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Preview: Molecular Human Reproduction - current issue

MHR: Basic science of reproductive medicine Current Issue





Published: Thu, 13 Jul 2017 00:00:00 GMT

Last Build Date: Sat, 05 Aug 2017 00:48:42 GMT

 






Resveratrol protects from lipopolysaccharide-induced inflammation in the uterus and prevents experimental preterm birth

2017-07-05

AbstractSTUDY QUESTIONIs resveratrol able to prevent the lipopolysaccharide (LPS)-induced preterm labor in 15-day pregnant BALB/c mice?SUMMARY ANSWERResveratrol prevented the LPS-induced onset of preterm labor in 64% of the cases and showed anti-inflammatory and tocolytic effects by downregulating COX-2 and iNOS expression and NOS activity, and by changing the uterine prostaglandin and endocannabinoid profiling.WHAT IS KNOWN ALREADYGenital tract infections by Gram-negative bacteria are a common complication in human pregnancy and have been shown to increase risk of preterm delivery. Bacterial LPS elicits a strong maternal inflammatory response that results in preterm delivery and fetal death in a murine model endotoxin-induced preterm labor.STUDY DESIGN, SIZE, DURATIONAn in vivo animal study was conducted. On Day 15 of pregnancy, mice received at 8:00 h a dose of vehicle (40% ethanol in saline solution) or resveratrol (3 mg/kg in vehicle) via oral gavage followed by two doses of LPS or vehicle administered intraperitoneally (i.p.), the first one at 10:00 h (0.17 mg/kg in 0.1 ml of sterile saline solution) and the second at 13:00 h (0.5 mg/kg in 0.1 ml of sterile saline solution). The mice were closely observed for any signs of morbidity (piloerection, decreased movement, and diarrhea), vaginal bleeding or preterm delivery. The beginning of preterm delivery was defined by early delivery of the first pup. Normal term labor occurs on Day 19 of gestation.PARTICIPANTS/MATERIALS, SETTING, METHODSTime of labor, pregnancy outcome and morphological features were evaluated after LPS and/or resveratrol administration. Uterine stripes were collected 5 h after the last LPS injection and prostaglandin and endocannabinoid profiling was analyzed by mass spectrometry. Nitric oxide synthase (NOS) activity was measured by radioconversion assay. Cyclooxygenase-2 (Cox-2) and 15-hydroxyprostaglandin dehydrogenase (15-Pgdh) mRNA levels were analyzed by RT-PCR whilst the protein expression of inducible nitric oxide synthase (iNOS), COX-1 and COX-2 were studied by western blot.MAIN RESULTS AND THE ROLE OF CHANCEIn vivo treatment of 15-day pregnant BALB/c mice with resveratrol prevented the LPS-induced preterm birth in 64% of the cases, whereas only 15% of mice with LPS alone escaped preterm birth. Treatment with resveratrol resulted in a reduced NOS activity (P < 0.05) in the uterus of LPS-treated mice. Similarly, resveratrol reduced the expression of LPS-induced pro-inflammatory agents such as iNOS (P < 0.05), COX-2 (P < 0.05), prostaglandin E2 (PGE2) (P < 0.05) and anandamide (AEA) (P < 0.05). Moreover, resveratrol administration resulted in changes in the uterine endocannabinoid profiling altered by LPS.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONSince our experimental design involves the use of mice, the extrapolation of the results presented here to humans is limited.WIDER IMPLICATIONS OF THE FINDINGSOur findings provide evidence for the tocolytic effects of resveratrol.STUDY FUNDING AND COMPETING INTEREST(S)Dr Ana María Franchi was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2013/0097) and by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2012/0061). Dr Heather B. Bradshaw was funded by NIH (DA006668). The authors have no competing interests.[...]



