malformations and tumours in to 10 000) (20). assay (SCSA) and the acridine their offspring. On the other hand, Sperm chromatin is extremely orange test (AOT) (186,187). The paternal exposure to low levels of compact and stable relative to Comet, Tunel, and AOT techniques non-mutagenic compounds, such

464 as lead, has altered learning and disorders, as well as to elucidate following our report of a significant mating behaviour in offspring, but underlying mechanisms. This is of association between infant birth has not led to obvious malformations great benefit to public health, as we weight and mother’s age at initial or tumours (197). Evidence of now are better positioned to identify exposure to PBBs, independent male-mediated reproductive and early sentinels of exposure to of the association with maternal developmental effects in humans toxicants and to intervene to reduce serum PBB levels (201), highlight has derived mostly from studies of human exposures. the need to consider the time- children born to men exposed to The capability of measuring the dependency of maternal exposures environmental toxicants (e.g. methyl dose of exposure to a toxic agent in and the potential for differential mercury, anaesthetic gases, lead, individuals greatly reduces exposure effects on pregnancy outcomes. solvents and pesticides) through their misclassification (e.g. recall bias), For example, the effects of in utero occupations. These studies have especially notable in scenarios exposure to a biological agent that been limited by the lack of objective where the individuals have little remains in maternal circulation and precise measures of exposure knowledge of their exposure. (due either to a single exposure and by the failure to adequately Biomarkers of exposure also enable event or long-standing cumulative account for exposures in the mother. more precise determinations of exposure) during critical periods of The mechanisms proposed for dose–response relationships, embryogenesis and organogenesis male-mediated reproductive toxicity which can vary across age and may be very different from in utero in humans involve direct effects gender. The recent focus on critical effects mediated by the mother’s from contaminated seminal fluid, as windows of susceptibility has initial exposure to the agent during well as both genetic and epigenetic also affected the risk assessment critical periods of her own childhood pathways. These pathways could process, as evidenced by the use and reproductive development (202). involve mutation, sperm of age-dependent adjustment Such complexities pose a significant DNA instability, suppression of factors (ADAFs) by the EPA in risk challenge to occupational and germ cell apoptosis, or interference assessment regarding early life environmental epidemiology, as well with genomic imprinting (197). As exposures to carcinogens (198). as to molecular epidemiology (203). available animal models may have For ages 0–2 years, the ADAF is As researchers further explore the reproductive systems and exposure 10, indicating a 10-fold increase underlying biological relationships regimens that poorly approximate in carcinogenic potency during among the growing number of conditions in humans, evidence this period. For ages 2–16 years, measurable biomarkers, they must for or against male-mediated the ADAF is 3, and for ages ≥ 16 take care to operationally define all reproductive toxicity and the years, the ADAF decreases to 1. At relevant variables, allow for time- hypothesized underlying molecular present, there is no ADAF for the dependencies, and be prepared mechanisms will largely depend on prenatal period, which is a limitation. to reach beyond conventional the validity and precision of future Studies in reproductive statistical modeling strategies that epidemiologic investigations. epidemiology that incorporate may be oversimplified or poorly biochemical markers of exposure specified (204,205). Strengths, limitations are only just beginning to address At the same time, epidemiologists and lessons learned the “windows of susceptibility” have become more alert to the issue. It is important to consider the periods of heightened susceptibility There have been tremendous independent and joint influences of during early human development; strides in the development and age at initial exposure and intensity there is growing awareness of the implementation of biomarkers in of exposure, as an environmental uncertainties and complexities reproductive health studies over agent may have irreversible, long- relating to the toxicokinetics of the past decade. These advances term reproductive health effects different environmental chemicals. have greatly enhanced our ability to in addition to acute or immediate There is greater appreciation that explore potential etiologies of and effects. Perhaps because the timing nonlinear dynamics may be involved increased susceptibility to adverse of exposure is an emergent area and that many interacting factors reproductive outcomes. In addition, of inquiry for epidemiologic studies can influence the rates of uptake, these new techniques have enabled of environmental chemicals, this biotransformation, metabolism us to identify precursor events and issue is not without controversy. and elimination. An especially more subtle manifestations of these Recent commentaries (199,200) important development is the Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 465 recognition among toxicologists populations of individuals that are comprehensive quality control that the methodologic challenges to expected to have a gradient of dioxin programme should be thoroughly valid exposure assessment are not exposure (due to the environmentally reviewed before selecting a unique to epidemiologic research. ubiquitous and persistent nature collaborating laboratory. If at all As underscored in a recent thought- of these compounds) are eagerly possible, studies applying novel provoking review (206), these awaited despite the methodologic biomarkers or high-throughput challenges extend to experimental and political challenges (210– technologies should incorporate a animal models as well. There is 212). One strategy that has been validation study comparing results room for substantial improvement proposed for biomarker studies of using the new test (for at least a in biomonitoring that incorporates TCDD and other environmental reasonably large random sample sensitive, repeated measures to chemicals that require either of participants) with results using a better reveal biological variability relatively large aliquots for each suitable gold standard methodology. both within and between subjects individual sample, expensive A validation study of a lower (whether human or animal). analytical methodologies, or both, is cost screening test for dioxin- Accurate exposure assessment to design statistically powerful and like compounds in serum (i.e. the for some of the most toxic cost-conserving protocols for the chemically activated luciferase environmental chemicals (e.g. pooling of individual blood or serum reporter gene expression (CALUX) dioxins and the most toxic congener, samples (213–218). assay) in a case–control study of 2,3,7,9 tetrachlorodibenzo-p- The ability to utilize easily neural tube defects in the children dioxin (TCDD)), requires potentially obtained biological specimens (e.g. of US veterans of the Viet Nam uncomfortable or invasive specimen urine samples and buccal swabs), War provides a striking example collection and highly expensive, rather than relying on collection of (219). Of interest was the potential technically complicated sample blood samples (often serially), will use of the CALUX assay to reduce preparation and analysis (207,208). greatly improve the acceptability costs in large-scale molecular This may partially explain why, of biomarker studies in large epidemiology studies that seek to for example, specific reproductive population-based studies. A major examine the association between health effects of dioxin congeners, limitation of biomarker studies is parental exposure to dioxin including TCDD, have been studied the reluctance of study participants congeners, especially TCDD, and extensively with biomarkers in to undergo serial blood draws, adverse pregnancy outcomes. To animal models, but infrequently in but as discussed above, there is assess the validity of the CALUX population-based epidemiologic considerable research focused assay, results were compared with studies (209). The rarity of many on the development of alternative results from the gold standard specific adverse reproductive biological media that would be of method of dioxin analysis—high health outcomes in the general huge benefit for researchers in this resolution gas chromatography/ population (e.g. narrowing the broad area. Use of home-based collection high resolution mass spectrometry class of reportable birth defects and storage protocols also improves (HRGC/HRMS). Figure 25.3 to diagnostic categories, such as compliance and reduces the costs shows a lack of correlation (and neural tube defects, or to single, associated with the transport and in the negative direction) between clinically-defined entities, such as processing of the samples. the CALUX results for dioxin-like spina bifida) has made conclusive Despite the allure of activity in serum (in TEQs) and the findings difficult to compile from inexpensive, high-throughput serum TCDD levels measured by the few cohorts of highly exposed technologies, the appropriate the gold standard, HRGC/HRMS, individuals that have been tested application of biomarker assays to on paired serum samples. A recent for serum TCDD levels and closely measure exposure, susceptibility, epidemiologic study of the Seveso, monitored for adverse reproductive and reproductive health effects in Italy cohort of residents exposed to outcomes (209). The expected large-scale epidemiologic research TCDD in 1976 (from an explosion number of cases of many of the will require painstaking validation, in a plant that manufactured single, well-defined reproductive comprehensive quality control 2,4,5-trichlorophenol) reported a health outcomes of interest in procedures, and active participation similar lack of correlation in results these exposed cohorts is relatively of collaborating laboratories in from serum analyses comparing small. Results from well-designed regular programmes of proficiency the CALUX assay with the gold epidemiologic studies in larger testing. Detailed results from a standard, HRGC/HRMS (220). Had

