The present and future of whole-exome sequencing in studying and treating human reproductive disorders

The causes of recurrent spontaneous abortion (RSA) and fetal malformations are multifactorial and unclear in most cases. Environmental, maternal, and genetic factors have been shown to contribute to these defects. Whole-exome sequencing (WES) is widely used to detect genetic variations associated with human diseases and has recently been successfully applied to unveil genetic causes of unexplained recurrent spontaneous abortion (URSA) and fetal malformations. Here, we review the current discovery and diagnosis strategies to identify the underlying pathogenic mutations of URSA and fetal malformations using WES technology and propose to further develop WES, both to advance our understanding of these diseases and to eventually lead to targeted therapies for reproductive disorders.     

Risks of hyperthermia associated with hot tub or spa use by pregnant women

There are a limited number of human studies linking hot tub or spa use during early pregnancy to increased risks for neural tube defects (NTDs) or spontaneous abortion. However, these data can be considered in the context of human studies that have demonstrated an association between high maternal fever in early pregnancy and NTDs. In addition, there is a large volume of animal literature suggesting that, regardless of the heat source, an elevated core maternal temperature at or above the threshold of 2 degrees C over baseline, as well as timing and duration of exposure, are the critical factors in conferring risk. Therefore, the potential for hot tub or spa use to increase core maternal body temperature to risky levels and thus increase the risk for NTDs is likely. A woman who knows or who may not yet be aware that she is pregnant should be advised of the recommended limits of exposure. She should also be aware of the possible variability in hot tub or spa temperature readings and be able to accurately monitor maximum water temperature in the hot tub or spa so that her body temperature can be maintained below 38.9 degrees C.     

Prenatal diagnosis--principles of diagnostic procedures and genetic counseling

The frequency of inherited malformations as well as genetic disorders in newborns account for around 3-5%. These frequency is much higher in early stages of pregnancy, because serious malformations and genetic disorders usually lead to spontaneous abortion. Prenatal diagnosis allowed identification of malformations and/or some genetic syndromes in fetuses during the first trimester of pregnancy. Thereafter, taking into account the severity of the disorders the decision should be taken in regard of subsequent course of the pregnancy taking into account a possibilities of treatment, parent's acceptation of a handicapped child but also, in some cases the possibility of termination of the pregnancy. In prenatal testing, both screening and diagnostic procedures are included. Screening procedures such as first and second trimester biochemical and/or ultrasound screening, first trimester combined ultrasound/biochemical screening and integrated screening should be widely offered to pregnant women. However, interpretation of screening results requires awareness of both sensitivity and predictive value of these procedures. In prenatal diagnosis ultrasound/MRI searching as well as genetic procedures are offered to pregnant women. A variety of approaches for genetic prenatal analyses are now available, including preimplantation diagnosis, chorion villi sampling, amniocentesis, fetal blood sampling as well as promising experimental procedures (e.g. fetal cell and DNA isolation from maternal blood). An incredible progress in genetic methods opened new possibilities for valuable genetic diagnosis. Although karyotyping is widely accepted as golden standard, the discussion is ongoing throughout Europe concerning shifting to new genetic techniques which allow obtaining rapid results in prenatal diagnosis of aneuploidy (e.g. RAPID-FISH, MLPA, quantitative PCR).     

Pregnancy outcomes after weekly oral administration of ethanol during gestation in the pig-tailed macaque (Macaca nemestrina)

Ethanol was orally administered once per week to gravid pig-tailed macaques (Macaca nemestrina) in doses of 0.3, 0.6, 1.2, 1.8, 2.5, 3.3, or 4.1 g/kg. A control group received a sucrose solution, isocaloric and isovolemic to the highest ethanol dose. Pregnancy was followed after 116 possible conceptions in 54 females. Peak plasma ethanol concentrations (PPECs) ranged from 24 +/- 6 mg/dl at the 0.3 g/kg dose to 549 +/- 71 mg/dl at the 4.1 g/kg dose. An increased rate of spontaneous abortion was related to ethanol exposure at and above 1.8 g/kg (mean PPEC = 205 mg/dl). Pregnancy failure in the first 30 days of gestation increased at doses above 2.5 g/kg. The effect on pregnancy outcome of weekly exposure to ethanol in this nonhuman primate is comparable to available data on humans. The methodology of this study represents an effective model for studying ethanol teratogenesis in a nonhuman primate.     

