Higher Concentrations of Essential Trace Elements in Women Undergoing IVF May Be Associated with Poor Reproductive Outcomes Following Single Euploid Embryo Transfer.

Essential trace elements are micronutrients whose deficiency has been associated with altered fertility and/or adverse pregnancy outcomes, while surplus may be toxic. The concentrations of eight essential trace elements were measured using inductively coupled mass spectrometry (ICP-MS) and assessed with respect to clinical in vitro fertilization (IVF) outcomes in a population of 51 women undergoing IVF with intracytoplasmic sperm injection (ICSI), pre-implantation genetic screening for aneuploidy (PGT-A), and single frozen euploid embryo transfer (SET/FET). Specifically, copper (Cu), zinc (Zn), molybdenum, selenium, lithium, iron, chromium, and manganese were quantified in follicular fluid and whole blood collected the day of vaginal oocyte retrieval (VOR) and in urine collected the day of VOR and embryo transfer. We found that the whole blood Cu/Zn ratio was significantly associated with superior responses to ovarian stimulation. Conversely, the whole blood zinc and selenium concentrations were significantly associated with poor ovarian response outcomes. Higher levels of whole blood zinc and selenium, urinary selenium, lithium, and iron had significant negative associations with embryologic outcomes following IVF. Regarding clinical IVF outcomes, higher urinary molybdenum concentrations the day of VOR were associated with significantly lower odds of implantation and live birth, while higher urinary Cu/Mo ratios on the day of VOR were associated with significantly higher odds of implantation, clinical pregnancy, and live birth. Our results suggest that essential trace element levels may directly influence the IVF outcomes of Spanish patients, with selenium and molybdenum exerting negative effects and copper-related ratios exerting positive effects. Additional studies are warranted to confirm these relationships in other human populations.

Deciphering the role of PGRMC2 in the human endometrium during the menstrual cycle and in vitro decidualization using an in vitro approach.

