Tissue microarray: an effective high-throughput method to study the placenta for clinical and research purposes.

OBJECTIVE: Tissue microarray (TMA) technology allows simultaneous examination of the expression of many molecular markers (protein, mRNA, DNA, etc.) with high-throughput. The application of this technology, to date, has been largely confined to the study of cancer. Placental pathology poses unique challenges because of the size of the organ, its complex anatomy, as well as its histological heterogeneity. The objective of this study was to assess the feasibility and efficiency of TMAs for immunohistochemistry and in situ hybridization of placental tissues. STUDY DESIGN: TMAs were constructed using an automated tissue arrayer. Standard 0.6-mm or 1-mm microarray needles were used. Villous parenchyma, basal plate, and chorioamniotic membranes were targeted in each block. Five mum-thick TMA sections underwent immunohistochemical analysis of both cytoplasmic and nuclear antigens using a panel of antibodies against a variety of cytoplasmic [cytokeratin-7, vascular endothelial growth factor (VEGF), and protein Z], membranous (endoglin), and nuclear (c-fos and c-jun) antigens. mRNA in situ hybridization for surfactant protein A (SP-A) and chromogenic in situ hybridization for the Y chromosome (DYZ1) were also performed. RESULTS: Validation of TMA immunoreactivity demonstrated comparable results with corresponding whole sections. When a two-tiered scoring system (positive/negative) was employed, there was agreement between two and three cores and whole tissue sections (kappa>0.7). When a three-tiered scoring system (negative, weak-positive, or strong-positive) was used, the data from three cores showed the highest agreement with whole tissue sections (kappa >0.7). In situ hybridization experiments for mRNA and DNA were also successful in that the signals were readily detectable. Successful transfer from the donor block to the recipient block differed according to the anatomical compartment. The transfer efficiency of villous parenchyma, basal plate, and chorioamniotic membranes were 96.9% (875/903), 76.7% (115/150), and 75.4% (224/297), respectively. CONCLUSION: TMA is a practical and effective tool for high-throughput molecular analysis of the human placenta. Duplicate and triplicate cores offer agreement with whole tissue sections for two-category distinction immunostaining. TMA also affords relevant results from in situ hybridization experiments for mRNA and DNA. The major advantages are the conservation of tissues and reagents, simultaneous comparison of molecular markers in different anatomical compartments of the placenta, and reduction of experimental error.     

Validation of tissue microarray technology using cervical adenocarcinoma and its precursors as a model system

The tissue microarray (TMA) technology has potentiated large-scale retrospective cohort studies using archival formalin-fixed, paraffin-embedded tissues. We used a large series of cervical adenocarcinomas to investigate TMA technology in assessment of immunohistochemical staining. A TMA was constructed using 273 archival paraffin blocks from 139 patients with 119 invasive and 20 adenocarcinoma in situ and 16 normal controls. Two paired cores were obtained from specific regions of donor blocks selected at histologic review and were arrayed into a recipient blocks. The novel array blocks and some whole donor blocks were sectioned and used for immunohistochemical analysis for carcinoembryonic antigen, cytokeratin 7, and cytokeratin 20 antibodies as potential diagnostic markers. We compared staining in the microarray disks with the whole tissue sections. Two paired TM cores were found to yield good immunohistochemical staining that was concordant with that of the whole section from which it originated in about 97% of cases, and the cores accurately represented the morphology of the tumor with respect to tumor typing and differentiation in all cases. Our results suggest that TMAs can be successfully used for immunohistochemical studies of cervical adenocarcinomas. The areas sampled from donor blocks must be selected by careful review of sections from the original blocks.     

The plasma level and gene expression of resistin in polycystic ovary syndrome

Objectives.?To investigate the relationship between plasma resistin, polycystic ovary syndrome (PCOS), and insulin resistance (IR). To compare the mRNA level of resistin in mononuclear cells and monocyte-derived macrophages in women with PCOS and controls.

Materials and methods.?Patients with PCOS and controls were enrolled and IR was considered as the stratified factor for subgroups. Fasting blood was collected to determine the levels of sex hormones, insulin, glucose, blood lipid, and resistin. Resistin gene expression was evaluated by quantitative real-time RT-PCR in mononuclear cells and monocyte-derived macrophages cultured with or without rosiglitazone for 96?h.

