Expression and role of peroxisome proliferator-activated receptors in the porcine early placenta trophoblast.

Peroxisome proliferator-activated receptors (PPARs) are members of a nuclear receptor family of ligand-dependent transcription factors. Three isoforms of PPAR named PPARα, PPARß/δ, and PPARγ have been described, each encoded by a separate gene: PPARA, PPARD, and PPARG, respectively. In the present study, we examined the profiles of PPAR and retinoid X receptor (RXR; PPAR heterodimer partner) mRNA expression and PPAR DNA binding activity in porcine trophoblast tissue collected on days 15, 20, 25, and 30 of pregnancy and in day-20 embryos. Placenta trophoblast cells isolated on day 25 of pregnancy were used to determine effects of (1) cytokines on PPAR and RXR mRNA expression and (2) PPAR agonists on prostaglandin (PG) E2 synthesis and the expression of genes involved in steroidogenesis, fatty acid binding, and PG transport, as well as on cell proliferation. The mRNA expression of PPARA and RXRB was greater in trophoblast tissue collected on days 25 and 30 of pregnancy compared with day 15 (P < 0.05), while DNA binding activity of PPARα decreased between day 15 and 25 (P < 0.05). Increased concentrations of PPARD and RXRA transcripts were observed in trophoblasts collected on day 20 compared to trophoblasts from days 15 and 30 (P < 0.05). Moreover, concentrations of DNA-bound PPARß/δ and PPARγ proteins increased in day-30 trophoblasts compared to day 15 (P < 0.01) and day 20 (P < 0.05), respectively. On day 20 of gestation, the mRNA expression of PPARD, PPARG, and RXRA and protein levels of PPARα and PPARγ isoforms were greater in trophoblast than embryonic tissue (P < 0.01). Interleukin 1ß and/or interferon γ, but not IL6 and leukemia inhibitory factor, upregulated PPAR and RXR mRNA expression in placenta trophoblast cells in vitro (P < 0.05). Rosiglitazone (a PPARγ agonist) stimulated prostaglandin E synthase mRNA expression in trophoblast cells and PGE2 accumulation in incubation medium (P < 0.05). Moreover, activation of PPAR isoforms differentially affected the expression of genes involved in steroidogenesis, fatty acid binding, and PG transport in studied cells. Finally, PPARα and PPARγ agonists stimulated trophoblast cell proliferation (P < 0.05), and this effect was abolished by the addition of a respective PPAR antagonist (P < 0.05). Overall, these results point to a role of PPAR isoforms in porcine placenta development and function.

Effect of oestrus synchronization with PGF2α/eCG/hCG on luteal P4 synthesis in early pregnant gilts.

Administration of hormones to synchronize oestrus is a useful tool in animal breeding. However, exogenous ovarian stimulation may be detrimental to reproductive function. This study was aimed to examine whether an oestrus synchronization with PGF2α/eCG/hCG could affect luteal P4 synthesis in early pregnant gilts. Corpora lutea (CLs) were collected on days 9, 12 and 16 of pregnancy from gilts with natural (n = 16) and synchronized (n = 18) oestrus and analysed for (i) the expre-ssion of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A polypeptide (CYP11A1), and 3ß-hydroxysteroid dehydrogenase (3ßHSD); (ii) the concentration of P4 in the luteal tissue and blood; and (iii) the expression of luteinizing hormone receptors (LHR) and oestrogen receptors (ERα and ERß). Additionally, the effect of LH on P4 secretion from CL slices collected from synchronized and naturally ovulated animals has been studied in vitro. PGF2α /eCG/hCG administration increased mRNA expression of StAR, CYP11A1, 3ßHSD, and LHR on day 9 and CYP11A1 and LHR on day 12 of pregnancy compared with the control group (p < 0.05). CYP11A1, 3ßHSD, LHR, ERα and ERß proteins were not affected by synchronization; only StAR protein increased in hormonally treated animals (p = 0.017). The concentration of P4 in luteal tissue was greater on day 9 (p < 0.01), but lower on day 16 (p < 0.05) in gilts with hormonally induced oestrus compared with control animals. Blood serum levels of P4 were lower in synchronized than control gilts (p < 0.001). Synchronization did not affect LH-stimulated P4 secretion from luteal slices; however, greater basal concentration of P4 in incubation medium was detected for CLs collected from synchronized than control gilts (p < 0.05). In conclusion, synchronization of oestrus with PGF2α/eCG/hCG protocol in gilts did not impair the expression of luteal P4 synthesis system, although decreased P4 concentration in the blood.

