Progesterone modulates extracellular heat-shock proteins and interlukin-1ß in human choriodecidual after Escherichia coli infection.

INTRODUCTION: Chorioamnionitis is an adverse condition in human pregnancy caused by many bacterial pathogens including Escherichia coli (E. coli); which has been associated with higher risk of preterm birth. We recently reported that human maternal decidua (MDec) tissue responds to E. coli infection by secreting extracellular heat-shock proteins (eHsp)-60, -70 and interlukin-1ß (IL-1ß). Previous studies have shown that progesterone (P4) regulates the immune response, but it is unknown whether P4 inhibits the secretion of eHsp. The aim of this investigation was to determine the role of P4 on the secretion of eHsp-27, -60, -70 and IL-1ß in MDec after 3, 6, and 24 h of E. coli infection. METHODS: Nine human feto-maternal interface (HFMi) tissues were included and mounted in the Transwell culture system. Only the maternal decidua (MDec) was stimulated for 3, 6 and 24 h with E. coli alone or in combination with progesterone and RU486. After each treatment, the HFMi tissue was recovered to determine histological changes and the culture medium recovered to evaluate the levels of eHsp-27, -60, -70 and IL-1ß by ELISA and mRNA expression by RT-PCR. RESULTS: No structural changes were observed in the HFMi tissue treated with P4 and RU486. However, stimulation with E. coli produces diffuse inflammation and ischemic necrosis. E. coli induced infection decreases, in time- and dose-dependent manner, eHsp-27 and increases eHsp-60, eHsp-70 and IL-1ß levels. In contrast, incubation of HFMi tissue with E. coli + P4 reversed eHsp and IL-1ß secretion levels relative to E. coli stimulation group but not relative to the control group. The same profile was observed on the expression of eHsp-27 and eHsp-60. DISCUSSION: we found that progesterone modulates the anti-inflammatory (eHsp-27) and pro-inflammatory (eHsp-60 and eHsp-70) levels of eHsp induced by E. coli infection in human choriodecidual tissue. eHsp-60 and eHsp-70 levels were not completely reversed; maintaining the secretion of IL-1ß, which has been associated with adverse events during pregnancy.

The Effect of Metformin and Carbohydrate-Controlled Diet on DNA Methylation and Gene Expression in the Endometrium of Women with Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is an endocrine disease associated with infertility and metabolic disorders in reproductive-aged women. In this study, we evaluated the expression of eight genes related to endometrial function and their DNA methylation levels in the endometrium of PCOS patients and women without the disease (control group). In addition, eight of the PCOS patients underwent intervention with metformin (1500 mg/day) and a carbohydrate-controlled diet (type and quantity) for three months. Clinical and metabolic parameters were determined, and RT-qPCR and MeDIP-qPCR were used to evaluate gene expression and DNA methylation levels, respectively. Decreased expression levels of HOXA10, GAB1, and SLC2A4 genes and increased DNA methylation levels of the HOXA10 promoter were found in the endometrium of PCOS patients compared to controls. After metformin and nutritional intervention, some metabolic and clinical variables improved in PCOS patients. This intervention was associated with increased expression of HOXA10, ESR1, GAB1, and SLC2A4 genes and reduced DNA methylation levels of the HOXA10 promoter in the endometrium of PCOS women. Our preliminary findings suggest that metformin and a carbohydrate-controlled diet improve endometrial function in PCOS patients, partly by modulating DNA methylation of the HOXA10 gene promoter and the expression of genes implicated in endometrial receptivity and insulin signaling.

First-trimester plasma extracellular heat shock proteins levels and risk of preeclampsia.