Infertility diagnosis has a significant impact on the transcriptome of developing blastocysts

2017-06-27

AbstractSTUDY QUESTIONIs the human blastocyst transcriptome associated with infertility diagnosis, specifically: polycystic ovaries (PCO), male factor (MF) and unexplained (UE)?SUMMARY ANSWERThe global blastocyst transcriptome was significantly altered in association with a PCO, MF and UE infertility diagnosis.WHAT IS KNOWN ALREADYInfertility diagnosis has an impact on the probability for a successful outcome following an IVF cycle. Limited information is known regarding the relationship between a specific infertility diagnosis and blastocyst transcription during preimplantation development.STUDY DESIGN, SIZE, DURATIONBlastocysts created during infertility treatment from patients with specific infertility diagnoses (PCO, MF and UE) were analyzed for global transcriptome compared to fertile donor oocyte blastocysts (control).PARTICIPANTS/MATERIALS, SETTING, METHODSSurplus cryopreserved blastocysts were donated with patient consent and institutional review board approval. Female patients were <38 years old with male patients <40 years old. Blastocysts were grouped according to infertility diagnosis: PCO (n = 50), MF (n = 50), UE (n = 50) and fertile donor oocyte controls (n = 50). Pooled blastocysts were lysed for RNA isolation followed by microarray analysis using the SurePrint G3 Human Gene Expression Microarray. Validation was performed on significant genes of interest using real-time quantitative PCR (RT-qPCR).MAIN RESULTS AND THE ROLE OF CHANCETranscription alterations were observed for all infertility etiologies compared to controls, resulting in differentially expressed genes: PCO = 869, MF = 348 and UE = 473 (P < 0.05; >2-fold). Functional annotation of biological and molecular processes revealed both similarities, as well as differences, across the infertility groups. All infertility etiologies displayed transcriptome alterations in signal transducer activity, receptor binding, reproduction, cell adhesion and response to stimulus. Blastocysts from PCO patients were also enriched for apoptotic genes while MF blastocysts displayed enrichment for genes involved in cancer processes. Blastocysts from couples with unexplained infertility displayed transcription alterations related to various disease states, which included mechanistic target of rapamycin (mTOR) and adipocytokine signaling. RT-qPCR validation confirmed differential gene expression for the following genes: BCL2 like 10 (BCL2L10), heat shock protein family A member 1A (HSPA1A), heat shock protein family A member 1B (HSPA1B), activating transcription factor 3 (ATF3), fibroblast growth factor 9 (FGF9), left-right determination factor 1 (LEFTY1), left-right determination factor 2 (LEFTY2), growth differentiation factor 15 (GDF15), inhibin beta A subunit (INHBA), adherins junctions associated protein 1 (AJAP1), cadherin 9 (CDH9) and laminin subunit alpha 4 (LAMA4) (P < 0.05; >2-fold).LARGE SCALE DATANot available due to participant privacy.LIMITATIONS, REASONS FOR CAUTIONBlastocyst samples for microarray analysis required pooling. While this allows for an overall average in each infertility etiology group and can reduce noise from sample-to-sample variation, it cannot give a detailed analysis of each blastocyst within the group.WIDER IMPLICATIONS OF THE FINDINGSUnderlying patient infertility diagnosis has an impact on the blastocyst transcriptome, modifying gene expression associated with developmental competence and implantation potential.STUDY FUNDING AND COMPETING INTEREST(S)No conflict of interest or outside funding provided.[...]



Improvement of implantation potential in mouse blastocysts derived from IVF by combined treatment with prolactin, epidermal growth factor and 4-hydroxyestradiol