Regulation of prostacyclin synthase expression and prostacyclin content in the pig endometrium.

Prostaglandins (PGs) are critical regulators of a number of reproductive processes, including embryo development and implantation. In the present study, prostacyclin (PGI(2)) synthase (PGIS) mRNA and protein expression, as well as 6-keto PGF(1α) (a PGI(2) metabolite) concentration, were investigated in the pig uterus. Endometrial tissue and uterine luminal flushings were obtained on Days 4 to 18 of the estrous cycle and pregnancy. Additionally, conceptuses were collected and examined for PGIS mRNA expression and 6-keto PGF(1α) concentration. Regulation of PGI(2) synthesis in the porcine endometrium by steroids, conceptus products, and cytokines was studied in vitro and/or in vivo. Endometrial PGIS protein level increased on Days 12 and 16 in pregnant but not in cyclic gilts. Moreover, higher PGIS protein expression on Day 12 of pregnancy was accompanied by a greater content of 6-keto PGF(1α) in the endometrium. The concentration of 6-keto PGF(1α) in uterine luminal flushings increased substantially on Days 16 and 18 in pregnant gilts and was higher than in cyclic animals. Greater PGIS mRNA expression and PGI(2) metabolite concentration were detected in Day 12 and 14 conceptuses, respectively. Incubation of endometrial explants with conceptus-conditioned medium resulted in upregulation of PGIS protein expression and increased PGI(2) secretion. Moreover, PGIS mRNA and protein expression were upregulated in the endometrium collected from gravid uterine horn on Day 14 of pregnancy. In summary, PGIS is differentially expressed in the endometrium of cyclic and pregnant gilts resulting in higher PGI(2) synthesis in pregnant animals. Porcine conceptuses are important regulators of endometrial PGIS expression and PGI(2) release during the implantation period.

Regulation of expression and role of leukemia inhibitory factor and interleukin-6 in the uterus of early pregnant pigs.

Cytokines, which are generally involved in the process of inflammation, may also play a critical role in conceptus implantation. We examined: (1) the expression profiles of leukemia inhibitory factor (LIF) and interleukin (IL)-6 mRNA and their protein content in the endometrium of cyclic and pregnant gilts on Days 10 to 18 after estrus; (2) the effect of conceptus-exposed medium on LIF and IL-6 synthesis in the endometrium; (3) the profiles of IL6R and LIFR mRNA expression in pig conceptuses collected on Days 10 to 18 of pregnancy; and (4) the effect of LIF and IL-6 on the attachment and proliferation of porcine trophoblast cells. The expression of LIF mRNA in the endometrium increased between Days 10 and 12 in both cyclic and pregnant gilts, and tended to be higher in Day 12 pregnant animals compared with nonpregnant ones. The LIF protein content in the uterine lumen peaked on Day 12 of pregnancy, and was higher than on Day 12 of the estrous cycle. Endometrial IL-6 mRNA expression was upregulated on Day 12 in pregnant gilts compared with nonpregnant animals. Moreover, a higher content of IL-6 protein was observed in pregnant than in cyclic gilts. The addition of conceptus-exposed medium resulted in up-regulation of LIF and IL6 mRNA expression, and increased IL-6 content in endometrial slices. In conceptuses, increased mRNA expression was detected on Days 10 to 14 for IL6R and on Day 14 for LIFR, when compared with other days studied. LIF and IL-6 stimulated the attachment and proliferation of trophoblast cells in vitro. In summary, LIF and IL-6 are important components of embryo-uterine interactions during early pregnancy in the pig, and may contribute to successful conceptus implantation.

Mechanisms for the establishment of pregnancy in the pig.

