Fetoplacental vascular effects of maternal adrenergic antihypertensive and cardioprotective medications in pregnancy.

Maternal cardiovascular diseases, including hypertension and cardiac conditions, are associated with poor fetal outcomes. A range of adrenergic antihypertensive and cardioprotective medications are often prescribed to pregnant women to reduce major maternal complications during pregnancy. Although these treatments are not considered teratogenic, they may have detrimental effects on fetal growth and development, as they cross the fetoplacental barrier, and may contribute to placental vascular dysregulation. Medication risk assessment sheets do not include specific advice to clinicians and women regarding the safety of these therapies for use in pregnancy and the potential off-target effects of adrenergic medications on fetal growth have not been rigorously conducted. Little is known of their effects on the fetoplacental vasculature. There is also a dearth of knowledge on adrenergic receptor activation and signalling within the endothelium and vascular smooth muscle cells of the human placenta, a vital organ in the maintenance of adequate blood flow to satisfy fetal growth and development. The fetoplacental circulation, absent of sympathetic innervation, and unique in its reliance on endocrine, paracrine and autocrine influence in the regulation of vascular tone, appears vulnerable to dysregulation by adrenergic antihypertensive and cardioprotective medications compared with the adult peripheral circulation. This semi-systematic review focuses on fetoplacental vascular expression of adrenergic receptors, associated cell signalling mechanisms and predictive consequences of receptor activation/deactivation by antihypertensive and cardioprotective medications.

Ex vivo perfusion of the human placenta to investigate pregnancy pathologies.

Pregnancy pathologies including gestational diabetes, intrauterine fetal growth restriction, and pre-eclampsia are common and significantly increase the risk of poor pregnancy outcomes. Research to better understand the pathophysiology and improve diagnosis and treatment is therefore crucial. The ex vivo placenta perfusion model offers a unique system to study pregnancy pathology without the risk of harm to mother or fetus. The presence of a maternal and fetal circulation and intact villus tree, facilitates investigations into maternal-fetal transfer, altered hemodynamics and vascular reactivity in the human placenta. It also provides a platform to test novel therapeutic agents. Here we review the key studies which have utilized the ex vivo placenta perfusion model to study different aspects of such pregnancy pathologies.

Ex vivo dual perfusion of an isolated human placenta cotyledon: Towards protocol standardization and improved inter-centre comparability.

Since the full development of the ex vivo dual perfusion model of the human placenta cotyledon, the technique has provided essential insight into how nutrients, lipids, gases, immunoglobulins, endocrine agents, pharmaceuticals, chemicals, nanoparticles, micro-organisms and parasites might traverse the maternofetal barrier. Additionally, the model has been instrumental in gaining a better understanding of the regulation of vascular tone, endocrinology and metabolism within this organ. The human placenta is unique amongst species in its anatomy and transfer modalities. This orthologous diversity therefore requires an appropriate consideration of placental transfer rates of compounds, particles and micro-organisms specific to humans. Different research centres have adapted this model with a wide variation in perfusion parameters, including in the establishment of perfusion, perfusate composition, gassing regime, cannulation method, flow rates, perfused tissue mass, and also in the application of quality control measures. The requirement to harmonise and standardise perfusion practice between centres is largely driven by the need to obtain consistency in our understanding of placental function, but also in the qualification of the model for acceptance by regulatory agencies in drug and toxicology testing. A pilot study is proposed, aiming to describe how existing inter-centre variation in perfusion methodology affects placental metabolism, protein synthesis, oxygen consumption, the materno-fetal transfer of key molecular markers, and placental structure.

Targeted Delivery of Epidermal Growth Factor to the Human Placenta to Treat Fetal Growth Restriction.