Results.?No significant difference of plasma resistin levels was found among PCOS-IR, PCOS-non-IR, control-IR, and control-non-IR groups. There were no significant differences in resistin mRNA expression between participants with and without PCOS and with and without IR. Resistin mRNA expression in monocyte-derived macrophages was higher than that in mononuclear cells (p?=?0.04), and could be reduced by rosiglitazone (p?<?0.001).

Conclusions.?Plasma resistin does not correlate with normal weight PCOS or IR. Resistin gene expression in mononuclear cells and monocyte-derived macrophages in PCOS and IR is the same as controls. Further researches on the role of resistin in the pathogenesis of PCOS or IR should concentrate on the tissue level.     

MCI-186 administered to the maternal circulation inhibits fetal brain injury resulting from total umbilical cord occlusion in the chronically instrumented fetal lamb

Objective.?To evaluate the transplacental effects of MCI-186 (edaravone), a potent hydroxyl radical scavenger, administered to the maternal circulation to inhibit fetal brain injury caused by umbilical cord occlusion.

Methods.?Nine chronically instrumented lambs were prepared. In three cases, 10-min persistent total umbilical cord occlusion (group A) was performed. Another three cases underwent occlusion and were administered 60 mg of MCI-186 through the maternal femoral vein prior to the end of occlusion (group B). The remaining three cases underwent sham operation (group C). On day 3 after insult, fetal brains were extirpated. Paraffin-embedded brain tissue sections were stained with hematoxylin and eosin, Bodian, Kluver–Barrera, and TUNEL. Neuronal cellular damage was evaluated by two pathologists blinded to the experimental conditions.

Results.?Group A displayed numerous cells with eosinophilic condensation of nuclear chromatin and proliferation of microglia in the hippocampus and basal ganglia. TUNEL-positive cells were observed in the periventricular area. Group B showed microglial proliferations, but no marked changes. No pathological changes were apparent in group C.

Conclusions.?MCI-186 administered to the maternal circulation could inhibit fetal brain injury resulting from hypoxia-reperfusion induced by umbilical cord occlusion.     

Interleukin-33 in the human placenta

Objective: Interleukin-33 (IL-33) is the newest member of the IL-1 cytokine family, a group of key regulators of inflammation. The purpose of this study was to determine whether IL-33 is expressed in the human placenta and to investigate its expression in the context of acute and chronic chorioamnionitis. Methods: Placental tissues were obtained from five groups of patients: 1) normal pregnancy at term without labor (n = 10); 2) normal pregnancy at term in labor (n = 10); 3) preterm labor without inflammation (n = 10); 4) preterm labor with acute chorioamnionitis and funisitis (n = 10); and 5) preterm labor with chronic chorioamnionitis (n = 10). Immunostaining was performed to determine IL-33 protein expression patterns in the placental disk, chorioamniotic membranes, and umbilical cord. mRNA expression of IL-33 and its receptor IL1RL1 (ST2) was measured in primary amnion epithelial and mesenchymal cells (AECs and AMCs, n = 4) and human umbilical vein endothelial cells (HUVECs, n = 4) treated with IL-1β (1 and 10?ng/ml) and CXCL10 (0.5 and 1 or 5?ng/ml). Results: 1) Nuclear IL-33 expression was found in endothelial and smooth muscle cells in the placenta, chorioamniotic membranes, and umbilical cord; 2) IL-33 was detected in the nucleus of CD14+ macrophages in the chorioamniotic membranes, chorionic plate, and umbilical cord, and in the cytoplasm of myofibroblasts in the Wharton’s jelly; 3) acute (but not chronic) chorioamnionitis was associated with the presence of IL-33+ macrophages in the chorioamniotic membranes and umbilical cord; 4) expression of IL-33 or IL1RL1 (ST2) mRNA in AECs was undetectable; 5) IL-33 mRNA expression increased in AMCs and HUVECs after IL-1β treatment but did not change with CXCL10 treatment; and 6) IL1RL1 (ST2) expression decreased in AMCs and increased in HUVECs after IL-1β but not CXCL10 treatment. Conclusions: IL-33 is expressed in the nucleus of placental endothelial cells, CD14+ macrophages, and myofibroblasts in the Wharton’s jelly. IL-1β can induce the expression of IL-33 and its receptor. Protein expression of IL-33 is detectable in macrophages of the chorioamniotic membranes in acute (but not chronic) chorioamnionitis.     