Preeclampsia (PE) occurs annually in 8% of pregnancies. Patients without risk factors represent 10% of these. There are currently no first-trimester biochemical markers that accurately predict PE. An increase in serum 60- and 70-KDa extracellular heat shock proteins (eHsp) has been shown in patients who developed PE at 34 weeks. We sought to determine whether there is a relationship between first-trimester eHsp and the development of PE. This was a prospective cohort study performed at a third level hospital in Mexico City from 2019 to 2020. eHsp levels were measured during the first-trimester ultrasound in singleton pregnancies with no comorbidities. First-trimester eHsp levels and biochemical parameters of organ dysfunction were compared between patients who developed preeclampsia and those who did not. All statistical analyses and model of correlation (r) between eHsp and clinical parameter were performed using bootstrapping R-software. p-values <0.05 were considered significant. The final analysis included 41 patients. PE occurred in 11 cases. eHsp-60 and eHsp-70 were significantly higher at 12 weeks in patients who developed PE (p = 0.001), while eHsp-27 was significantly lower (p = 0.004). Significant differences in first-trimester eHsp concentration suggest that these are possible early biomarkers useful for the prediction of PE.

Differential proMMP-2 and proMMP-9 secretion in human pre-implantation embryos at day 5 of development.

Morphological development is the most common non-invasive criterion used to select in vitro human embryos for implantation. With this criterion, however, embryos in cellular arrest go unnoticed. A more accurate criterion is needed to improve the success rate of implantation. Extracellular matrix metalloproteases type 2 (MMP-2) and MMP-9 are key markers of embryonic development and the implantation process, according to various animal studies. The first objective of this study was to examine proMMP-2 and proMMP-9 activity in the culture media of human embryos with good morphological development. Secondly, the results of proMMP-2 and proMMP-9 activity in the culture medium were compared between pregnant and non-pregnant. Forty-two patients were approved by the Ethics and Research Committees of the Instituto Nacional de Perinatología in México City hospital, based on institutional inclusion criteria for in vitro fertilization. On day 5 of development, embryos were transferred to patients, and the culture media secretion profile of proMMP-2 and proMMP-9 activity was determined by substrate gel zymography. After analysis of embryo sac development, each patient was assigned to the pregnant (n=17) or non-pregnant (n=25) group. Our results demonstrate that proMMP-2 was active in the culture media corresponding to all 17 women achieving full-term pregnancy and proMMP-9 in the media corresponding to 11 of these women. Contrarily proMMP-2 and proMMP-9 were active in the culture media corresponding to 3 and 11 of the 25 non-pregnant patients, respectively. The clinical implications of this study suggest the activity evaluation of proMMP-2 and proMMP-9 in embryonic culture media on day 5 of development appears to be a reliable indicator of the quality of embryos and their capacity to establish a pregnancy.

MiR-191-5p is upregulated in culture media of implanted human embryo on day fifth of development.

BACKGROUND: Morphological features are the most common criteria used to select human embryos for transfer to a receptive uterine cavity. However, such characteristics are not valid for embryos in cellular arrest. Even aneuploid embryos can have normal morphology, and some euploid embryos have aberrant morphology. The aim of this study was to quantify the expression profile of hsa-miR-21-3p, -24-1-5p, -191-5p, and -372-5p in culture media on day 5 of in vitro embryo development, and compare the profiles of two groups of media classified by outcome: successful (n = 25) or unsuccessful (n = 25) implantation pregnancy. METHODS: Fifty patients were accepted in the Department of Reproductive Biology of a Hospital in México City, based on the Institutional inclusion criteria for in vitro fertilization. Embryos were transferred to the women on day 5 of cultivation, and the culture media were collected. RNA was isolated from each culture medium with TRIzol reagent, and microRNA (miRNA) expression was detected through RT-PCR with specific primers. Expression bands were quantified by reading optical density. RESULTS: There was a 5.2-fold greater expression of hsa-miR-191-5p in the pregnancy-related culture media (p ≤ 0.001) and a 1.6-fold greater level of hsa-miR-24-1-5p (p = 0.043) in the media corresponding to non-pregnant women. No significant difference existed between the two groups hsa-miR-21-3p (p = 0.38) or hsa-miR-372-5p (p = 0.41). CONCLUSIONS: Regarding adequate in vitro embryo development, hsa-miR-191-5p could possibly represent a positive biomarker, while has-miR-24-1-5p may indicate poor prognosis. This former miRNA modulates IGF2BP-1 and IGF2R, associated with the implantation window. On the other hand, hsa-miR-24-1-5p may be related to a poor prognosis of human embryo development.