Establishment of pregnancy in pigs requires continuous function of corpora lutea and endometrial preparation for embryo implantation. Progesterone regulates expression of many proteins necessary for endometrial remodelling and embryo-maternal communications. Attaining the uterine receptivity involves progesterone priming and loss of progesterone receptors in the uterine epithelium before days 10-12 after oestrus. Spermatozoa and oocytes in oviduct alter secretion of specific proteins that exert beneficial effect on gametes and embryos. Moreover, an appropriate leucocyte activation and maintenance of delicate cytokine balance within the oviduct and uterus are important for early pregnancy. This early local immune response is rather mediated by seminal plasma components. These components also influence prostaglandin (PG) synthesis in the oviduct that is important for gamete and embryo transport. Pregnancy establishment requires the biphasic pattern of oestrogen secretion by conceptuses on days 11-12 and 15-30. Conceptus affects lipid signalling system consisting of prostaglandins and lysophosphatic acid. PG synthesis is changed by conceptus signals in favour of luteoprotective PGE(2) . Additionally, existence of PGE(2) positive feedback loop in the endometrium contributes to increased PGE(2) /PGF(2α) ratio during the peri-implantation period. PGE(2) through endometrial PGE(2) receptor (PTGER2) elevates the expression of enzymes involved in PGE(2) synthesis. Higher PGE(2) secretion in uterine lumen coincides with the elevated expression of HOXA10 transcription factor critical for implantation. A stable adhesion between conceptus and endometrium requires reduction in mucin-1 on the apical surface of epithelium and integrin activation by extracellular matrix proteins. Furthermore, growth factors, cytokines and its receptors are involved in embryo-maternal interactions.

Effect of conceptus secretions on HOXA10 and PTGS2 gene expression, and PGE2 release in co-cultured luminal epithelial and stromal cells of the porcine endometrium at the time of early implantation.

Homeobox A10 (HOXA10) gene expression was demonstrated in the endometrium of adult porcine uteri, however there is little information concerning the role of this gene in the pig. Objectives of the present study were to examine: 1) the expression of HOXA10 in the endometrium of cyclic and early pregnant gilts; 2) the effect of estradiol (E(2)) and progesterone (P(4)) on HOXA10 expression in porcine luminal epithelial (LE) and stromal (ST) cells in vitro; 3) the effect of E(2) and conceptus-exposed medium (CEM) on HOXA10 and prostaglandin endoperoxide synthase (PTGS2) gene expression and prostaglandin (PG) E(2) secretion from LE and ST cells in a co-culture model. The abundance of HOXA10 mRNA was increased on day 15 of pregnancy in comparison to day 15 of the estrous cycle. Moreover, increased HOXA10 mRNA level was detected in ST cells after E(2) and P(4) treatment. E(2) stimulated the expression of HOXA10 in LE cells cultured on collagen and pre-treated with steroids, but not in LE on plastic surfaces. Addition of CEM to LE cells cultured in collagen-coated inserts of the co-culture system resulted in elevated HOXA10 and PTGS2 gene expression and PGE(2) secretion in these cells, but not in ST cells cultured in basal compartments. ST cells directly treated with E(2) or CEM showed higher levels of HOXA10 and PTGS2 expression. Blocking of estrogen receptors with ICI-182,780 did not influence the stimulatory effect of CEM. We conclude that HOXA10 expression in the porcine endometrium is closely related to the implantation process and stimulated by conceptus products. Moreover, the co-culture system of LE and ST cells is a promising model for the study of endometrial response to conceptus-derived factors.

Effect of luteinizing hormone and tumour necrosis factor-alpha on VEGF secretion by cultured porcine endometrial stromal cells.

To cope with rising demands for increased blood supply during pregnancy, the vasculature of the uterus undergoes several adaptive changes, including increased permeability, angiogenesis and vasodilatation. Although it is clear that vascular endothelial growth factor (VEGF) plays a paramount role in achieving these adaptations, little is known about regulation of VEGF expression in endometrium during pregnancy. Thus, we have investigated whether luteinizing hormone (LH) and tumour necrosis factor-alpha (TNFalpha) may affect VEGF secretion by stromal cells during early pregnancy in pigs. Real-time reverse transcription/polymerase chain reaction (RT/PCR) of VEGF120 and VEGF164 gene expression revealed significantly higher levels of VEGF164 mRNA in cultured stromal cells (p < 0.0001). The LH-stimulated secretion of VEGF was detected after 24 and 48 h of treatment when doses 50 and 100 ng/ml were used (p < 0.05 and p < 0.01, respectively). The TNFalpha-induced secretion of VEGF by stromal cells was detected only after 24-h treatment with the highest dose used in the experiment (50 ng/ml; p < 0.05). Although the influence of LH on VEGF secretion was more visible compared with TNFalpha, both factors may be considered as potential modulators of adaptive changes in uterine vasculature occurring during pregnancy in the pig.