Terbutaline inhibits corticotropin-releasing hormone (CRH) expression in human trophoblast cells

Objective.?To evaluate the effect of beta-adrenergic agonists on the regulation of the expression of the placental corticotropin-releasing hormone (CRH) gene.

Study design.?Term placentae were collected at the time of elective cesarean section, and trophoblast cells were harvested, isolated, and cultured. The isolated trophoblasts were plated, cultured and subsequently treated with cortisol, terbutaline, RU486, or vehicle control. CRH expression, mRNA abundance of CRH, and the housekeeping gene beta-actin were evaluated by Northern blot analysis.

Results.?Exposure of the trophoblasts to terbutaline (10^?8 M) inhibited the expression of the CRH gene as depicted by Northern blot analysis. Co-addition of terbutaline (10^?8 M) and RU486 (10^?6 M) did not block the stimulatory effects of RU486 on placental trophoblast cells.

Conclusion.?The beta-adrenergic agonist terbutaline inhibits the expression of CRH in human trophoblasts. This finding may provide insight into the mechanism of action of terbutaline as a tocolytic agent.     

Histological chorioamnionitis in preterm prelabor rupture of the membranes is associated with increased expression of galectin-3 by amniotic epithelium

Purpose: Gal-3, which can regulate immune responses upon infection and inflammation, was not studied so far in intrauterine infection leading to preterm prelabor rupture of the membranes (PPROM), although gal-1 was reported to be implicated in the process. Gal-3 mRNA and protein expression in amnion and its changes during histological chorioamnionitis were studied here.

Materials and methods: Fetal membranes were obtained from women with PPROM with (n?=15) and without histological chorioamnionitis (n?=15) during second and third trimester. Immunohistochemical reactivity was evaluated semiquantitatively and analyzed using t-test. Galectin profile of amniotic epithelia was determined by polymerase chain reaction (PCR) and change assessed in gal-3 in PPROM with (n?=5) or without histological chorioamnionitis (n?=5) by real-time PCR.

Results: Human amniotic epithelium was found to express gal-1, gal-3, gal-7 and gal-8 mRNA. Gal-3 mRNA and protein is increased in fetal membranes and in the amniotic epithelium in patients with chorionamnionitis.

Conclusion: Histological chorioamnionitis is associated with increased gal-3 expression and strong immunoreactivity of the amnion. Gal-3 may participate in the regulation of the inflammatory responses to chorioamniotic infection and/or direct interaction with pathogens.     

Raised serum levels of matrix metalloproteinase-9 in women with polycystic ovary syndrome and its association with insulin-like growth factor binding protein-1

Background.?It has been suggested in recent studies that matrix metalloproteinases (MMPs) may be implicated in the pathogenesis of polycystic ovary syndrome (PCOS) through regulating ovarian tissue remodeling. In addition to degrading the extracellular matrix, MMPs exhibit the ability to cleave insulin-like growth factor binding protein-1 (IGFBP-1), the major regulator of insulin-like growth factor-I (IGF-I) in serum. The present study aimed to investigate the possible role of MMPs in the pathophysiology of PCOS.

Methods.?Serum levels of MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), IGF-I and IGFBP-1 were measured in 42 patients with PCOS and 30 healthy women with regular menstruation, matched for age and body mass index. Correlation between IGFBP-1 and other parameters in the PCOS group was analyzed by Pearson's linear correlations.

Results.?Serum MMP-9 concentrations and MMP-9/TIMP-1 ratios were significantly higher in PCOS women than in controls. Serum levels of IGFBP-1 were markedly lower in the PCOS group. There was a negative correlation between serum IGFBP-1 and MMP-9 in women with PCOS.

Conclusion.?Our results raise the possibility that MMPs may be implicated in the pathophysiology of PCOS either by regulating ovarian tissue remodeling or indirectly by facilitating IGF-I bioavailability through proteolysis of IGFBP-1.     