Placental dysfunction is the underlying cause of pregnancy complications such as fetal growth restriction (FGR) and pre-eclampsia. No therapies are available to treat a poorly functioning placenta, primarily due to the risks of adverse side effects in both the mother and the fetus resulting from systemic drug delivery. The use of targeted liposomes to selectively deliver payloads to the placenta has the potential to overcome these issues. In this study, we assessed the safety and efficacy of epidermal growth factor (EGF)-loaded, peptide-decorated liposomes to improve different aspects of placental function, using tissue from healthy control pregnancies at term, and pregnancies complicated by FGR. Phage screening identified a peptide sequence, CGPSARAPC (GPS), which selectively homed to mouse placentas in vivo, and bound to the outer syncytiotrophoblast layer of human placental explants ex vivo. GPS-decorated liposomes were prepared containing PBS or EGF (50-100 ng/mL), and placental explants were cultured with liposomes for up to 48 h. Undecorated and GPS-decorated liposomes containing PBS did not affect the basal rate of amino acid transport, human chorionic gonadotropin (hCG) release or cell turnover in placental explants from healthy controls. GPS-decorated liposomes containing EGF significantly increased amino acid transporter activity in healthy control explants, but not in placental explants from women with FGR. hCG secretion and cell turnover were unaffected by EGF delivery; however, differential activation of downstream protein kinases was observed when EGF was delivered via GPS-decorated vs. undecorated liposomes. These data indicate that targeted liposomes represent a safe and useful tool for the development of new therapies for placental dysfunction, recapitulating the effects of free EGF.

Ex vivo dual perfusion of an isolated cotyledon of human placenta: History and future challenges.

This is an introductory chapter for the Special Issue: Ex vivo dual perfusion of an isolated cotyledon of the human placenta. It covers the early stages of development of this technique in the laboratory of Professor Maurice Panigel. It was successfully further developed by Henning Schneider during his stay in Paris as a research assistant. Together with Professor Joseph Dancis in New York they managed to overcome initial problems of acceptance. Its value was finally confirmed by studies performed by several groups in different parts of the world and it became a valuable additional tool for research on the human placenta.

Pulsatility effects of flow on vascular tone in the fetoplacental circulation.

INTRODUCTION: The regulation of vascular tone in the fetoplacental circulation is governed by endocrine and mechanical forces yielding a relaxed basal state in normal pregnancy. Flow mediated vasodilation, induced by shear stress and endothelial nitric oxide signalling, is key to driving vasorelaxation in this circulation. The pulsatile property of blood flow, as opposed to the flow rate, could provide an additional factor in this regulation, but its effects and signalling have never been explored in the fetoplacental microvasculature. METHODS: Here, we studied the effects of non-pulsatile and pulsatile flow modalities on vascular resistance in the fetoplacental microcirculation of the human placenta using an ex vivo perfusion model; and examined a potential role for nitric oxide. We also explored whether the placental Doppler velocimetry waveform is sustained within subchorial arteries in vivo. RESULTS: Pulsatile flow reduced basal impedance to flow during steady state perfusion compared to non-pulsatile flow, signalled through enhanced nitric oxide production. Doppler velocimetry waveforms were visible within the subchorial arteries in vivo. CONCLUSION: This work suggests that the pulsatile property of flow through the fetoplacental circulation is sensed by the fetoplacental vasculature to mediate a signalling response and provide additional vasodilation of this microcirculation. We speculate that in pregnancy disease, altered amplitude and frequency of the subchorial pulse might impact on vascular function in a compromised high-resistance placental microcirculation.

Quantifying the impact of tissue metabolism on solute transport in feto-placental microvascular networks.

The primary exchange units in the human placenta are terminal villi, in which fetal capillary networks are surrounded by a thin layer of villous tissue, separating fetal from maternal blood. To understand how the complex spatial structure of villi influences their function, we use an image-based theoretical model to study the effect of tissue metabolism on the transport of solutes from maternal blood into the fetal circulation. For solute that is taken up under first-order kinetics, we show that the transition between flow-limited and diffusion-limited transport depends on two new dimensionless parameters defined in terms of key geometric quantities, with strong solute uptake promoting flow-limited transport conditions. We present a simple algebraic approximation for solute uptake rate as a function of flow conditions, metabolic rate and villous geometry. For oxygen, accounting for nonlinear kinetics using physiological parameter values, our model predicts that villous metabolism does not significantly impact oxygen transfer to fetal blood, although the partitioning of fluxes between the villous tissue and the capillary network depends strongly on the flow regime.

Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction?