Pseudomonas aeruginosa induces spatio-temporal secretion of IL-1ß, TNFα, proMMP-9, and reduction of epithelial E-cadherin in human alveolar epithelial type II (A549) cells.

Pseudomonas aeruginosa, is an opportunistic bacterium with a high prevalence in diverse pulmonary infections. Although several genes are involved in the system of resistance and evasion of the immunological response of the host, little is known about the inflammatory, degradative, and cell-binding response induced by P. aeruginosa in human lung alveolar epithelial cells. The purpose of this study was to determine the cytokine expression (IL-1ß and TNFα), pro matrix metalloproteinases activation (proMMP-2 and proMMP-9), and the effects on the cell-binding adhesion protein (E-cadherin) in an in vitro model of human lung alveolar epithelial cells. A549 cells were stimulated with a different number of colony-forming units of P. aeruginosa for 3, 6, and 24 hours. Subsequently, the culture medium was collected, IL-1ß and TNFα levels were evaluated by ELISA; proMMP-2 and -9 levels were determined by substrate gel zymography, and the MMP-9 and E-cadherin assessed by immunostaining of A549 cells. Our results demonstrated that P. aeruginosa induces mainly the secretion of TNFα, increases actMMP-9 level, and significantly reduces the level of E-cadherin in the A549 cells. In summary, the inflammatory/degradative process induced by P. aeruginosa modulates the expression of the E-cadherin protein. The probable clinical implications of this study suggest the use of inhibitors that reduce the degradative activity of proMMP-9 which will be further explored in the next phase of this study.

Expression of Membrane Progesterone Receptors in Eutopic and Ectopic Endometrium of Women with Endometriosis.

Endometriosis is one of the most frequent gynecological diseases in reproductive age women, but its etiology is not completely understood. Endometriosis is characterized by progesterone resistance, which has been explained in part by a decrease in the expression of the intracellular progesterone receptor in the ectopic endometrium. Progesterone action is also mediated by nongenomic mechanisms via membrane progesterone receptors (mPRs) that belong to the class II members of the progesterone and adipoQ receptor (PAQR) family. The aim of the present study was to evaluate the expression at mRNA and protein levels of mPR members in the eutopic and ectopic endometrium of women with endometriosis. Total RNA and total protein were isolated from control endometrium (17 samples), eutopic endometrium (17 samples), and ectopic endometrium (9 samples). The expression of PAQR7 (mPRα), PAQR8 (mPRß), and PAQR6 (mPRδ) at mRNA and protein levels was evaluated by RT-qPCR and Western blot, whereas PAQR5 (mPRγ) gene expression was evaluated by RT-qPCR. Statistical analysis between comparable groups was performed using one-way ANOVA followed by Tukey's multiple comparisons test with a confidence interval of 95 %. The analysis of gene expression showed that PAQR7 and PAQR5 expression was lower in both eutopic and ectopic endometrium as compared to the endometrium of women without endometriosis, whereas the expression of PAQR8 and PAQR6 was only reduced in eutopic endometrium. Furthermore, mPRα and mPRß protein content was decreased in the ectopic endometrium of women with endometriosis. Our results demonstrate a decrease in the expression and protein content of mPRs in eutopic and ectopic endometrium of patients with endometriosis, which could contribute to the progesterone resistance observed in patients with this disease.

Secretion of heat shock -60, -70 kD protein, IL-1ß and TNFα levels in serum of a term normal pregnancy and patients with pre-eclampsia development.