Differential expression pattern of genes encoding for anti-microbial peptides in the fetal membranes of patients with spontaneous preterm labor and intact membranes and those with preterm prelabor rupture of the membranes

Objective.?Increased amniotic fluid concentrations of anti-microbial peptides, components of the innate immune system, have been reported in patients with preterm labor (PTL) with intact membranes and intra-amniotic infection and/or inflammation (IAI), as well as in patients with preterm prelabor rupture of the membranes (PPROM). This study was designed to confirm these results using a targeted approach, detecting DEFA1, DEFB1, GNLY, and S100A9 gene expression in the choriamniotic membranes in pregnancies complicated with PTL and intact membranes or PPROM, with and without histologic chorioamnionitis.

Study design.?Human fetal membranes were obtained from patients in the following groups: (1) PTL with intact membranes (n?=?15); (2) PTL with intact membranes with histologic chorioamnionitis (n?=?12); (3) PPROM (n?=?17); and (4) PPROM with histologic chorioamnionitis (n?=?21). The mRNA expression of α-defensin-1, β-defensin-1, calgranulin B and granulysin in the fetal membranes was determined by qRT-PCR.

Results.?(1) The expression of α-defensin-1 mRNA in the fetal membranes was higher in patients with PTL and intact membranes with histologic chorioamnionitis, than those without chorioamnionitis (19.4-fold, p?<?0.001); (2) Among patients with histologic chorioamnionitis, patients with PTL and intact membranes had a higher α-defensin-1 mRNA expression than those with PPROM (5.5-fold, p?=?0.003); (3) Histologic chorioamnionitis was associated with a higher calgranulin B mRNA expression in the chorioamniotic membranes of patients with both PTL and intact membranes (7.9-fold, p?=?0.03) and PPROM (7.6-fold, p?<?0.0001); (4) The expression of calgranulin B mRNA in the fetal membranes was higher in patients with PTL and intact membranes without histologic chorioamnionitis than in those with PPROM without histologic chorioamnionitis (2.7-fold, p?=?0.03); (5) There were no differences in the expression of β-defensin-1 and granulysin in the chorioamniotic membranes between the study groups even in the presence of histologic chorioamnioniotis.

Conclusions.?(1) Among patients with histologic chorioamnionitis, the mRNA expression of α-defensin-1 and calgranulin B in the fetal membranes of patients with PTL and intact membranes as well as that of calgranulin B in the fetal membranes of patients with PPROM is higher than in the membranes of those without histologic chorioamnionitis; (2) histologic chorioamnionitis is associated with differences in the pattern of α-defensin-1 mRNA expression in the fetal membranes in patients with PTL and intact membranes and those with PPROM.     

Cardiac flow parameters with conventional and pulsed tissue Doppler echocardiography imaging in patients with polycystic ovary syndrome

Background.?Polycystic ovary syndrome (PCOS) is independently associated with the major cardiovascular risk factors. The aim of this study was to examine the echocardiographic profiles of patients with PCOS using conventional echocardiographic methods and tissue Doppler imaging.

Methods.?For this study, we have registered 48 women with PCOS and 21 healthy control subjects who were mathced with respect to age and body mass index. Standard two-dimensional and M-mode measurement, transmitral valve flows and tissue Doppler imaging of mitral and tricuspid anulus were recorded.

Results.?In PCOS and control groups, left ventricular and atrium diameters, ejection fraction, mitral E/A ratio, deceleration time and isovolumic relaxation time were similar. There were no significant differences between patients with PCOS and control subjects with respect to tissue Doppler profiles.

Conclusion.?Patients with PCOS execute echocardiographic measures of cardiac function that are similar to those of healthy women.     

Different expression of estrogen receptors α and β in human myometrium and leiomyoma during the proliferative phase of the menstrual cycle and after GnRHa treatment

Uterine leiomyomas (uterine fibroids) are sex-steroid dependent benign tumors. Limited knowledge is available regarding the role of estrogen and their receptors in the regulation of fibroids in premenopausal women ,and in their shrinkage after treatment with a gonadotropin-releasing hormone analogue (GnRHa). The expression of the two subtypes of the estrogen receptor (ER) ,ERα and ERβ, was studied in leiomyoma and homologous myometrium from women in the proliferative phase of the menstrual cycle and from women treated with GnRHa. The mRNA levels of ERα and ERβ were monitored by solution hybridization and in situ hybridization ,and receptor proteins were detected and localized by immuno-histochemistry. Both ERα and ERβ were present in the leiomyomas and homologous myometrium. The ERα mRNA level in the leiomyomas was higher than in the surrounding myometrium. The ERβ mRNA level was lower than that of ERα in both groups. ERβ immunoreactivity was lower in leiomyomas when compared with the myometrium after GnRHa treatment ,while ERα was higher in the leiomyomas. The present results imply that the increased ratio of ERα/ERβ observed in the fibroids after GnRHa treatment could reflect or be the cause of the shrinkage of the leiomyoma ,which is the clinical outcome of this treatment.     