STUDY QUESTION: What is the human endometrial non-classical progesterone receptor (PGR) membrane component 2 (PGRMC2) expression pattern throughout the menstrual cycle and what role does it play during decidualization? SUMMARY ANSWER: Endometrial PGRMC2 expression fluctuates during the human menstrual cycle and is abundantly expressed in human endometrial stromal cells (hEnSCs) during in vitro decidualization, process where PGRMC2 is involved in embryo implantation-related pathways. WHAT IS KNOWN ALREADY: The endometrial response to progesterone is mediated by the classical and non-classical PGRs. We previously demonstrated that PGR membrane component 1 (PGRMC1) is critical for endometrial function, embryo implantation, and future placentation, however, the role(s) of PGRMC2, which is structurally similar to PGRMC1, have not been studied in the human endometrium. STUDY DESIGN, SIZE, DURATION: This prospective study comprehensively evaluated the endometrial expression of PGRMC2 throughout the human menstrual cycle and during in vitro decidualization of hEnSCs (isolated from 77 endometrial biopsies that were collected from 66 oocyte donors), using immunohistochemistry, RT-qPCR, western blot, transcriptomic, and proteomic analyses. In addition, functional analysis was carried out to validate the implication of PGRMC2 in hEnSCs during embryo invasion using an in vitro outgrowth model. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vitro decidualization of hEnSCs was induced using co-treatment with cAMP and medroxyprogesterone 17-acetate progestin, and evaluated by measuring prolactin by ELISA and F-actin immunostaining. RT-qPCR was employed to compare expression with other PGRs. To reveal the function of PGRMC2 during the decidualization process, we specifically knocked down PGRMC2 with siRNAs and performed RNA-seq and quantitative proteomics techniques (SWATH-MS). The common differentially expressed genes (DEGs) and proteins (DEPs) were considered for downstream functional enrichment analysis. Finally, to verify its implication in the trophoblast invasion, an outgrowth model was carried out where hEnSCs with silenced PGRMC2 were co-cultured with human trophoblastic spheroids (JEG-3) following in vitro decidualization. MAIN RESULTS AND THE ROLE OF CHANCE: In contrast to PGRMC1 and classical PGRs, endometrial PGRMC2 gene expression was significantly lower during the late- versus mid-secretory phase (P < 0.05). Accordingly, the elevated PGRMC2 protein abundance observed in the endometrial epithelial glands throughout the menstrual cycle dropped in the late secretory phase, when abundance decreased in all endometrial compartments. Nevertheless, PGRMC2 protein increased during the mid-secretory phase in stromal and glandular cells, and PGRMC2 mRNA (P < 0.0001) and protein (P < 0.001) levels were significantly enhanced in the membranes/organelles of decidualized hEnSCs, compared to non-decidualized hEnSCs. Notably, PGRMC1 and PGRMC2 mRNA were significantly more abundant than classical PGRs throughout menstrual cycle phases and in decidualized and non-decidualized hEnSCs (P < 0.05). RNA-seq and proteomics data revealed 4687 DEGs and 28 DEPs, respectively, in decidualized hEnSCs after PGRMC2 silencing. While functional enrichment analysis showed that the 2420 upregulated genes were mainly associated with endoplasmic reticulum function, vesicular transport, morphogenesis, angiogenesis, cell migration, and cell adhesion, the 2267 downregulated genes were associated with aerobic respiration and protein biosynthesis. The protein enrichment analysis showed that 4 upregulated and 24 downregulated proteins were related to aerobic respiration, cellular response, metabolism, localization of endoplasmic reticulum proteins, and ribonucleoside biosynthesis routes. Finally, PGRMC2 knockdown significantly compromised the ability of the decidualized hEnSCs to support trophoblast expansion in an outgrowth model (P < 0.05). LARGE-SCALE DATA: Transcriptomic data are available via NCBI's Gene Expression Omnibus (GEO) under GEO Series accession number GSE251843 and proteomic data via ProteomeXchange with identifier PXD048494. LIMITATIONS, REASONS FOR CAUTION: The functional analyses were limited by the discrete number of human endometrial biopsies. A larger sample size is required to further investigate the potential role(s) of PGRMC2 during embryo implantation and maintenance of pregnancy. Further, the results obtained in the present work should be taken with caution, as the use of a pure primary endometrial stromal population differentiated in vitro does not fully represent the heterogeneity of the endometrium in vivo, nor the paracrine communications occurring between the distinct endometrial cell types. WIDER IMPLICATIONS OF THE FINDINGS: The repression of endometrial PGRMC2 during the late- versus mid-secretory phase, together with its overexpression during decidualization and multiple implications with embryo implantation not only highlighted the unknown roles of PGRMC2 in female reproduction but also the potential to exploit PGRMC2 signaling pathways to improve assisted reproduction treatments in the future. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Instituto de Salud Carlos III (ISCIII) granted to F.D. (PI20/00405 and PI23/00860), co-funded by the European Union. Y.M.-L. was supported by a predoctoral research grant from Generalitat Valenciana (ACIF/2019/262). R.G.-M. was supported by Generalitat Valenciana (CIAPOT/2022/15). P.d.C. was supported by a predoctoral grant for training in research into health (PFIS FI20/00086) from the Instituto de Salud Carlos III. I.D.-H. was supported by the Spanish Ministry of Science, Innovation and Universities (FPU18/01550). A.P. was supported by the Instituto de Salud Carlos III (PFIS FI18/00009). This research was also supported by IVI Foundation-RMA Global (1911-FIVI-103-FD). The authors declare no conflict of interest.

Identification of miR-30c-5p microRNA in Serum as a Candidate Biomarker to Diagnose Endometriosis.

The diagnosis of endometriosis by laparoscopy is delayed until advanced stages. In recent years, microRNAs have emerged as novel biomarkers for different diseases. These molecules are small non-coding RNA sequences involved in the regulation of gene expression and can be detected in peripheral blood. Our aim was to identify candidate serum microRNAs associated with endometriosis and their role as minimally invasive biomarkers. Serum samples were obtained from 159 women, of whom 77 were diagnosed with endometriosis by laparoscopy and 82 were healthy women. First, a preliminary study identified 29 differentially expressed microRNAs between the two study groups. Next, nine of the differentially expressed microRNAs in the preliminary analysis were evaluated in a new cohort of 67 women with endometriosis and 72 healthy women. Upon validation by quantitative real-time PCR technique, the circulating level of miR-30c-5p was significantly higher in the endometriosis group compared with the healthy women group. The area under the curve value of miR-30c-5p was 0.8437, demonstrating its diagnostic potential even when serum samples registered an acceptable limit of hemolysis. Dysregulation of this microRNA was associated with molecular pathways related to cancer and neuronal processes. We concluded that miR-30c-5p is a potential minimally invasive biomarker of endometriosis, with higher expression in the group of women with endometriosis diagnosed by laparoscopy.