2017-06-27

AbstractSTUDY QUESTIONCan supplementation of medium with prolactin (PRL), epidermal growth factor (EGF) and 4-hydroxyestradiol (4-OH-E2) prior to embryo transfer improve implantation potential in mouse blastocysts derived from IVF?SUMMARY ANSWERCombined treatment with PRL, EGF and 4-OH-E2 improves mouse blastocyst implantation rates, while alone, each factor is ineffective.WHAT IS KNOWN ALREADYBlastocyst dormancy during delayed implantation caused by ovariectomy is maintained by continued progesterone treatment in mice, and estrogen injection rapidly activates blastocysts to implantation-induced status in vivo. While the expression of many proteins is upregulated in implantation-induced blastocysts, selective proteolysis by proteasomes, such as estrogen receptor α (ESR1), occurs in implantation-induced blastocysts to achieve implantation-competent status. It is worth evaluating the proteins expressed during these periods to identify humoral factors that might improve the implantation potential of IVF-derived blastocysts because the poor quality of embryos obtained by IVF is one of the major causes of implantation failure.STUDY DESIGN, SIZE, DURATIONSuperovulated oocytes from ICR mice were fertilized with spermatozoa and then cultured in vitro in potassium simplex optimized medium (KSOM) without phenol red (KSOM-P) for 90–96 h. Blastocysts were treated with PRL (10 or 20 mIU/mL), EGF (5 or 10 ng/mL) or 4-OH-E2 (1 or 10 nM) in KSOM-P for 24 h.PARTICIPANTS/MATERIALS, SETTING, METHODSLevels of breast cancer 1 (BRCA1), EGF receptor (EGFR, also known as ERBB1), ERBB4, tubulointerstitial nephritis antigen-like 1 (TINAGL1) and ESR1 protein were examined with immunohistochemical analysis using immunofluorescence methods and confocal laser scanning microscopy. For embryo transfer, six blastocysts were suspended in HEPES-buffered KSOM-P medium and transferred into the uteri of recipient mice on the morning of Day 4 (0900–1000 h) of pseudopregnancy (Day 1 = vaginal plug). The number of implantation sites was then recorded on Day 6 using the blue dye method.MAIN RESULTS AND THE ROLE OF CHANCEPRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the trophectoderm (TE). While PRL treatment resulted in an increase in EGFR, EGF increased both EGFR and ERBB4 in the blastocyst TE. TINAGL1 in the TE was enhanced by 4-OH-E2, which also increased localization of this protein to the basement membrane. Treatment with PRL, EGF or 4-OH-E2 alone did not improve blastocyst implantation rates. Combined treatment with PRL, EGF and 4-OH-E2 resulted in increased levels of EGFR, ERBB4, TINAGL1 and BRCA1 in the TE, whereas ESR1 was not upregulated in the treated blastocysts. Furthermore, combined treatment with PRL, EGF and 4-OH-E2 improved blastocyst implantation rates versus control (P = 0.009).LARGE SCALE DATANot applicable.LIMITATIONS, REASONS FOR CAUTIONOur studies were carried out in a mouse model, and the conclusions were drawn from limited results obtained from one species. Whether the increase in EGFR, ERBB4 and TINAGL1 protein in the TE improves implantation potential of blastocysts needs to be further studied experimentally by assessing other expressed proteins. The influence of combined supplementation in vitro of PRL, EGF and 4-OH-E2 on implantation also requires further examination and optimization in human blastocysts before it can be considered for clinical use in ART.WIDER IMPLICATIONS OF THE FINDINGSEnhanced implantation potential by combined treatment with PRL, EGF and 4-OH-E2 appears to result in the upregulation of at least two distinct mechanisms, namely signaling via EGF receptors and basement membrane formation during the peri-implantation period in mice. While PRL, EGF and 4-OH-E2 each promoted BRCA1 protein level in the TE, treatment with each alone did not improve blastocyst implantation. Therefore, BRCA1 protein appears to be unnecessary for the attachment reaction in blastocysts in mice Combined supplementation of PRL, EGF and 4-OH-E2 m[...]