The extracellular heat shock proteins (eHsp) family act as molecular chaperones regulating folding, transporting protein and are associated with immune modulation in different physiological and pathological processes. They have been localized in different gestational tissues and their concentration in amniotic fluid and serum has been determined. In the present study, we proposed to determine the concentration of eHsp-60, -70, IL-1ß and TNFα in the serum of pregnant patients with 34 weeks of gestation with and without clinical evidences of preeclampsia (PE). Our results indicate significant increase of these markers in patients with PE with respect to healthy pregnant patients without active labor. Finally, the concentration of eHsp-60 and -70 correlated positively with the hepatic dysfunction markers uric acid, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) glutamic pyruvic transaminase (GPT), and inflammatory IL-1ß and TNFα response. In conclusion, our results demonstrate a strong associated between Hsp and marker of hepatic dysfunction.

Expresión de microRNAs asociados con corioamnionitis en suero materno / Expression of microRNAs associated with the development of chorioamnionitis in maternal serum

Resumen OBJETIVO Determinar el perfil de expresión de los miRNA-21, -106, -126 y -146 y la cuantificación de IL-1β en el suero de pacientes embarazadas sanas, a término, con y sin trabajo de parto activo y en pacientes con evidencias clínicas de corioamnionitis. MATERIALES Y MÉTODOS Estudio analítico, longitudinal y prolectivo efectuado en pacientes que ingresaron para control, seguimiento obstétrico y terminación del embarazo al Instituto Nacional de Perinatología de la Ciudad de México entre febrero de 2015 y agosto de 2016. Variables de estudio: pacientes embarazadas sanas, a término, con y sin trabajo de parto activo y pacientes con evidencias clínicas de corioamnionitis, edad materna y semanas de gestación. A cada paciente se le tomaron cinco mililitros de sangre periférica para centrifugación y recuperación de suero. La obtención del ARN se efectuó a partir de 500 µL de suero, al que se añadió el mismo volumen del reactivo de TRIzol. El cADN se sintetizó a partir de 3 ng de ARN mediante el ensayo de retrotranscripción. La expresión de los miRNAs se efectuó mediante reacción en cadena de la polimerasa (PCR) con iniciadores específicos. Los resultados se reportan en media ± desviación estándar y el análisis estadístico se llevó a cabo mediante la prueba de Mann-Whitney, con una diferencia significativa de p < 0.05. RESULTADOS Se estudiaron 45 pacientes embarazadas que se dividieron en tres grupos: 1) pacientes sanas a término, sin trabajo de parto activo (n = 15); 2) pacientes con trabajo de parto activo (n = 15); y 3) pacientes con evidencias clínicas de corioamnionitis (n = 15). En las embarazadas sanas, con trabajo de parto activo, el perfil de expresión del miR-126 se incrementó 1.23 veces (p ≤ 0.001) en tanto que el miR-21 y miR-146 disminuyeron 2.1 ( p ≤ 0.001) y 0.73 veces (p ≤ 0.001), respectivamente, y la concentración de IL-1β se incrementó 1.8 veces (p = 0.014) en relación con las pacientes sanas sin trabajo de parto. El perfil de expresión de estos miRNAs y de IL-1β cambió en las pacientes con evidencias clínicas de corioamnionitis. El miR-126 y miR-146 se incrementaron 1.31 (p ≤ 0.001) y 1.1 veces (p = 0.05); en tanto que el miR-21 disminuyó 1.4 veces (p ≤ 0.05) y la concentración de la IL-1β se incrementó 2.8 veces (p = 0.002) con respecto a las pacientes sin trabajo de parto activo. El miR-106 no mostró diferencias significativas entre los tres grupos de estudio. CONCLUSIONES En su conjunto, los resultados de este ensayo sugieren que el perfil de expresión entre miR-21, miR-126 y miR-146 podría considerarse marcador molecular de corioamnionitis. Para que esto pueda tomarse como patrón de referencia deberán emprenderse más ensayos que corroboren este patrón y evalúen su eficacia.