Proteomic Profiling Identifies Candidate Diagnostic Biomarkers of Hydrosalpinx in Endometrial Fluid: A Pilot Study.

Hydrosalpinx is a fluid occlusion and distension of the fallopian tubes, often resulting from pelvic inflammatory disease, which reduces the success of artificial reproductive technologies (ARTs) by 50%. Tubal factors account for approximately 25% of infertility cases, but their underlying molecular mechanisms and functional impact on other reproductive tissues remain poorly understood. This proteomic profiling study applied sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to study hydrosalpinx cyst fluid and pre- and post-salpingectomy endometrial fluid. Among the 967 proteins identified, we found 19 and 17 candidate biomarkers for hydrosalpinx in pre- and post-salpingectomy endometrial fluid, respectively. Salpingectomy significantly affected 76 endometrial proteins, providing insights into the enhanced immune response and inflammation present prior to intervention, and enhanced coagulation cascades and wound healing processes occurring one month after intervention. These findings confirmed that salpingectomy reverses the hydrosalpinx-related functional impairments in the endometrium and set a foundation for further biomarker validation and the development of less-invasive diagnostic strategies for hydrosalpinx.

Bioaccumulation of Non-Essential Trace Elements Detected in Women's Follicular Fluid, Urine, and Plasma Is Associated with Poor Reproductive Outcomes following Single Euploid Embryo Transfer: A Pilot Study.

This study aims to determine the association of non-essential trace elements present in follicular fluid, plasma, and urine with reproductive outcomes of women undergoing intracytoplasmic sperm injection (ICSI), preimplantation genetic testing for aneuploidies (PGT-A) and single frozen euploid embryo transfer (SET/FET). This single-center, prospective cohort study included sixty women undergoing ICSI with PGT-A and SET/FET between 2018 and 2019. Urine, plasma and follicular fluid samples were collected on the vaginal oocyte retrieval day to simultaneously quantify ten non-essential trace elements (i.e., Ba, Sr, Rb, Sn, Ti, Pb, Cd, Hg, Sb, and As). We found several associations between the levels of these non-essential trace elements and clinical IVF parameters. Specifically, the increased levels of barium in follicular fluid were negatively associated with ovarian function, pre-implantation development and embryo euploidy, while elevated strontium concentrations in this biofluid were negatively associated with impaired blastulation and embryo euploidy. Elevated plasma strontium levels were negatively associated with ovarian function, fertilization and blastulation. Enhanced presence of other trace elements in plasma (i.e., rubidium and arsenic) were associated with a diminished ovarian function and limited the number of recovered oocytes, mature oocytes and zygotes, respectively. Fully adjusted models suggested significantly lower odds of achieving a live birth when increased concentrations of barium and tin were found in urine.

Phytoestrogens Present in Follicular Fluid and Urine Are Positively Associated with IVF Outcomes following Single Euploid Embryo Transfer.

The impact and safety of phytoestrogens, plant-derived isoflavones with estrogenic activity predominantly present in soy, on female reproductive health and IVF outcomes continues to be hotly debated. In this prospective cohort study, 60 women attending IVI-RMA New Jersey undergoing IVF with single frozen embryo transfer (SET/FET) of good-quality euploid blastocyst after PGT-A analysis were recruited. Concentrations of two phytoestrogens (daidzein and genistein) in follicular fluid (FF) and urine (U) were measured by UPLC-MSMS, both collected on vaginal oocyte retrieval day. These measurements correlated with IVF clinical outcomes. In models adjusted for age, BMI, race/ethnicity, and smoking status, higher FF phytoestrogen concentrations were significantly associated with higher serum estradiol, enhanced probability of implantation, clinical pregnancy, and live birth. Moreover, higher urine phytoestrogen concentrations were significantly associated with improved oocyte maturation and fertilization potential and increased probability of clinical pregnancy and live birth. Finally, higher FF and urine phytoestrogen concentrations were associated with a higher probability of live birth from a given IVF cycle. Our results suggest that dietary phytoestrogens improved reproductive outcomes of women undergoing IVF treatment. However, additional prospective studies are needed to optimize the use of phytoestrogens to further enhance reproductive outcomes and/or protect against reproductive insults.

The Impact of Essential Trace Elements on Ovarian Response and Reproductive Outcomes following Single Euploid Embryo Transfer.