Postmarketing surveillance for human teratogenicity: a model approach.

BACKGROUND: Most congenital defects associated with prenatal exposures are notable for a pattern of major and minor malformations, rather than for a single major malformation. Thus, traditional epidemiological methods are not universally effective in identifying new teratogens. The purpose of this report is to outline a complementary approach that can be used in addition to other more established methods to provide the most comprehensive evaluation of prenatal exposures with respect to teratogenicity. METHODS: We describe a multicenter prospective cohort study design involving dysmorphological assessment of liveborn infants. This design uses the Organization of Teratology Information Services, a North American network of information providers who also collaborate for research purposes. Procedures for subject selection, methods for data collection, standard criteria for outcome classification, and the approach to analysis are detailed. RESULTS: The focused cohort study design allows for evaluation of a spectrum of adverse pregnancy outcomes ranging from spontaneous abortion to functional deficit. While sample sizes are typically inadequate to identify increased risks for single major malformations, the use of dysmorphological examinations to classify structural anomalies provides the unique advantage of screening for a pattern of malformation among exposed infants. CONCLUSIONS: As the known human teratogens are generally associated with patterns of structural defects, it is only when studies of this type are used in combination with more traditional methods that we can achieve an acceptable level of confidence regarding the risk or safety of specific exposures during pregnancy.     

Emerging data on the use of anti-tumor necrosis factor-alpha medications in pregnancy

Anti-tumor necrosis factor (TNF) α medications are used for the treatment of a number of autoimmune diseases. Evaluation of pregnancy safety for these medications is complicated by the contribution of the underlying maternal disease to adverse pregnancy outcomes, such as preterm delivery and reduced birth weight. Placental transport of these medications is thought to be minimal in the first trimester, thereby providing some reassurance regarding theoretical risks for congenital malformations. Available human exposure data are sparse; however, to date there has been no convincing evidence to support an increased risk for a specific pattern of major congenital malformations with any of the drugs in this group for which some data is currently available. As a result of the improvement of symptoms during pregnancy in some women with autoimmune diseases, it may be possible to discontinue treatment before or shortly after conception. However, in some cases the benefits of treatment and concerns for disease flares in pregnancy have warranted continued treatment during pregnancy. Because of the relatively long half-life of these medications, and theoretical concerns for immune compromise of the infant following exposure in the latter two trimesters, some clinicians recommend discontinuation of treatment in the third trimester to avoid potentially prolonged infant exposure in the postpartum period. Currently ongoing controlled cohort studies for some of the TNF blocker medications will help to provide more definitive answers for clinicians and patients. Birth Defects Research (Part A) 94:607-611, 2012. ? 2012 Wiley Periodicals, Inc.     

Dietary calcium supplementation to lower blood lead levels in pregnancy and lactation

Pregnancy and lactation are states known to be accompanied by physiologically up regulated bone resorption in response to the calcium demands of the developing fetus and nursing infant. The role of calcium supplements in altering maternal responses to fetal demand for calcium is not fully understood. Exposure to the toxicant lead is known to pose a major hazard to fetal neurodevelopment and growth. Since >95% of maternal lead is stored in the bone, mobilization of cumulative maternal lead stores into the circulation represents an endogenous source of exposure, which may pose a significant hazard for the fetus and infant. Maternal dietary calcium supplementation has been associated with reductions in lead levels in both animal and human studies when administered during pregnancy and lactation. Therefore, supplementation of the maternal diet with calcium may represent an important secondary prevention strategy aimed not only at reducing circulating levels of lead in the mother but also at reducing lead exposure to the developing fetus and nursing infant.     