Abstract OBJECTIVE To determine the expression profile of miRNA-21, -106, -126 and -146 and to quantify the secretion of IL-1β in the serum of healthy pregnant patients at term with and without active labor and in patients with clinical evidences of chorioamnionitis. MATERIALS AND METHODS Analytical, longitudinal, and prolective study carried out in patients admitted for control, obstetric, and pregnancy resolution into the National Institute of Perinatology in Mexico City between February 2015 and August 2016. Study variables: healthy pregnant patients at term with and without active labor and patients with clinical evidence of chorioamnionitis, maternal and gestational age. Five milliliters of peripheral blood were taken from each patient, which was centrifuged, and the serum recovered. RNA was obtained from 500 μL of serum to which the same volume of TRIzol reagent was added. The cDNA was synthesized with 3 ng of RNA by the reverse transcription assay. The expression of the microRNAs was performed by polymerase chain reaction (PCR) with specific primers. The results are presented as mean ± standard deviation and statistical analysis was performed using the Mann-Whitney test with a significant difference of p<0.05. RESULTS In healthy pregnant patients with active labor, it was observed that the expression profile of miR-126 increased 1.23-fold (p≤0.001); while the miR-21 and miR-146 decreased 2.1- (pp≤0.001) and 0.73-fold (p≤0.001) respectively and the concentration of IL-1β increased 1.8-fold (p = 0.014) with respect to the healthy patients without labor. The expression profile of these miRNAs and IL-1β change in patients with clinical evidence of chorioamnionitis. The miR-126 and miR-146 increased 1.31- (p≤0.001) and 1.1-fold (p = 0.05); while miR-21 decreased 1.4-fold (p≤0.05) and the concentration of IL-1β increased 2.88-fold (p = 0.002) with respect to patients without active labor. The miR-106 did not show significant differences between the three study groups. CONCLUSIONS These results suggest that miRNA-21, -126 and -146 could be considered as molecular markers in the development of chorioamnionitis; however, more tests should be carried out to corroborate this pattern and evaluate its efficiency.

Tissue-specific human beta-defensins (HBD)-1, HBD-2 and HBD-3 secretion profile from human amniochorionic membranes stimulated with Candida albicans in a two-compartment tissue culture system.

BACKGROUND: During intrauterine infection, amniochorionic membranes represent a mechanical and immunological barrier against dissemination of infection. Human beta defensins (HBD)-1, HBD-2, and HBD-3 are key elements of innate immunity that represent the first line of defense against different pathogen microorganisms associated with preterm labor. The aim of this work was to characterize the individual contribution of the amnion (AMN) and choriodecidua (CHD) regions to the secretion of HBD-1, HBD-2 and HBD-3, after stimulation with Candida albicans. METHODS: Full-thickness human amniochorionic membranes were obtained after delivery by elective cesarean section from women at 37-40 wk of gestation with no evidence of active labor. The membranes were cultured in a two-compartment experimental model in which the upper compartment is delimited by the amnion and the lower chamber by the choriodecidual membrane. One million of Candida albicans were added to either the AMN or the CHD face or to both and compartmentalized secretion profiles of HBD-1, HBD-2, and HBD-3 were quantified by ELISA. Tissue immunolocalization was performed to detect the presence of HBD-1, -2, -3 in tissue sections stimulated with Candida albicans. RESULTS: HBD-1 secretion level by the CHD compartment increased 2.6 times (27.30 [20.9-38.25] pg/micrograms protein) when the stimulus with Candida albicans was applied only on this side of the membrane and 2.4 times (26.55 [19.4-42.5] pg/micrograms protein) when applied to both compartments simultaneously. HBD-1 in the amniotic compartment remained without significant changes. HBD-2 secretion level increased significantly in the CHD when the stimulus was applied only to this region (2.49 [1.49-2.95] pg/micrograms protein) and simultaneously to both compartments (2.14 [1.67- 2.91] pg/micrograms protein). When the stimulus was done in the amniotic compartment HBD-2 remained without significant changes in both compartments. HBD-3 remained without significant changes in both compartments regardless of the stimulation modality. Localization of immune-reactive forms of HBD-1, HBD-2, and HBD-3 was carried out by immunohistochemistry confirming the cellular origin of these peptides. CONCLUSION: Selective stimulation of amniochorionic membranes with Candida albicans resulted in tissue-specific secretion of HBD-1 and HBD-2, mainly in the CHD, which is the first region to become infected during an ascending infection.