Essential trace elements are required in extremely small amounts and obtained through diet. This research focuses on detecting major trace elements in different biofluids of sixty women undergoing ICSI with PGT-A and SET/FET at IVI-RMA, New Jersey, and assessing their impact on their IVF outcomes. Urine, plasma, and follicular fluid samples were collected on the vaginal oocyte retrieval day to measure the concentrations of eight essential trace elements (copper, zinc, molybdenum, lithium, selenium, manganese, chromium, and iron) using ICP-MS. After analysis, ovarian response and preimplantation outcomes had significant positive associations with both copper alone and the copper/zinc ratio in the follicular fluid and plasma, in addition to plasma manganese. Alternatively, elevated follicular fluid lithium concentrations were significantly associated with poor preimplantation outcomes while the urinary molybdenum concentration was significantly associated with a lower probability of implantation, clinical pregnancy, and live birth. Urinary lithium and chromium concentrations were significantly associated with a lower probability of achieving a live birth. Our results suggest that the essential trace elements present in follicular fluid, plasma, and urine of women are directly associated with their reproductive outcomes, with copper and manganese exerting positive effects and lithium and molybdenum exerting negative effects.

Association of blood cadmium and lead levels with self-reported reproductive lifespan and pregnancy loss: The national health and nutrition examination survey 1999-2018.

Cadmium and lead are known to interfere with the endocrine function. Thus, hormonally regulated processes such as menarche, menopause and pregnancy are likely influenced by chronic exposure to these metals. In US post-menopausal women, who already completed their reproductive lifespan, we evaluated the association between blood cadmium and lead levels with self-reported reproductive lifespan and personal history of pregnancy loss. We selected 5317 post-menopausal women participating in the National Health and Nutrition Examination Survey (NHANES), 1999-2018. Blood cadmium and lead levels were measured by inductively coupled plasma mass spectrometry. Reproductive lifespan was defined as the number of years between self-reported age at menarche and menopause. Personal history of pregnancy loss was defined as number of self-reported pregnancy losses out of the self-reported number of pregnancies. The fully adjusted mean difference in reproductive lifespan (95% confidence interval [CI]) comparing the 80th to the 20th percentiles of blood cadmium and lead distributions was, respectively, 0.50 (0.10, 0.91) and 0.72 (0.41, 1.03) years. Ever smoker showed stronger association of blood lead with reproductive lifespan. For self-reported pregnancy loss, the corresponding fully adjusted relative prevalence (95% CI) was 1.10 (0.93, 1.31) for cadmium and 1.10 (1.00, 1.21) for lead, and remained similar after additional adjustment for reproductive lifespan. In never smokers, the relative prevalence was 1.07 (1.04, 1.11) and 1.16 (1.05, 1.28) for blood cadmium and lead, respectively. These findings suggest that blood cadmium and lead exposures increase reproductive lifespan and prevalence of pregnancy loss in the general population. Additional studies are needed to improve the understanding of mechanisms and prevention potential of metals-related pregnancy outcomes.

Antioxidant Supplementation Alleviates Mercury-Induced Cytotoxicity and Restores the Implantation-Related Functions of Primary Human Endometrial Cells.

Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.

Comparative single-cell transcriptomic profiles of human androgenotes and parthenogenotes during early development.

OBJECTIVE: To unravel the differential transcriptomic behavior of human androgenotes (AGs) and parthenogenotes (PGs) throughout the first cell cycles, analyze the differential expression of genes related to key biologic processes, and determine the time frame for embryonic genome activation (EGA) in AGs and PGs. DESIGN: Laboratory study. SETTING: Private fertility clinic. PATIENT(S): Mature oocytes were retrieved from healthy donors and subjected to artificial oocyte activation using calcium ionophore and puromycin to generate PGs (n = 6) or enucleated and subjected to intracytoplasmic sperm injection to generate AGs (n = 10). INTERVENTION(S): Uniparental constructs at different early stages of development were disaggregated into constituent single cells (we suggest the terms parthenocytes and androcytes) to characterize the single-cell transcriptional landscape using next-generation sequencing. MAIN OUTCOMES MEASURE(S): Transcriptomic profiles comparison between different stages of early development in AGs and PGs. RESULT(S): The uniparental transcriptomic profiles at the first cell cycle showed 68 down-regulated and 26 up-regulated differentially expressed genes (DEGs) in PGs compared with AGs. During the third cell cycle, we found 60 up-regulated and 504 down-regulated DEGs in PGs compared with AGs. In the fourth cell cycle, 1,771 up-regulated and 1,171 down-regulated DEGs were found in PGs compared with AGs. The AGs and PGs had reduced EGA profiles during the first 3 cell cycles, and a spike of EGA at the fourth cell cycle was observed in PGs. CONCLUSION(S): Transcriptomic analysis of AGs and PGs revealed their complementary behavior until the fourth cell cycle. Androgenotes undergo a low wave of transcription during the first cell cycle, which reflects the paternal contribution to cell cycle coordination, mechanics of cell division, and novel transcription regulation. Maternal transcripts are most prominent in the third and fourth cell cycles, with amplification of transcription related to morphogenic progression and embryonic developmental competence acquisition. Regarding EGA, in PGs, a primitive EGA begins at the 1-cell stage and gradually progresses until the 4-cell stage, when crucial epigenetic reprogramming (through methylation) is up-regulated. In addition, our longitudinal single-cell transcriptomic analysis challenges that the zygote and early cleavage stages are the only totipotent entities, by revealing potential totipotency in cleavage-stage AGs and implications of paternal transcripts.