MMP-2 and TIMP-2 Expression Correlates with Poor Prognosis in Cervical Carcinoma—A Clinicopathologic Study Using Immunohistochemistry and mRNA in Situ Hybridization

Objective. The spread of malignant neoplasms is closely associated with matrix and basement membrane degradation, mediated by various classes of proteolytic enzymes. Matrix metalloproteinases (MMP) appear to have a key role in the sequence of events that lead to local invasion and metastasis. The present study evaluated the role of matrix metalloproteinase-2 (MMP-2), tissue inhibitor of metalloproteinases-2 (TIMP-2), and membrane-type metalloproteinase (MT1-MMP) in cervical neoplasia.Methods. We have analyzed 49 uterine cervical squamous cell carcinomas, 10 cases of high-grade cervical intraepithelial neoplasia (CIN II–III), and 10 control cervices for the presence of MMP-2, TIMP-2, and MT1-MMP using in situ hybridization. MMP-2 protein expression was evaluated using immunohistochemistry. Results were analyzed for possible correlation with disease outcome.Results. MMP-2, TIMP-2, and MT1-MMP mRNA were localized to both stromal and tumor cells. However, an intense signal for MMP-2 was detected almost exclusively in tumor cells and was uniformly absent from CIN lesions and control cervices. Conversely, intense signals for TIMP-2 and MT1-MMP were detected in both stromal and tumor cells of invasive carcinomas, more often for the former. As with MMP-2, they were absent from CIN lesions. MMP-2 protein expression was enhanced in tumor cells compared to CIN cases and controls, significantly compared to the latter (P = 0.01). The presence of both MMP-2 and TIMP-2 mRNA in tumor cells correlated with advanced stage (P = 0.003 for MMP-2, P = 0.002 for TIMP-2) and with poor survival (P = 0.003 for MMP-2, P = 0.002 for TIMP-2) in univariate analysis. In addition, their presence in tumor cells intercorrelated (P = 0.002). In multivariate survival analysis, MMP-2 presence retained its association with survival (P = 0.004), in addition to patient age (P = 0.027) and advanced stage (P = 0.0002).Conclusions. Both MMP-2 and TIMP-2 have a key role in extracellular matrix invasion in cervical carcinoma, largely through their elaboration by tumor cells. The presence of mRNA for both proteins is interrelated and is associated with poor survival.     

Increased Placental Telomerase mRNA in Hypertensive Disorders of Pregnancy

Objective.?We assessed hTERT mRNA levels in normal versus preeclamptic placental samples, examining hTERT expression levels in different clinical manifestations of hypertensive disorder of pregnancy.?Methods.?We performed a single-site, prospective case-control study of hTERT mRNA levels in placentas from term and preterm pregnancies with hypertensive disorders compared with unaffected pregnancies. Placental biopsies were collected from 61 patients (preeclamptic: 32; non-preeclamptic (control): 29). Total RNA from placenta was isolated and reversely transcribed to c‐DNA. A probe-specific real-time quantitative PCR assay was employed to determine the relative expressional levels of hTERT mRNA levels in these placentas from both unaffected and affected pregnancies with different categories of hypertensive disorders including preeclampsia, severe preeclampsia, eclampsia and HELLP syndrome (Hemolysis, Elevated Liver function tests, Low Platelet).?Results.?The average ratio of hTERT mRNA levels was 1.73 in the preeclamptic group and 1.02 for control group (p < 0.0001). The hTERT expression levels were elevated for each of the different categories of hypertensive disorders of pregnancy compared with control: HELLP syndrome 1.86, severe preeclampsia 1.81, eclampsia 1.71 and mild preeclampsia 1.63. In addition, hTERT levels were higher in severe than mild preeclampsia (p < 0.01). Conclusions.?Elevated hTERT mRNA expression is observed in placentas from pregnancies with different clinical manifestations of hypertensive disorders of pregnancy. The patho-physiological significance of this finding awaits further studies.     