Minor chromosomal variants and major chromosomal anomalies in couples with recurrent abortion.

One hundred three women with prior histories of recurrent spontaneous abortion and 81 of their mates were karyotyped with Q-banding during 1976-1980. Recurrent abortion was defined as two or more spontaneous pregnancy losses; no couple with a previous malformed fetus or child was included. These cases were reviewed in order to examine the possible contributions of minor polymorphic chromosomal variants and major chromosomal abnormalities to recurrent spontaneous pregnancy loss. Balanced translocations were detected in four women and two men in the study; mosaic X aneuploidy was noted in one woman. Quantitative (1 qh, 9qh, 16qh, Yqh) and qualitative (3c, 4c, 13p, 13s, 14p, 14s, 15p, 15s, 21p, 21s, 22p, 22s) heterochromatic polymorphisms were blindly assessed and compared with a control group. Cases and controls did not differ in the frequency of any qualitative polymorphisms or in the length of any quantitative polymorphism. Thus, while major parental cytogenetic aberrations are significantly associated with fetal wastage, these data suggest that minor polymorphic chromosomal variants do not play an important role in the etiology of recurrent spontaneous abortion.     

Spontaneous pregnancy loss mediated by abnormal maternal inflammation in rats is linked to deficient uteroplacental perfusion

Abnormal maternal inflammation during pregnancy is associated with spontaneous pregnancy loss and intrauterine fetal growth restriction. However, the mechanisms responsible for these pregnancy outcomes are not well understood. In this study, we used a rat model to demonstrate that pregnancy loss resulting from aberrant maternal inflammation is closely linked to deficient placental perfusion. Administration of LPS to pregnant Wistar rats on gestational day 14.5, to induce maternal inflammation, caused fetal loss in a dose-dependent manner 3-4 h later, and surviving fetuses were significantly growth restricted. Pregnancy loss was associated with coagulopathy, structural abnormalities in the uteroplacental vasculature, decreased placental blood flow, and placental and fetal hypoxia within 3 h of LPS administration. This impairment in uteroplacental hemodynamics in LPS-treated rats was linked to increased uterine artery resistance and reduced spiral arteriole flow velocity. Pregnancy loss induced by LPS was prevented by maternal administration of the immunoregulatory cytokine IL-10 or by blocking TNF-α activity after treatment with etanercept (Enbrel). These results indicate that alterations in placental perfusion are responsible for fetal morbidities associated with aberrant maternal inflammation and support a rationale for investigating a potential use of immunomodulatory agents in the prevention of spontaneous pregnancy loss.     

The Need to Update Reference Values for Lead in Zaragoza, Spain

Stricter regulations and controls for environmental lead have resulted in significantly lower frequency and reduced severity of chronic and acute lead intoxication. The aim of the present study was to update established reference ranges for lead in whole blood of healthy adults residing in Zaragoza and its region in northeastern Spain. The mean blood level was 2.94 +/- 2.02 microg/dl, with a median of 2.43 microg/dl (n = 156). For women, the mean was 2.29 +/- 1.64 microg/dl (n = 73) and for men 3.51 +/- 2.16 microg/dl (n = 83). The difference between genders is statistically significant (p < 0.005). Our results confirm an ongoing decline in blood lead levels in the studied region, which in 1989 were found to average 13.17 +/- 3.47 microg/dl. In addition, there is a clear need to take into account gender differences when defining normal ranges for lead.     

Antibodies to rubellavirus, herpes simplex virus and Toxoplasma gondii in serial serum samples of pregnant and non-pregnant women.

By testing serial serum samples of 213 pregnant women for rubellavirus, of 196 for herpes simplex virus and of 134 for Toxoplasma gondii, it was found that during pregnancy there was a fall in the humoral antibody level. Presence and titre of antibodies were lower in sera of pregnant than of non-pregnant women. Alteration of the humoral antibody level during pregnancy may influence serological studies aimed at clarifying the role of infections in fetal malformations. Serial serum samples (4 samples from each pregnant woman involved) should be tested for obtaining reliable data regarding the frequency of infections during pregnancy.     