Endometrial Cells Acutely Exposed to Phthalates In Vitro Do Not Phenocopy Endometriosis.

Environmental factors that have been linked to an increased endometriosis risk include exposure to di-(2-ethylhexyl)-phthalate (DEHP), an endocrine disruptor. This study aims to investigate whether DEHP in vitro exposure in primary endometrial stromal cells (EnSC), primary endometrial epithelial cells (EnEC), and the human endometrial adenocarcinoma cell line Ishikawa properly mimics alterations described in the eutopic endometrium of women with endometriosis. Primary EnSC and EnEC, isolated from six fertile egg donors, and Ishikawa cells were exposed to DEHP (0.1, 1, and 10 µM) and were assessed for viability, endometriosis markers (IL-6, VEGF-A, HOXA10, EZH2, and LSD1), steroid receptor gene expressions (ER-1, ER-2, PR-T, PR-B, and PGRMC1), and invasive capacity. Viability after 72 h of DEHP exposure was not significantly affected. None of the endometriosis markers studied were altered after acute DEHP exposure, nor was the expression of steroid receptors. The invasive capacity of EnSC was significantly increased after 10 µM of DEHP exposure. In conclusion, acute DEHP exposure in primary endometrial cells does not fully phenocopy the changes in the viability, expression of markers, or steroidal receptors described in endometriosis. However, the significant increase in EnSC invasiveness observed after DEHP exposure could be a link between DEHP exposure and increased endometriosis likelihood.

Mercury impairs human primary endometrial stromal cell function†.

Heavy metal exposures could compromise endometrial cells. Although studies assessed mercury toxicity in cell lines, limited data are available on the concentration of mercury that damage human endometrial stromal cells (hEnSCs) and alter endometrial function. This research aims to study the effects of mercury exposure on cell viability and functional features of hEnSCs. Primary hEnSCs were isolated from 23 endometrial biopsies obtained from healthy donors. After in vitro mercury exposure cell viability of hEnSCs was evaluated via tetrazolium salt metabolism and oxidative stress was assessed by 2', 7'-dichlorofluorescin diacetate assay. hEnSCs were decidualized in vitro in the presence of mercury (0, 25, 50, 75, 250, and 350 nM). Decidualization was evaluated based on prolactin and insulin-like growth factor-binding protein (IGFBP1) secretion and cytoskeletal rearrangement (F-actin staining). Cell proliferation and apoptosis were evaluated by Ki67 immunostaining and TUNEL assay. Mercury doses of 250 nM (P = 0.028) and 500 nM (P = 0.026) increased reactive oxygen species production in hEnSCs after 24 h. Cell viability significantly decreased after 48 h and 72 h (P < 0.05) of mercury exposure at 500 nM. After in vitro decidualization and mercury treatment, decidual hEnSCs showed a dose-dependent decrease in prolactin and IGFBP1 secretion, particularly at 350 nM (P = 0.016). Cell proliferation was decreased in hEnSCs treated with 350 nM mercury (P < 0.001); an increase in apoptosis followed a dose-dependent trend in non-decidual and decidual hEnSCs. These findings support that mercury-induced damage could be due to an increase in ROS production.

Deciphering the Role of PGRMC1 During Human Decidualization Using an In Vitro Approach.