The effects of α-zearalanol and estradiol benzoate on expression of c-myc,c-fos and epidermal growth factor receptor mRNAs in breast tissues implanted into nude mice

This study was designed to evaluate the effects of a novel phytoestrogen, α-zearalanol (α-ZAL), and estradiol benzoate (B-E₂), on c-myc, c-fos, and EGFR expression in normal human breast tissues implanted into nude mice. A xenograft-model, pieces of normal human breast tissue implanted subcutaneously into 9–10-week-old athymic nude mice, was established. The mice were divided into five groups subjected to the following treatments: normal saline (Controls); α-ZAL at 1 and 5?mg/kg; and estradiol benzoate (B-E₂) at 1 and 5?mg/kg. Treatment was given every other day, and human breast tissues were removed for experiments after treatment for 30 days. The expression of c-myc, c-fos, and EGFR mRNAs were determined by in situ hybridization. α-ZAL decreased expression of c-myc (p?<?0.05). About 1?mg/kg α-ZAL increased EGFR expression (p?<?0.05) and two dosage of α-ZAL increased c-fos expression (p?<?0.01) compared with control. B-E₂ significantly increased expression of c-myc, c-fos, and EGFR mRNAs expression compared with controls (p?<?0.01). The extents of the increases in EGFRmRNA expression induced by α-ZAL and in c-fos mRNA by 5?mg/kg α-ZAL were lower than those induced by B-E₂ (p?<?0.01). These data suggest that the phytoestrogen α-ZAL may be safer than estrogen on breast.     

Evidence of the activation of unfolded protein response in granulosa and cumulus cells during follicular growth and maturation

Abstract

The objective of the present study is to investigate whether unfolded protein response (UPR), activated by endoplasmic reticulum (ER) stress, in granulosa cells (GC) and cumulus cells (CC) is involved in the process of follicular growth and maturation. First, to examine the presence of UPR in growing follicles, the expression of spliced form of X-box-binding protein 1 (XBP1(S)) and heat shock 70?kDa protein 5 (HSPA5) mRNA, typical UPR genes, in mice ovaries were examined by in situ hybridization. GC of later stage than large secondary follicles expressed both XBP1(S) and HSPA5 mRNA, which was accompanied with the activation of ER stress sensor proteins, inositol-requiring enzyme 1 (IRE1) and double-stranded RNA-activated protein kinase-like ER kinase (PERK), confirmed by immunohistochemistry. Next, to examine the association between the fertilization capacity of oocytes and the expression levels of UPR genes in surrounding CC, human CC were collected from patients undergoing ICSI. The expression levels of XBP1(S) mRNA, quantified by RT-PCR, in CC enclosing human oocytes achieving fertilization were two-fold higher, than those in CC enclosing oocytes without fertilization capacity. In conclusion, UPR is activated during follicular growth and suggested to be involved in the process of follicular growth and maturation.     

Normal prenatal ultrasound findings in a case with de novo mosaic small supernumerary marker chromosome 18 – how to counsel?

Introduction.?We report on a prenatally diagnosed de novo small supernumerary marker chromosome (sSMC) derived form chromosome 18. Molecular cytogenetic studies led to information about the clinical relevance of the sSMC-induced chromosomal imbalance. As prenatal ultrasound was normal, detailed information with respect to prenatal counseling of the parents was necessary. In general, detection of an sSMC requires as much information on the exact genetic content with its possible impact on the phenotype as achievable.

Material and methods.?Amniocentesis was performed in a 37-year-old Gravida IV Para II with a history of an induced abortion due to a prenatally diagnosed trisomy 21. Fluorescence in situ hybridization quick test gave hint on a possible mosaic trisomy 18, whereas the conventional banding cytogenetic analysis revealed an sSMC. The amount of euchromatin was estimated to be less than 5 MB.

Conclusions.?sSMC are rare, being present in less than 0.08% of all pregnancies. Going together with an abnormal ultrasound, counseling of the parents is relatively easy to perform. In cases of normal prenatal ultrasound, profound knowledge about the surplus genetic content is necessary for the estimation of the fetal outcome prognosis. In the present case, detailed molecular cytogenetics techniques led the parents to continue the pregnancy.