Septo-optic dysplasia as a manifestation of valproic acid embryopathy.

BACKGROUND: The use of valproic acid during pregnancy has been associated with adverse fetal outcomes, including major and minor congenital malformations, intrauterine growth retardation (IUGR), hyperbilirubinemia, hepatotoxicity, transient hyperglycemia, and fetal and neonatal distress. In addition, intrauterine exposure to valproic acid has been associated with an increased risk of central nervous system abnormalities, primarily neural tube defects. Optic nerve hypoplasia has been reported in association with other prenatal anticonvulsant exposures, but the occurrence of septo-optic dysplasia as a manifestation of valproic acid embryopathy has not been reported previously. RESULTS: We report on a woman who received Depakote (valproic acid) throughout her pregnancy for the treatment of a seizure disorder. The patient presented with features typical of valproic acid embryopathy, including bitemporal narrowing, hypertelorism, short palpebral fissures, epicanthal folds, microphthalmia, a flat broad nasal bridge, small mouth, hypoplastic nails, mild clinodactyly, and camptodactyly. MRI showed hypoplasia of the optic chiasm and absence of the septum pellucidum. CONCLUSIONS: We report the first case of septo-optic dysplasia associated with maternal exposure to valproic acid throughout pregnancy. This case expands the clinical phenotype of valproate embryopathy.     

Effect of vitamin A and beta carotene supplementation on women's health

A large field trial conducted in Nepal confirmed a beneficial effect of vitamin A and beta carotene supplementation on maternal mortality. Maternal deaths during pregnancy or within 12 weeks of delivery occurred at rates of 704/100,000 pregnancies (51/7241) in women who received a placebo, 426/100,000 pregnancies (33/7747) in women who received a single oral vitamin A supplement per week, and 361/100,000 pregnancies (26/7201) among those who received weekly oral beta carotene. The relative risks of pregnancy-related mortality were 0.60 (95% confidence interval (CI), 0.37-0.97) for vitamin A and 0.51 (95% CI, 0.30-0.86) for beta carotene. Before widespread vitamin A and beta carotene supplementation programs are implemented, further evaluation is required of the possible hazards to women of childbearing age and their offspring. For example, exposure to vitamin A during pregnancy has been linked to fetal malformations and schizophrenia.     

Effects of feed restriction during organogenesis on embryo-fetal development in rabbit

BACKGROUND: Appropriate maternal nutrition and body weight gain during pregnancy is well established as a major factor in healthy prenatal development in humans. Given the role of nutrition and body weight gain in normal development, pharmaceuticals intended to reduce appetite and promote weight loss will generate developmental toxicity data that may be challenging to interpret. To aid with this, the effects of feed restriction, and subsequent reduction in maternal body weight gain, on embryo-fetal development was investigated in the rabbit. METHODS: Groups of 15 pregnant New Zealand White rabbits were offered 150 (control), 110, 75, 55, 35, and 15 g feed/day from gestation day (GD) 7-19. Cesarean sections were carried out on GD 29 and fetuses were examined for external, visceral, and skeletal development. RESULTS: Maternal body weights at the end of the feed restriction period (GD 20) were 0.97, 0.98, 0.93, 0.94, and 0.86 x control for the 110, 75, 55, 35, and 15 g feed/day groups, respectively. Only at 15 g feed/day was there a net maternal body weight loss (the GD 20 body weight was 0.93 x the GD 6 body weight) at the end of the feed restriction period. Six does aborted in the 15 g feed/day group; there were no other abortions associated with feed restriction. Fetal body weight was significantly reduced at 75, 55, 35, and 15 g feed/day (0.95, 0.90, 0.86, and 0.84 x control, respectively). There were no external or visceral malformations or variations, and no skeletal malformations associated with feed restriction. The incidence of fetuses with sternebrae 5 or 6 unossified was increased at feed levels < or = 75 g/day. At a feed level of 35 g/day there was an increase in unossified metatarsals and metacarpals, and an increase in the number of fetuses with a reduced number of caudal vertebrae ossified. Although these findings were not increased at a feed level of 15 g/day, the lack of dose response was likely due to increased abortion and subsequent decrease in fetuses available for evaluation at 15 g feed/day. CONCLUSION: These data demonstrate that feed restriction to feed levels that produce substantial reductions in maternal body weight gain can result in developmental toxicity expressed by abortion, reduced fetal weight, and alterations in ossification. Abortion only occurred when feed was restricted to an amount that produced maternal body weight loss (15 g feed/day) whereas reduced fetal weight and increased incidence of fetuses with unossified sternebrae, metatarsals, metacarpals, or caudal vertebrae were noted at feed levels of < or = 75 g/day. There were no fetal malformations associated with feed restriction.     