CONTEXT: Non-classical membrane progesterone receptor (mPRs) and progesterone receptor membrane component 1 (PGRMC1) expression have been detected in endometrium, but their role in decidualization had not yet been investigated. We previously demonstrated PGRMC1 downregulation in receptive endometrium and that its overexpression inhibits decidualization. Furthermore, during decidualization, PGRMC1 mainly interacts with proteins involved in biosynthesis, intracellular transport, and mitochondrial activity. OBJECTIVE: To determine PGRMC1 and mPRs signaling role during decidualization. METHODS: Isolated primary endometrial stromal cells (EnSC) were decidualized in vitro in the presence of classic stimuli (E2 + P4), PGRMC1 inhibitor (AG205), or membrane-impermeable P4 (P4-BSA). Endometrial biopsies were obtained from 19 fertile oocyte donors attending the IVI-Valencia in vitro fertilization (IVF) clinic. EnSC decidualization was evaluated by prolactin ELISA and F-actin immunostaining. Progesterone receptor localization was evaluated by immunofluorescence. EnSC transcriptomic profiles were analyzed by microarray technology. RESULTS: PGRMC1 inhibition during EnSC decidualization (AG205dEnSC) does not interfere with EnSC cytoskeletal rearrangements and prolactin secretion. However, global transcriptional profiling revealed more differentially expressed genes in AG205dEnSC than in dEnSC, compared with nondecidualized EnSC (ndEnSC). In silico analysis showed that PGRMC1 inhibition upregulated more genes related to metabolism, molecular transport, and hormonal biosynthesis compared with control dEnSC. EnSC decidualized in the presence of P4-BSA showed a similar behavior as ndEnSC in terms of morphological features, absence of prolactin secretion, and transcriptomic pattern. CONCLUSION: Our findings associate PGRMC1 to hormonal biosynthesis, metabolism, and vesicular transport-important cellular functions for dEnSC supporting pregnancy. Activation of membrane P4 receptor signaling alone was unable to induce downstream effects needed for proper decidualization.

Differential sperm proteomic profiles according to pregnancy achievement in intracytoplasmic sperm injection cycles: a pilot study.

PURPOSE: To describe the proteomic profiles in semen samples and define the differences in sperm proteomic profiles among samples that ultimately achieved pregnancy (P) via intracytoplasmic sperm injection (ICSI) in an oocyte donation program and those that were unsuccessful (NP). METHODS: Prospective, analytical, observational nested case and control study evaluating the proteomic profile of spermatozoa from patients' ejaculates where pregnancies were (group pregnant (P), n= 4) or were not (group non-pregnant (NP), n=4) achieved after ICSI in an oocyte donation program aiming to standardize female factor. Proteins were separated and analyzed by means of SWATH-MS) and compared between P/NP groups to identify sperm biomarkers of fertility/infertility. Proteins are available via ProteomeXchange. RESULTS: We identified and quantified 2228 proteins, with 37 significantly higher in the P group and 16 higher in NP. Enrichment analysis revealed that the increased proteins in P group sperm were related to motility, anaerobic metabolism, and protein biosynthesis functions, while the increased proteins in the NP group were involved in protein biosynthesis, protein folding, aerobic metabolism, and signal transduction, all of which are functions not previously described as influencing sperm success. Some proteins identified (e.g., SLC2A3, or CD81) are located in the cell membrane and thus may be employed to select spermatozoa by magnetic-activated cell sorting (MACS). CONCLUSION(S): This work revealed differences in the proteomic profiles of sperm samples successful in achieving pregnancy and those that were not, expanding our understanding of sperm function and infertility-related molecular markers, and enabling the future development of male fertility diagnostic tools and therapies.

Novel nonclassic progesterone receptor PGRMC1 pulldown-precipitated proteins reveal a key role during human decidualization.

OBJECTIVE: To investigate PGRMC1-precipitating proteins in human endometrial stromal cells (ESC) to understand its role during in vitro decidualization. DESIGN: Prospective observational study. SETTING: Academic fertility center. PATIENT(S): Fifteen fertile oocyte donors. INTERVENTION(S): Isolated ESCs decidualized in vitro and used in pulldown assays. MAIN OUTCOME MEASURE(S): GST-PGRMC1-precipitated proteins identified in nondecidualized ESC (ndESC) and ESC decidualized via a long (8 days) or short (4 days) decidualization protocol (dESC). RESULT(S): Using pulldown assays and mass spectrometry, decidualization was evaluated by prolactin secretion (ELISA) and cytoskeleton morphology (F-actin staining). The protein interactions were validated by colocalization and coimmunoprecipitation. The pulldown and mass spectrometry analysis identified 21, 24, and 24 new significant GST-PGRMC1-precipitated proteins in ndESC, long dESC, and short dESC, respectively, compared with controls. The functional annotation analysis categorized these proteins mainly into endomembrane system and mitochondria cellular components, both related to adenosine triphosphate (ATP) generation and transport activity, protein biosynthesis and posttranslational processing, vesicle trafficking, and protection against oxidative stress activities. Monoamine oxidase B (MAOB) and B-cell receptor-associated protein 31 (BAP31) were identified in dESC from both decidualization protocols. PGRMC1-MAOB/BAP31 interactions were confirmed by immunofluorescence and coimmunoprecipitation in dESC. CONCLUSION(S): Novel GST-PGRMC1-precipitated proteins discovered in ESC suggest that this protein is implicated in deep remodeling of ESC during decidualization and aggregates mainly with proteins involved in biosynthesis, intracellular transport, and mitochondrial activity.