Cell free hemoglobin impairs vascular function and blood flow in adult cardiovascular disease. In this study, we investigated the hypothesis that free fetal hemoglobin (fHbF) compromises vascular integrity and function in the fetoplacental circulation, contributing to the increased vascular resistance associated with fetal growth restriction (FGR). Women with normal and FGR pregnancies were recruited and their placentas collected freshly postpartum. FGR fetal capillaries showed evidence of erythrocyte vascular packing and extravasation. Fetal cord blood fHbF levels were higher in FGR than in normal pregnancies ( P < 0.05) and the elevation of fHbF in relation to heme oxygenase-1 suggests a failure of expected catabolic compensation, which occurs in adults. During ex vivo placental perfusion, pathophysiological fHbF concentrations significantly increased fetal-side microcirculatory resistance ( P < 0.05). fHbF sequestered NO in acute and chronic exposure models ( P < 0.001), and fHbF-primed placental endothelial cells developed a proinflammatory phenotype, demonstrated by activation of NF-κB pathway, generation of IL-1α and TNF-α (both P < 0.05), uncontrolled angiogenesis, and disruption of endothelial cell flow alignment. Elevated fHbF contributes to increased fetoplacental vascular resistance and impaired endothelial protection. This unrecognized mechanism for fetal compromise offers a novel insight into FGR as well as a potential explanation for associated poor fetal outcomes such as fetal demise and stillbirth.-Brook, A., Hoaksey, A., Gurung, R., Yoong, E. E. C., Sneyd, R., Baynes, G. C., Bischof, H., Jones, S., Higgins, L. E., Jones, C., Greenwood, S. L., Jones, R. L., Gram, M., Lang, I., Desoye, G., Myers, J., Schneider, H., Hansson, S. R., Crocker, I. P., Brownbill, P. Cell free hemoglobin in the fetoplacental circulation: a novel cause of fetal growth restriction?

Human placental oxygenation in late gestation: experimental and theoretical approaches.

The placenta is crucial for life. It is an ephemeral but complex organ acting as the barrier interface between maternal and fetal circulations, providing exchange of gases, nutrients, hormones, waste products and immunoglobulins. Many gaps exist in our understanding of the detailed placental structure and function, particularly in relation to oxygen handling and transfer in healthy and pathological states in utero. Measurements to understand oxygen transfer in vivo in the human are limited, with no general agreement on the most appropriate methods. An invasive method for measuring partial pressure of oxygen in the intervillous space through needle electrode insertion at the time of Caesarean sections has been reported. This allows for direct measurements in vivo whilst maintaining near normal placental conditions; however, there are practical and ethical implications in using this method for determination of placental oxygenation. Furthermore, oxygen levels are likely to be highly heterogeneous within the placenta. Emerging non-invasive techniques, such as MRI, and ex vivo research are capable of enhancing and improving current imaging methodology for placental villous structure and increase the precision of oxygen measurement within placental compartments. These techniques, in combination with mathematical modelling, have stimulated novel cross-disciplinary approaches that could advance our understanding of placental oxygenation and its metabolism in normal and pathological pregnancies, improving clinical treatment options and ultimately outcomes for the patient.

Knowledge needed about the exchange physiology of the placenta.

There is now a basic understanding of the driving forces and mechanisms underlying rates of solute exchange across the placenta but there are still major gaps in knowledge. Here we summarise this basic understanding, whilst highlighting gaps in knowledge. We then focus on two particular areas where more knowledge is needed: (1) the electrical potential difference (PD) across the placenta and (2) the paracellular permeability of the placenta to hydrophilic solutes. In many species a PD has been recorded between a catheter in a maternal blood vessel and one in a fetal vessel. However, the key question is whether this PD is the same as that across the placental exchange barrier. We addressed this in the human placenta using microelectrodes to measure the PD in isolated villi in vitro; the transtrophoblast PD so measured had a median value of -3 mV (range 0-15 mV). There have been no subsequent studies to validate this measurement. The syncytiotrophoblast of haemochorial placentas lacks any obvious extracellular water filled paracellular space between the syncytial nuclei. However, in mouse, rat, guinea pig and human there is an inverse relationship between the rate of diffusion of inert hydrophilic solutes across the placenta and their molecular size. The simplest explanation is that a paracellular route exists but its morphological identity is still uncertain. Areas of syncytial denudation could provide a paracellular route but this has not been proven. Answers to these and similar questions are required to fully understand the exchange physiology of the normal placenta and how this is affected in pathology.