Effect of estrus induction on prostaglandin content and prostaglandin synthesis enzyme expression in the uterus of early pregnant pigs.

Prostaglandins (PGs) play a pivotal role in maternal recognition of pregnancy and implantation in pigs. In the present study, PGE(2), PGF(2alpha), and PGFM (PGF(2alpha) metabolite) content, as well as PGE(2) synthase (mPGES-1) and PGF(2alpha) synthase (PGFS) expression was investigated in early pregnant gilts with natural (n=21) and PMSG/hCG-stimulated (n=19) estrus. Endometrial tissue samples, uterine luminal flushings (ULFs), and blood serum were collected on days 10-11, 12, and 15 after insemination. Additionally, day 15 conceptuses were collected for mPGES-1 and PGFS protein expression. Effect of estrus induction was observed on day 15 of pregnancy, when the content of PGE(2) in the uterine lumen was fourfold lower in gonadotropin-stimulated gilts in comparison to controls (P<0.001). Decreased PGE(2) content in ULFs of gonadotropin-treated pigs was preceded by lower endometrial mPGES-1 gene expression in hormonally-stimulated animals in comparison to control gilts (P<0.01). On the other hand, estrus induction with PMSG/hCG resulted in higher PGE(2) accumulation in the endometrial tissue on day 15 of pregnancy (P<0.01). Furthermore, PGF(2alpha) content in the endometrium and PGFM levels in blood serum were lower in gonadotropin-treated gilts, especially on day 12 after insemination when compared to control gilts (P<0.01). Finally, PGFS expression in day 15 conceptuses was decreased in animals with hormonally-induced estrus. We conclude that PMSG/hCG stimulation of prepubertal gilts to induce estrus results in changes of PG production and secretion during early pregnancy, which, in turn, may affect conceptus development, implantation, and the course of pregnancy.

Endometrial and conceptus expression of HoxA10, transforming growth factor beta1, leukemia inhibitory factor, and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus.

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) beta1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72h later. Gilts in the control group (n=23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P<0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P<0.05) and 15 (P<0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFbeta, LIF, and PGHS-2 on day 12 of pregnancy (P<0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.

Antiluteolytic mechanisms and the establishment of pregnancy in the pig.

Extended exposure of progesterone and conceptus estrogen influences the vascular compartment of the uterus and expression of many factors, such as prostaglandins (PGs), growth factors, extracellular matrix and adhesion molecules, cytokines and transcription factors. One of the supportive mechanisms by which the conceptus inhibits luteolysis is by changing PG synthesis in favor of luteoprotective PGE2. Alteration in PG synthesis may result from increased PGE synthase (mPGES-1) expression in the trophoblast and endometrium on days 10-13 of pregnancy with simultaneous down-regulation of PGF synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1). Conceptus and endometrial, rather than luteal, synthesis of PGE2, is involved in the process of maternal recognition of pregnancy. However, complex (direct and indirect) actions of estrogen on the CL, including decreased luteal VEGF soluble receptor on day 12 of pregnancy, are important for luteal maintenance. Moreover, conceptus signals affect another lipid signaling component - lysophosphatidic acid receptor (LPA3), as well as HoxA10 and Wnt in the endometrium, to create the appropriate uterine environment for establishment of pregnancy and implantation.

Role of tumour necrosis factor alpha in stimulation of prostaglandins F(2alpha) and E(2) release by cultured porcine endometrial cells.