Phytoestrogen exposure alters endometrial stromal cells and interferes with decidualization signaling.

OBJECTIVE: To investigate whether phytoestrogens (genistein and daidzein) alter in vitro decidualization of human endometrial stromal cells (ESCs). DESIGN: Isolated primary ESCs were exposed to phytoestrogens and decidualized in vitro. SETTING: Academic fertility center. PATIENT(S): Twenty fertile oocyte donors attending the IVI Valencia clinic. INTERVENTION(S): Treatment of ESC with phytoestrogens at 0, 10, 20, 50, and 100 µM. MAIN OUTCOME MEASURE(S): The ESC proliferation was analyzed by MTS assay. In vitro decidualization was induced in the presence of phytoestrogens by medroxyprogesterone acetate/cyclic adenosine 3':5' monophosphate and evaluated by prolactin (PRL) ELISA and F-actin immunostaining. The Ki67 proliferative marker was analyzed by immunofluorescence. The ESC apoptosis was assessed by annexin V/propidium iodide detection using flow cytometry. Estrogen (ERß) and P receptor (PR) localization were evaluated by immunofluorescence. RESULT(S): The ESC exposed to 0, 19, 20, 50, and 100 µM of genistein, daidzein, and genistein + daidzein showed a dose-dependent proliferation decrease. After 48-96 hours of culture, this reduction was significant in the presence of 50 µM of phytoestrogens versus 10 µM untreated ESC. The ESC decidualized in the presence of phytoestrogens did not rearrange their cytoskeletons and showed a significant decrease in PRL secretion compared with untreated decidualized ESCs (dESCs). However, phytoestrogens did not alter proliferative status or the percentage of viable/apoptotic cells in dESC compared with untreated dESC. During decidualization, phytoestrogens induced the same nuclear translocation of ERß and PR as the control dESC. CONCLUSION(S): This study reveals that high doses of phytoestrogens could affect the in vitro decidualization process.

Sistema de trabajo con los cuadros directivos del Estado y Gobierno cubanos / Work system with the directive cadres of the Cuban State and Government

En este artículo se exponen las consideraciones relacionadas con la política de cuadros de dirección, aprobadas en la Primera Conferencia Nacional del Partido Comunista de Cuba. Además se analizan el sistema de trabajo con los cuadros directivos del Estado y del Gobierno cubanos y la vigencia de dicho tema a través del pensamiento de Raúl Castro Ruz. Finalmente se concluye con una frase de este líder de la Revolución, que resume todo el trabajo necesario a realizar con los cuadros para lograr mayores resultados, y que debe ser guía de acción para todos los dirigentes.

In this work the considerations related to the policy of direction cadres, approved in the First National Conference of the Cuban Communist Party are exposed. Besides, the work system with the directive cadres of the Cuban State and Government and the validity of this topic through Raúl Castro Ruz `s thought are analyzed. Finally it is concluded with a sentence of this Revolution leader that summarizes the whole necessary work to be carried out with the cadres to achieve higher results, and that should be action guide to all the leaders.