IFPA meeting 2017 workshop report: Clinical placentology, 3D structure-based modeling of placental function, placental bed, and treating placental dysfunction.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2017 there were four themed workshops, all of which are summarized in this report. These workshops discussed new knowledge and technological innovations in the following areas of research: 1) placental bed; 2) 3D structural modeling; 3) clinical placentology; 4) treatment of placental dysfunction.

Ex Vivo Dual Perfusion of the Human Placenta: Disease Simulation, Therapeutic Pharmacokinetics and Analysis of Off-Target Effects.

In recent years ex vivo dual perfusion of the human placental lobule is seeing an international renaissance in its application to understanding fetal health and development. Here, we discuss the methods and uses of this technique in the evaluation of (1) vascular function, (2) transplacental clearance, (3) hemodynamic and oxygenation changes associated with pregnancy complications on placental structure and function, and (4) placental toxicology and post-perfusion evaluation of tissue architecture.

In Vitro Human Placental Studies to Support Adenovirus-Mediated VEGF-DΔNΔC Maternal Gene Therapy for the Treatment of Severe Early-Onset Fetal Growth Restriction.

Severe fetal growth restriction (FGR) affects 1 in 500 pregnancies, is untreatable, and causes serious neonatal morbidity and death. Reduced uterine blood flow (UBF) is one cause. Transduction of uterine arteries in normal and FGR animal models using an adenovirus (Ad) encoding VEGF isoforms increases UBF and improves fetal growth in utero. Understanding potential adverse consequences of this therapy before first-in-woman clinical application is essential. The aims of this study were to determine whether Ad.VEGF-DΔNΔC (1) transfers across the human placental barrier and (2) affects human placental morphology, permeability and primary indicators of placental function, and trophoblast integrity. Villous explants from normal term human placentas were treated with Ad.VEGF-DΔNΔC (5 × 107-10 virus particles [vp]/mL), or virus formulation buffer (FB). Villous structural integrity (hematoxylin and eosin staining) and tissue accessibility (LacZ immunostaining) were determined. Markers of endocrine function (human chorionic gonadotropin [hCG] secretion) and cell death (lactate dehydrogenase [LDH] release) were assayed. Lobules from normal and FGR pregnancies underwent ex vivo dual perfusion with exposure to 5 × 1010 vp/mL Ad.VEGF-DΔNΔC or FB. Perfusion resistance, para-cellular permeability, hCG, alkaline phosphatase, and LDH release were measured. Ad.VEGF-DΔNΔC transfer across the placental barrier was assessed by quantitative polymerase chain reaction in DNA extracted from fetal-side venous perfusate, and by immunohistochemistry in fixed tissue. Villous explant structural integrity and hCG secretion was maintained at all Ad.VEGF-DΔNΔC doses. Ad.VEGF-DΔNΔC perfusion revealed no effect on placental permeability, fetoplacental vascular resistance, hCG secretion, or alkaline phosphatase release, but there was a minor elevation in maternal-side LDH release. Viral vector tissue access in both explant and perfused models was minimal, and the vector was rarely detected in the fetal venous perfusate and at low titer. Ad.VEGF-DΔNΔC did not markedly affect human placental integrity and function in vitro. There was limited tissue access and transfer of vector across the placental barrier. Except for a minor elevation in LDH release, these test data did not reveal any toxic effects of Ad.VEGF-DΔNΔC on the human placenta.

Image-Based Modeling of Blood Flow and Oxygen Transfer in Feto-Placental Capillaries.

During pregnancy, oxygen diffuses from maternal to fetal blood through villous trees in the placenta. In this paper, we simulate blood flow and oxygen transfer in feto-placental capillaries by converting three-dimensional representations of villous and capillary surfaces, reconstructed from confocal laser scanning microscopy, to finite-element meshes, and calculating values of vascular flow resistance and total oxygen transfer. The relationship between the total oxygen transfer rate and the pressure drop through the capillary is shown to be captured across a wide range of pressure drops by physical scaling laws and an upper bound on the oxygen transfer rate. A regression equation is introduced that can be used to estimate the oxygen transfer in a capillary using the vascular resistance. Two techniques for quantifying the effects of statistical variability, experimental uncertainty and pathological placental structure on the calculated properties are then introduced. First, scaling arguments are used to quantify the sensitivity of the model to uncertainties in the geometry and the parameters. Second, the effects of localized dilations in fetal capillaries are investigated using an idealized axisymmetric model, to quantify the possible effect of pathological placental structure on oxygen transfer. The model predicts how, for a fixed pressure drop through a capillary, oxygen transfer is maximized by an optimal width of the dilation. The results could explain the prevalence of fetal hypoxia in cases of delayed villous maturation, a pathology characterized by a lack of the vasculo-syncytial membranes often seen in conjunction with localized capillary dilations.