[Perinatal result of an institutional assisted reproduction program]. / Resultado perinatal de un programa de reproducción asistida institucional.

BACKGROUND: Assisted reproductive technology manipulates masculine gametes, embryos and implantation. It also aids the known or unknown factor of sterility without having the base problem correction as a target. In vitro fertilization and embryo transfer are among these techniques. OBJECTIVE: To describe the overall outcome and the final perinatal offspring after in vitro fertilization cycle in an institutional third level hospital. MATERIALS AND METHODS: IVF cycles were retrospectively analyzed from October 1999 to May 2004. Several variables were described like: age, fertilization rate, implantation and pregnancy rate, fetal status, time of gestation during labor, miscarriage follicle-stimulating hormone rate and take-home baby rate. Patients underwent hypophyseal supression with GnRH analog, using a long luteal phase protocol and stimulated with recombinant FSH. Overall data is expressed as average +/- standard deviation and percentage. RESULTS: 365 cycles were analyzed in 314 patients, average age was of 34 +/- 3.7 years, tubal factor was diagnosed in 63.3%, fertilization rate was of 60.4%, implantation rate of 37.1%, per transfer pregnancy rate of 25.1%, per transfer live born rate of 21.7%, multiple pregnancy rate of 29.3%, miscarriage rate of 28% and ectopic pregnancy rate of 4.8%. In 87.8% of the cases caesarean operation was made; multiple pregnancy offspring weighted more than 1250 g in 70% of them; 70.5% was born after 32 weeks of pregnancy; 90% was born live and with good prognosis. Glucose metabolism alterations were the most frequent maternal complication. CONCLUSION: Although the results obtained were similar to those of assisted reproduction centers, it suggests improving multiple pregnancy rate and abortion rate.

[Estrogen role in the luteal phase support in in vitro fertilization with embryo transfer cycles]. / Papel de los estrógenos en el soporte de la fase lútea en ciclos de fertilización in vitro con transferencia de embriones.

OBJECTIVE: To evaluate the effectiveness of estradiol administration for luteal phase support and to describe the progesterone and estradiol behavior in vitro fertilization-embryo transfer luteal phase. MATERIAL AND METHODS: Patients undergoing in vitro fertilization-embryo transfer with controlled ovarian hyperstimulation and using gonadotropin releasing hormone agonist. They were divided at random into two groups: group 1 would receive progesterone alone, and group 2 would take estrogen and progesterone. Serum concentrations of estradiol and progesterone were measured on days 7 and 14 post-embryo transfer. RESULTS: We examined 52 patients; 24 received progesterone alone and 28 took estrogen and progesterone. Significantly higher estradiol and progesterone concentrations on day 14 were found in pregnant women. It was not on day 7. A significant increment of estrogen was found in the estrogen and progesterone group. Progesterone did not increase significantly. Pregnancy rate was the same in both groups. CONCLUSIONS: For patients undergoing in vitro fertilization-embryo transfer, the addition of estradiol to the progesterone support regimen does not have beneficial effects in terms of pregnancy rate. On day 7 neither progesterone nor estradiol are good predictors of pregnancy.