Resultados de un trienio del Programa Ramal Gestión, formación y desarrollo del capital humano para la salud / Results during three years the Branch Program Management, formation and development of the human capital for health

Introducción: Luego de 3 años de iniciado el Programa Ramal Gestión Formación y Desarrollo del capital humano para la salud, se realiza un estudio descriptivo del comportamiento de los resultados. En cada una de las convocatorias del 2009 al 2011, se realizó un exhaustivo análisis del comportamiento tras la evaluación de los proyectos recepcionados procedentes de las diferentes instituciones de salud a nivel nacional. En cada convocatoria se exploraron: proyectos presentados y aprobados, según línea del programa y por tipo de centro y provincia. La información se obtuvo por revisión documental de las estadísticas del programa, resultados anuales e informes de los evaluadores. Se tuvo en cuenta la opinión de un grupo de expertos del programa, para identificar de forma cualitativa los factores que influyen en la calidad y cantidad de los proyectos. Entre los resultados se encuentra con un número total de 224 proyectos enviados, y aprobados 50 percent, en diferentes líneas temáticas entre las que se destacan los procesos formativos de recursos humanos y el desarrollo profesional posgraduado. Las provincias con mayor número de proyectos presentados y aprobados son: Pinar del Rio, Villa Clara, Sancti Spíritus y Granma y los principales resultados demuestran la baja calidad metodológica en su elaboración

Introduction: After three years of the Branch Program of Management, Formation and Development of Human Resources in Health, a descriptive study of the results was carried out. In each of the official calls, a thorough analysis of the behavior of the program was made after evaluating the projects sent by several health institutions nationwide. There were also explored the submitted and approved projects according to the line of program and per type of center and province. The information from the documentary review of the program statistics, annual results and the evaluators' reports was obtained. The opinion of a group of experts in the program was taken into account, in order to qualitatively identify the factors affecting the quality and the quantity of the projects. Among the results were a total number of 224 delivered programs, of which 50 por ciento was approved, in various thematic lines in which the formative processes of human resource and the postgraduate professional development stand out. The provinces with the highest number of projects submitted and approved were Pinar del Rio, Villa Clara, Sancti Spiritus and Granma, and the main results showed the low methodological quality of the preparation of these projects

Comportamiento del Programa Ramal Gestión, Formación y Desarrollo del capital humano para la salud / Behaviour of the Branch Program Management, formation and development of the human capital for health

El Programa Ramal Gestión, Formación y Desarrollo del Capital Humano para la Salud fue aprobado por el CITMA en el 2008 y realizó su primera convocatoria en el 2009. Con el objetivo de describir el comportamiento de este programa en el período 2009-2010, se realizó un estudio descriptivo. En cada convocatoria se exploraron: proyectos presentados y aprobados, proyectos presentados según línea del programa y los proyectos por tipo de centro y provincia. La información se obtuvo por revisión documental de las estadísticas del programa e informes de los evaluadores, además se trabajó con un grupo de expertos del programa, seleccionada la muestra por conveniencia, en la cual por técnica cualitativa fueron identificados los factores que han influido en la calidad y cantidad de los proyectos y fueron actualizadas las prioridades de investigación del programa. Como resultados se obtuvieron que en la primera convocatoria (2009), en 8 líneas temáticas se recibieron 119 proyectos, de ellos aprobados el 69,7 por ciento, resultaron con mayor número de proyectos enviados las provincias de Pinar del Río, La Habana y Ciego de Ávila. En la segunda convocatoria (2010), fueron procesados 53 proyectos con 43,3 por ciento de aprobados, y se mantiene con mayor número de envíos la provincia Pinar del Río. Se concluye que existen dificultades identificadas que han influido en el desarrollo del programa como son: baja cantidad y calidad, poca divulgación, desconocimiento del programa ramal, convocatoria con plazos de entrega muy cortos y baja calidad en la elaboración de los proyectos

The branch program called Management, formation and development of the human capital for health care was approved by the Ministry of Science, Technology and Environment in 2008 and it was announced for the first time in 2009. A descriptive study was conducted to describe the behaviour of this program in the 2009-2010 period. Each announcement explored submitted and approved projects, submitted projects according to the program´s lines and projects per type of center and province. Data were gathered from documentary review of the program´s statistics and the reports of evaluators; additionally, a group of experts in the program participated. The sample was selected by convenience in which the qualitative technique managed to identify the influential factors on the quality and quantity of projects; also the research priorities of the program were updated. The results yielded that the in first announcement (2009) with 8 thematic lines, 119 projects were received of which 69,7 percent was approved. The highest numbers of projects came from Pinar del Río, La Habana and Ciego de Avila provinces. In the second announcement (2010), 53 projects were processed and 43,3 percent of them approved and once again Pinar del Río was the first province in terms of number of projects sent. It was concluded that there are difficulties that influenced in the development of this program such as low quantity and quality, poor dissemination of information, the branch program was mostly unknown, the announcements gave very short periods of time for project submission and low quality in preparation of projects