An international network (PlaNet) to evaluate a human placental testing platform for chemicals safety testing in pregnancy.

The human placenta is a critical life-support system that nourishes and protects a rapidly growing fetus; a unique organ, species specific in structure and function. We consider the pressing challenge of providing additional advice on the safety of prescription medicines and environmental exposures in pregnancy and how ex vivo and in vitro human placental models might be advanced to reproducible human placental test systems (HPTSs), refining a weight of evidence to the guidance given around compound risk assessment during pregnancy. The placental pharmacokinetics of xenobiotic transfer, dysregulated placental function in pregnancy-related pathologies and influx/efflux transporter polymorphisms are a few caveats that could be addressed by HPTSs, not the specific focus of current mammalian reproductive toxicology systems. An international consortium, "PlaNet", will bridge academia, industry and regulators to consider screen ability and standardisation issues surrounding these models, with proven reproducibility for introduction into industrial and clinical practice.

Quantitative assessment of placental morphology may identify specific causes of stillbirth.

BACKGROUND: Stillbirth is frequently the result of pathological processes involving the placenta. Understanding the significance of specific lesions is hindered by qualitative subjective evaluation. We hypothesised that quantitative assessment of placental morphology would identify alterations between different causes of stillbirth and that placental phenotype would be independent of post-mortem effects and differ between live births and stillbirths with the same condition. METHODS: Placental tissue was obtained from stillbirths with an established cause of death, those of unknown cause and live births. Image analysis was used to quantify different facets of placental structure including: syncytial nuclear aggregates (SNAs), proliferative cells, blood vessels, leukocytes and trophoblast area. These analyses were then applied to placental tissue from live births and stillbirths associated with fetal growth restriction (FGR), and to placental lobules before and after perfusion of the maternal side of the placental circulation to model post-mortem effects. RESULTS: Different causes of stillbirth, particularly FGR, cord accident and hypertension had altered placental morphology compared to healthy live births. FGR stillbirths had increased SNAs and trophoblast area and reduced proliferation and villous vascularity; 2 out of 10 stillbirths of unknown cause had similar placental morphology to FGR. Stillbirths with FGR had reduced vascularity, proliferation and trophoblast area compared to FGR live births. Ex vivo perfusion did not reproduce the morphological findings of stillbirth. CONCLUSION: These preliminary data suggest that addition of quantitative assessment of placental morphology may distinguish between different causes of stillbirth; these changes do not appear to be due to post-mortem effects. Applying quantitative assessment in addition to qualitative assessment might reduce the proportion of unexplained stillbirths.

Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction.

Increased vascular resistance and reduced fetoplacental blood flow are putative aetiologies in the pathogenesis of fetal growth restriction (FGR); however, the regulating sites and mechanisms remain unclear. We hypothesised that placental vessels dictate fetoplacental resistance and in FGR exhibit endothelial dysfunction and reduced flow-mediated vasodilatation (FMVD). Resistance was measured in normal pregnancies (n = 10) and FGR (n = 10) both in vivo by umbilical artery Doppler velocimetry and ex vivo by dual placental perfusion. Ex vivo FMVD is the reduction in fetal-side inflow hydrostatic pressure (FIHP) following increased flow rate. Results demonstrated a significant correlation between vascular resistance measured in vivo and ex vivo in normal pregnancy, but not in FGR. In perfused FGR placentas, vascular resistance was significantly elevated compared to normal placentas (58 ± 7.7 mmHg and 36.8 ± 4.5 mmHg, respectively; 8 ml min(-1) ; means ± SEM; P < 0.0001) and FMVD was severely reduced (3.9 ± 1.3% and 9.1 ± 1.2%, respectively). In normal pregnancies only, the highest level of ex vivo FMVD was associated with the lowest in vivo resistance. Inhibition of NO synthesis during perfusion (100 µm l-NNA) moderately elevated FIHP in the normal group, but substantially in the FGR group. Human placenta artery endothelial cells from FGR groups exhibited increased shear stress-induced NO generation, iNOS expression and eNOS expression compared with normal groups. In conclusion, fetoplacental resistance is determined by placental vessels, and is increased in FGR. The latter also exhibit reduced FMVD, but with a partial compensatory increased NO generation capacity. The data support our hypothesis, which highlights the importance of FMVD regulation in normal and dysfunctional placentation.