Cystic fibrosis Δf508 mutation screening in Brazilian women with altered fertility

Cystic Fibrosis (CF) is an autosomal recessive disease, caused by mutations in the Cystic Fibrosis Transmembrane Regulator gene (CFTR). The most frequent mutation in CF is ΔF508. The disease is clinically characterized by elevated concentrations of sweat chlorides and abnormally thick mucus. It affects organs such as lung, pancreas, gastrointestinal and reproductive tract. Women with CF commonly present delayed puberty and amenorrhea due to malnutrition. Our objective was to screen the presence of ΔF508 mutation in 24 women with altered fertility. Nine of these women presented reduced fertility without a known cause, four showed polycystic ovaries and two had early menopause. One woman with early menopause was a carrier of the ΔF508 mutation. Our study demonstrates that it is possible that the frequency of CF mutations among patients with altered fertility may be higher than expected. Previous data showed that fibrocystic women can show reduced fertility, maternal mortality associated with pregnancy and increased incidence of spontaneous abortion. We therefore recommend that women with reduced fertility undertake genetic tests for a better evaluation of pregnancy risks and clinical monitoring.     

Treatment of idiopathic thrombocytic purpura in pregnancy by high-dose intravenous immunoglobulin

ITP in pregnancy may lead to fetal thrombocytopenia caused by the transplacental passage of maternal antiplatelet antibody. The most hazardous complication in the infant is intracranial hemorrhage. In addition ITP in pregnancy is reported to be associated with an increased abortion rate and an elevated fetal morbidity and mortality. Therefore obstetric management must aim at increasing maternal and fetal platelets. Several therapeutic approaches to the treatment of ITP in pregnancy are evaluated. Two cases of ITP in pregnancy are reported. Administration of high-dose intravenous immunoglobulin is introduced as a new therapy for ITP in pregnancy. The rapid reversal of thrombocytopenia following immunoglobulin G administration suggests that it is useful especially as emergency treatment for ITP in pregnancy.     

Spontaneous abortions in hospital staff engaged in sterilising instruments with chemical agents.

Spontaneous abortions in hospital sterilising staff were analysed using data from a postal questionnaire and a hospital discharge register. The study included all the sterilising staff employed in Finnish hospitals in 1980; the controls were nursing auxiliaries. Data from the questionnaire showed that the frequency of spontaneous abortions was 11.3% for the sterilising staff and 10.6% for the nursing auxiliaries. When the staff were concerned in sterilising procedures during their pregnancy the frequency was 16.7% compared with 5.6% for the non-exposed pregnancies. Adjustment for age, parity, decade of pregnancy, smoking habits, and intake of coffee and alcohol did not affect the differences. The increased frequency of spontaneous abortion correlated with exposure to ethylene oxide but not with exposure to glutaraldehyde or to formaldehyde. Analysis of spontaneous abortions from the hospital discharge register confirmed the findings. Thus the results from the two independent analyses suggest that exposure to ethylene oxide in hospitals may carry a risk of spontaneous abortion among sterilising staff.