The present studies were undertaken to examine the effect of tumour necrosis factor (TNF) alpha on prostaglandins (PGs) F(2alpha) and E(2) release by cultured porcine endometrial cells harvested on days 13-16 after oestrus in comparison to stimulation with oxytocin (OT) and luteinizing hormone (LH). A time-dependent effect of TNFalpha (10 ng/ml) on PGF(2alpha) release was observed in stromal and luminal epithelial cells. Moreover, TNFalpha increased PGF(2alpha) secretion from both endometrial cell types with effective concentrations of 1 (p < 0.05), 10 and 50 ng/ml (p < 0.01). The effect of TNFalpha (10 ng/ml) on endometrial PGF(2alpha) and PGE(2) release was compared with OT (100 nmol/l) and LH (100 ng/ml). All factors affected PGF(2alpha) secretion from stromal cells, however, the stimulation tended to be more potent after OT and LH (p < 0.01) than after TNFalpha (p < 0.05) treatment. In epithelial cells, only TNFalpha was able to stimulate PGF(2alpha) release (p < 0.001). PGE(2) secretion from stromal cells increased after incubation with TNFalpha and OT (p < 0.05). Only LH stimulated PGE(2) release from epithelium (p < 0.001), and its action was very effective when compared with TNFalpha or OT (p < 0.01). Summarizing, TNFalpha induces both PGs secretion from cultured porcine endometrium, but preferentially stimulates PGF(2alpha) release from luminal epithelial cells. Therefore, similarly to OT and LH, TNFalpha may be considered as a potential modulator of endometrial PGF(2alpha) production during luteolysis in the pig.

Inhibition of luteolysis and embryo-uterine interactions during the peri-implantation period in pigs.

Inhibition of luteolysis and establishment of pregnancy in pigs results from oestrogen secretion by the conceptuses and requires progesterone produced by the corpus luteum (CL). An integral part of maternal recognition of pregnancy in the pig is the redirection of prostaglandin (PG) F2alpha secretion from endocrine (blood) to exocrine (uterus) direction and an increase of PGE2 synthesis in both the endometrium and conceptus. Uterine and conceptus PGE2 synthases play an integrated role in establishing the PGE2:PGF2alpha ratio necessary for luteal maintenance. The luteolytic or luteotrophic changes in the CL are synchronised with the release of maternal pituitary and ovarian hormones. The presence of uterine oxytocin (OT) and luteinising hormone (LH) receptors are important for the luteolytic effect of PGF2alpha. Conceptus oestrogen secretion coincides with autocrine and paracrine dialogue between the multiple conceptuses and uterine biological compounds and their receptors in trophoblast and endometrium.

Expression of cyclooxygenase-1 and -2 in the porcine endometrium during the oestrous cycle and early pregnancy.

Cyclooxygenase (COX) is the rate-limiting enzyme that catalyses the initial step in prostaglandins (PGs) production. In the present studies, endometrial COX-1 and COX-2 expression throughout the oestrous cycle and early pregnancy was analysed in pigs using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry. There were no changes in messenger RNA (mRNA) and protein expression for COX-1 in cyclic pigs. In pregnant animals, mRNA levels of this enzyme increased on days 22-25 (p < 0.001). However, no upregulation of COX-1 protein was detected. Quantification of COX-2 mRNA expression during the oestrous cycle revealed significant increases on days 10-12 and 14 (p < 0.001 and p < 0.01 vs days 2-4, respectively). Protein levels were also increased on day 14 when compared with days 2-12 and 18-20 after oestrus. In pregnant animals, the patterns of both COX-2 mRNA and protein expression were similar. Messenger RNA levels were higher on days 16 and 22-25 (p < 0.01 vs day 10). Moreover, the protein content tended to increase on days 16 and 22-25. COX-1 and COX-2 were localized in the luminal and glandular epithelium as well as in the uterine stroma. In contrast to COX-1, a positive immunostaining reaction for COX-2 was detected only on days 12-16 after ovulation and on days 14-16 of pregnancy. In conclusion, these results indicate specific patterns of COX-1 and COX-2 expression in the porcine endometrium throughout the oestrous cycle and early pregnancy. COX-2 rather than COX-1 seems to be the primary enzyme responsible for modulated PGs production at the time of luteolysis in cyclic and during implantation in pregnant animals.