Hypoxic treatment of human dual placental perfusion induces a preeclampsia-like inflammatory response.

Preeclampsia is a human pregnancy-specific disorder characterized by a placental pro-inflammatory response in combination with an imbalance of angiogenic factors and clinical symptoms, including hypertension and proteinuria. Insufficient uteroplacental oxygenation in preeclampsia due to impaired trophoblast invasion during placentation is believed to be responsible for many of the molecular events leading to the clinical manifestations of this disease. We investigated the use of hypoxic treatment of the dual placental perfusion system as a model for preeclampsia. A modified perfusion technique allowed us to achieve a mean soluble oxygen tension within the intervillous space (IVS) of 5-7% for normoxia and <3% for hypoxia (as a model for preeclampsia). We assayed for the levels of different inflammatory cytokines, oxidative stress markers, as well as other factors, such as endothelin (ET)-1 that are known to be implicated as part of the inflammatory response in preeclampsia. Our results show a significant increase under hypoxia in the levels of different inflammatory cytokines, including IL-6 (P=0.002), IL-8 (P<0.0001), TNF-α (P=0.032) and IFN-γ (P=0.009) at 360 min in maternal venous samples (n=6). There was also a significant increase in ET-1 levels under hypoxia both on the maternal side at 30 min (P=0.003) and fetal side at 360 min (P=0.036) (n=6). Other markers of oxidative stress, including malondialdehyde and 8-iso-protaglandin F2α (P=0.009) also show increased levels. Overall, these findings indicate that exposure of ex vivo dually perfused placental tissue to hypoxia provides a useful model for mimicking the inflammatory response characteristic of preeclampsia. This would therefore provide a powerful tool for studying and further delineating the molecular mechanisms involved in the underlying pathophysiology of preeclampsia.

Adapting in vitro dual perfusion of the human placenta to soluble oxygen tensions associated with normal and pre-eclamptic pregnancy.

For decades, superoxic ex vivo dual perfusion of the human placental lobule has been used as a model to study the physiology and metabolism of the placenta. The aim of this study was to further develop the technique to enable perfusion at soluble oxygen concentrations similar to those in normal pregnancy (normoxia) and in pre-eclampsia (PE; hypoxia). Our design involved reducing the mean soluble oxygen tension in the maternal-side intervillous space (IVS) perfusate to 5-7% and <3% for normoxia and hypoxia, respectively, while providing a more ubiquitous delivery of perfusate into the IVS, using 22 maternal-side cannulae. We achieved quasi-steady states in [O2](fetal venous (soluble)), which were statistically different between the two adaptations at t=150 to t=240 min of dual perfusion (2.1, 1.2, 2.8 and 0.4, 0.0, 1.5%; median, 25th, 75th percentiles, n=20 and 24 readings in n=5 and n=6 lobules, normoxic and hypoxic perfusion, respectively; P<0.001, Mann-Whitney U-test). Lactate dehydrogenase (LDH) levels in fetal and maternal venous outflow perfusates were unaffected by the adaptations. There was also no difference in tissue lactate release between the two adaptations. Glucose consumption from the fetal circulation and maternal-side 'venous' pyruvate release were higher under normoxic conditions, indicative of a greater metabolic flux through glycolysis. Furthermore, there was greater release of the hypoxic-sensitive marker, macrophage inflammatory protein-1α, into the maternal venous perfusate in the hypoxic model. Also, during hypoxic perfusion, we found that fetal-side venous placental growth factor (PlGF) levels were higher compared with normoxic perfusion. We conclude that these ex vivo adapted methods of placental perfusion provide a means of studying aspects of placental metabolism in relation to normal oxygenation and hypoxia-associated pregnancy disease.