The transcriptome of human oocytes is related to age and ovarian reserve

2017-06-23

Abstract
STUDY QUESTION
How does the human oocyte transcriptome change with age and ovarian reserve?
SUMMARY ANSWER
Specific sets of human oocyte messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) are affected independently by age and ovarian reserve.
WHAT IS KNOWN ALREADY
Although it is well established that the ovarian reserve diminishes with increasing age, and that a woman's age is correlated with lower oocyte quality, the interplay of a diminished reserve and age on oocyte developmental competence is not clear. After maturation, oocytes are mostly transcriptionally quiescent, and developmental competence prior to embryonic genome activationrelies on maternal RNA and proteins.
STUDY DESIGN, SIZE, DURATION
A total of 36 vitrified/warmed MII oocytes from 30 women undergoing oocyte donation were included in this study, processed and analyzed individually.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Total RNA from each oocyte was independently isolated, amplified, labeled, and hybridized on HTA 2.0 arrays (Affymetrix). Data were analyzed using TAC software, in four groups, each including nine oocytes, according to the woman's age and antral follicular count (AFC) (mean ± SD): Young with High AFC (YH; age 21 ± 1 years and 24 ± 3 follicles); Old with High AFC (OH; age 32 ± 2 years and 29 ± 7 follicles); Young with Low AFC (YL; age 24 ± 2 years and 8 ± 2 follicles); Old with Low AFC (OL; age 34 ± 1 years and 7 ± 1 follicles). qPCR was performed to validate arrays.
MAIN RESULTS AND THE ROLE OF CHANCE
We identified a set of 30 differentially expressed mRNAs when comparing oocytes from women with different ages and AFC. In addition, 168 non-coding RNAs (ncRNAs) were differentially expressed in relation to age and/or AFC. Few mRNAs have been identified as differentially expressed transcripts, and among ncRNAs, a set of Piwi-interacting RNAs clusters (piRNAs-c) and precursor microRNAs (pre-miRNAs) were identified as increased in high AFC and old groups, respectively. Our results indicate that age and ovarian reserve are associated with specific ncRNA profiles, suggesting that oocyte quality might be mediated by ncRNA pathways.
LARGE SCALE DATA
Data can be found via GEO accession number GSE87201.
LIMITATIONS, REASONS FOR CAUTION
The oldest woman included in the study was 35 years old, thus our results cannot readily be extrapolated to women older than 35 or infertile women.
WIDER IMPLICATIONS OF THE FINDINGS
We show, for the first time, that several non-coding RNAs, usually regulating DNA transcription, are differentially expressed in relation to age and/or ovarian reserve. Interestingly, the mRNA transcriptome of in vivo matured oocytes remains remarkably stable across ages and ovarian reserve, suggesting the possibility that changes in the non-coding transcriptome might regulate some post-transcriptional/translational mechanisms which might, in turn, affect oocyte developmental competence.
STUDY FUNDING AND COMPETING INTEREST(S)
This work was supported by intramural funding of Clinica EUGIN and by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia. J.H. and A.S. are employees of Affymetrix, otherwise there are no competing interests.



Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines

2017-06-17

AbstractSTUDY QUESTIONIs extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation?SUMMARY ANSWERExtracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling.WHAT IS KNOWN ALREADYIn order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process.STUDY DESIGN, SIZE, DURATIONBovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA-AM (intracellular Ca2+ chelator, 50 μM), EGTA (10 μM) and Probenecid (MRPs general inhibitor, 500 μM). In addition, assays for binding to oviductal epithelial cells and IVF were carried out to test the effect of cAMP compared with other known capacitant agents such as heparin (60 μg/ml) and bicarbonate (40 mM).PARTICIPANTS/MATERIALS, SETTING, METHODSStraws of frozen bovine semen (20–25 × 106 spermatozoa/ml) were kindly provided by Las Lilas, CIALE and CIAVT Artificial Insemination Centers. The methods used in this work include western blot, immunohistochemistry, flow cytometry, computer-assisted semen analysis, live imaging of Ca2+ and fluorescence scanning. At least three independent assays with bull samples of proven fertility were carried.MAIN RESULTS AND THE ROLE OF CHANCEIn the present study, we elucidate the molecular events induced by extracellular cAMP. Our results showed that external cAMP induces sperm capacitation, depending upon the action of PLC. Downstream, this enzyme increased ERK1-2 activation through PKC and elicited a rise in sperm Ca2+ levels (P < 0.01). Moreover, extracellular cAMP-induced capacitation also depended on the activity of sAC and PKA, and increased tyrosine phosphorylation, indicating that the nucleotide exerts a broad range of responses. In addition, extracellular cAMP-induced sperm hyperactivation and concomitantly increased the proportion of spermatozoa with high mitochondrial activity (P < 0.01). Finally, cAMP increased the in vitro fertilization rate compared to control conditions (P < 0.001).LARGE SCALE DATANone.LIMITATIONS, REASONS FOR CAUTIONThis is an in vitro study performed with bovine cryopreserved spermatozoa. Studies in other species and with fresh samples are needed to extrapolate these data.WIDER IMPLICATIONS OF THE FINDINGSThese findings strongly suggest an important role of extracellular cAMP [...]