Nongonadal LH receptors, their involvement in female reproductive function and a new applicable approach.

Luteinising hormone (LH) and human chorionic gonadotropin (hCG) share a common receptor in gonadal cells. The receptors have also been detected in several nongonadal but reproduction-associated tissues of pigs, cattle, and other species including the uterus (myometrium, endometrium), oviduct, cervix, blood vessels, mammary gland and other tissues. The main role of LH/hCG receptors in the myometrium is stimulation of growth and hyperplasia, and relaxation of uterine motility; hCG also boosts blood flow in the uterine artery. LH and hCG can increase production of prostaglandins in the endometrium, oviduct, and blood vessels. We suggest that the preovulatory surge of LH plays an important role in controlling oviductal contractions. Awareness of LH binding to many tissues of the female reproductive tract and integration with embryonic factors may lead to the elaboration of new strategies for improved reproductive efficiency in domestic species. Mammary glands also possess LH/hCG receptors through which gonadotropins can affect the metabolism of steroid hormones and could play an inhibitory role in mammary carcinogenesis and in the growth of breast tumours. A novel approach to target and ablate carcinoma cells through LH receptors is described.

Prostaglandins f and e secretion by porcine epithelial and stromal endometrial cells on different days of the oestrous cycle.

The endometrial tissue of the uterus plays a key role in reproduction and is a source of hormones and factors responsible for the proper physiological function of reproductive tract during the oestrous cycle and pregnancy. In this study, we investigated the pattern of PGF(2alpha) and PGE(2) secretion from cultured porcine endometrial cells at different days of the oestrous cycle. Epithelial and stromal cells were isolated by differential enzymatic digestion on days 6-8, 10-12 and 14-16. After attachment cells were incubated for 3 and 24 h to estimate PGF(2alpha) and PGE(2) output. The purity of culture was 85-90% for epithelial and 95-98% for stromal cells as determined by immunofluorescent staining. Release of PGF(2alpha) and PGE(2) was affected by cell type, days of the oestrous cycle and the time of incubation. After 3 h of incubation epithelial cells secreted more PGF(2alpha) than PGE(2) during all studied periods of the oestrous cycle (p < 0.01 and p < 0.001, respectively), whereas stromal cells released more PGE(2) (p < 0.01) on days 10-12 and 14-16. Longer incubation of stromal cells revealed that PGF(2alpha) output tended to overcome PGE(2) on days 10-16. The lowest secretion of prostaglandins was observed on days 6-8 in both cell types. The highest secretion of PGF(2alpha) from epithelium was measured on days 10-12 after 24 h of incubation when compared with other days studied (p < 0.001). In stromal cells, PGE(2) output increased on consecutive days studied (p < 0.001) after 3 h of incubation. The differential properties of endometrial cell types seem to play an important role in the profile of PGF(2alpha) and PGE(2) release before and during luteolysis. Described endometrial cells culture might serve as the model for further studies on the hormonal regulation of prostaglandin production in the pig.

Laryngeal manifestations of gastroesophageal reflux disease in children.

Although the association between gastroesophageal reflux disease (GERD) and laryngeal disorders in adults is well established there is still a lack of information concerning the true extent of the laryngeal complications of GERD in children. The aim of this study was to determine the laryngeal status of children with diagnosed GERD. We sought to identify the initial appearance of their larynges and then to determine the clinical response to antireflux therapy. GERD was recognized in 90/100 children examined. Using 24-h pH monitoring we found that most of the patients experienced episodes of gastroesophageal reflux during the daytime when they were in an upright position. The hallmark of GERD affecting the larynx in our group was posterior laryngitis, which is characterized by erythema of the mucous membrane overlying the arytenoid cartilages and the posterior mucosal wall of the glottis. The findings regarding the effectiveness of therapy were that, in children with severe laryngeal alterations, voice quality improved significantly after 12 weeks of antireflux treatment (p < 0.001) and laryngeal status was significantly better after 6 weeks of treatment (p < 0.001). This study provides evidence that gastroesophageal reflux in children is the underlying cause of inflammatory and morphological lesions, and that antireflux treatment is effective in reducing or eliminating these lesions.