466 Figure 25.3. Correlation between log-transformed serum values of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), measured by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS), and serum values of dioxin-like activity measured by the chemically activated luciferase gene expression (CALUX) assay

Y axis = log TCDD values; X axis = log CALUX values; P=0.389. the validity of this CALUX assay acquisition and ethical utilization fertilization occurs would enhance been assumed (e.g. on the basis of biological samples, there are our understanding of fecundability of reports of its validity in matrices longstanding constraints posed by and fertility as related to other than human blood (221)), the the conventional approaches to environmental and lifestyle factors. significant three-fold association data analysis that measure gene– Identification of additional genetic between paternal serum TCDD level environment interactions on a susceptibility markers will open (measured by the gold standard multiplicative rather than additive multiple avenues of research in both methodology) and the occurrence scale (e.g. relative risk versus risk the genetic screening area, as well of neural tube defects in the children difference) and require extremely as research into gene–environment of US Viet Nam War veterans (219) large sample sizes (222–226). interactions in the etiology of would have been missed. adverse reproductive outcomes. The number of studies examining Future directions All of the advances will result in biomarkers of genetic susceptibility and challenges the increasing necessity to develop for adverse reproductive outcomes safeguards for the confidentiality is growing rapidly, with important In addition to the development of and ethical use of the data obtained. implications for prenatal screening, biomarkers using easily obtained It is also critical that effective means as well as for the detection of gene– biological samples, research into of communication of study results to environment interactions. In addition the identification of biomarkers participants be developed, so that to new challenges regarding the that would enable detection when important information regarding their Unit 5 Chapter 25 Chapter Unit 5 • Chapter 25. Disorders of reproduction 467 health status is provided to them methodologic rigor, replication socioeconomic exigency of modern, while also taking into consideration and sustained scientific vigilance. multidisciplinary, multicentre situations in which the interpretation Recent rulings by the US federal epidemiologic studies (e.g. genome- of the data and their relevance to Vaccine Injury Compensation wide association studies (26–33)) reproductive health may not yet be Program, highlighted by the Hannah data sharing has broad and complex understood. Poling case alleging vaccine- ethical implications for study To be sure, the vast and induced autism, raise concern for participants, study investigators and growing array of biomarkers the temptation to accept biologically other stakeholders ranging from and measurements that can be plausible molecular mechanisms corporate interests to the scientific made at the molecular level fuels on the perceived elegance of the community and the population at hope and raises expectations argument over the weight of the large. For future studies aiming to for breakthrough discoveries in empirical evidence (227). advance the fields of reproductive reproductive epidemiology, with the Future molecular epidemiology and molecular epidemiology, the potential for rapid translation and studies of reproductive and ethical challenges of data sharing significant health benefits. At the developmental health will be must be weighed against the growing same time, exciting developments shaped also by the increasing demand for scientific synergies and in molecular epidemiology must be pressures to register clinical public health benefit (229). balanced by advances in research research (228) and to share data design and data analysis that foster within a limited time frame (229). A

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