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Background: A significant association between endometrial vascularity and pregnancy has been shown in previous research, while poor vascularization was attributed to repeated implantation failure (RIF). One possible approach to enhance angiogenesis for successful implantation is endometrial scratching (ES). Objective: The purpose was to investigate endometrial responses to scratching by profiling angiogenesis-related gene expression in unexplained RIF participants. Materials and Methods: In this randomized controlled trial study, 20 infertile women with unexplained RIF were assigned to 2 groups by the balanced block randomization method (n = 10/each group): the intervention group (group A) (who received ES in the follicular phase) and the control group (group B). Endometrial biopsy was performed in the secretory phase. Gene expression profiling was performed using a polymerase chain reaction-array kit for human-angiogenic growth factors. The implantation and clinical pregnancy rates were also assessed. Results: Among the angiogenesis-promoting genes, FGF1, FGF13, FGF2, TGFA, ANG, ANGPT1, and VEGFA were significantly upregulated (p < 0.05). IL12A (an angiogenesis-inhibiting cytokine) was significantly upregulated (p < 0.01). In contrast, 15 genes with angiogenesis-related functions, including CXCL11, CXCL13, CXCL3, CXCL5, CXCL6, EREG, FIGF, FST, IL10, LEP, PPBP, PROK1, RHOB, TNF, and TYMP, were downregulated after ES. No significant differences were observed between the intervention (group A) and control (group B) groups in terms of implantation (43.75% vs. 28.57%) or clinical pregnancy rates (75% vs. 57.1%). Conclusion: ES induced significant alterations in the expression of angiogenesis-related genes, with notable up/downregulation of key angiogenic/antiangiogenic factors. These findings enhance our understanding of the molecular responses triggered by ES, underscoring the potential influence of ES on the complex processes of angiogenesis crucial for implantation.
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Among the primary objectives of contemporary assisted reproductive technology research are achieving the births of healthy singletons and improving overall fertility outcomes. Substantial advances have been made in refining the selection of single embryos for transfer, with the aim of maximizing the likelihood of successful implantation. The principal criterion for this selection is embryo morphology. Morphological evaluation systems are based on traditional parameters, including cell count and fragmentation, pronuclear morphology, cleavage rate, blastocyst formation, and various sequential embryonic assessments. To reduce the incidence of multiple pregnancies and to identify the single embryo with the highest potential for growth, invasive techniques such as preimplantation genetic screening are employed in in vitro fertilization clinics. However, new approaches have been suggested for clinical application that do not harm the embryo and that provide consistent, accurate results. Noninvasive technologies, such as time-lapse imaging and omics, leverage morphokinetic parameters and the byproducts of embryo metabolism, respectively, to identify noninvasive prognostic markers for competent single embryo selection. While these technologies have garnered considerable interest in the research community, they are not incorporated into routine clinical practice and still have substantial room for improvement. Currently, the most promising strategies involve integrating multiple methodologies, which together are anticipated to increase the likelihood of successful pregnancy.
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Objective: This study investigated the metabolic status of the spent culture media from embryos of patients with repeated implantation failure (RIF) undergoing in vitro fertilization-intracytoplasmic sperm injection cycles in comparison with the embryos from healthy fertile women. Methods: Metabolite levels in spent culture media were assessed and compared between embryos from RIF patients (n=35) and oocyte donors as controls (n=15). Protein levels of insulin-like growth factor 1 (IGF-1) were determined using Western blotting. Concentrations of glucose, pyruvate, and lactate were measured using spectrophotometry. Ionic colorimetric assay kits were utilized to analyze the concentrations of sodium, chloride, calcium, and magnesium ions. High-performance liquid chromatography was employed to measure the concentrations of glutamic acid, aspartic acid, methionine, phenylalanine, and histidine. Results: Glucose consumption and lactate secretion were higher in the control group than in the RIF group. The magnesium concentration was significantly higher in the control group than in the RIF group, but glutamic acid and aspartic acid concentrations were lower in the control group than in the RIF patients (p<0.05). The levels of IGF-1, sodium, calcium, chloride, methionine, histidine, and phenylalanine did not show statistically significant differences between the two groups. Conclusion: The metabolic profile of the culture medium of the embryos in the RIF group differed from that of the control group. These findings suggest potential factors that may affect implantation capacity in RIF patients and provide a new perspective on embryo selection.
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Sperm must pass a complex route in the female reproductive tract (FRT) to reach the fertilization site and join the oocyte. Thus, it should employ several mechanisms to survive against the female immune system, fertilize the oocyte, and successfully transmit paternal genes to the next generation. In addition to self-protection, sperm may be involved in the immune tolerance to the developing embryo and regulating the FRT for embryo implantation and subsequent pregnancy. Hence, this review intends to summarize the mechanisms that protect sperm in the FRT: including immunomodulatory factors that are carried by seminal plasma, cell-to-cell and molecular interaction of sperm with epithelial and immune cells of the FRT, high regulated secretions of inflammatory factors such as cytokines, chemokines, and growth factors, inducing immune tolerance to paternal antigens, and specialized expression of cell receptors and binding proteins. In most of these events sperm induces the FRT to protect itself by modulating immune responses for its own benefit. However, not all sperm in the semen are able to trigger the survival mechanisms and only high-quality sperm will overcome this challenge. A clear understanding of the molecular mechanisms that maintain sperm viability and function in the FRT can lead to new knowledge about infertility etiology and a new approach in assisted reproductive technologies for the preparation and selection of the best sperm based on the criteria that physiologically happen in-vivo.
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Tolerancia Inmunológica , Espermatozoides , Humanos , Femenino , Espermatozoides/inmunología , Espermatozoides/metabolismo , Masculino , Animales , Embarazo , Genitales Femeninos/inmunología , Genitales Femeninos/metabolismo , Semen/inmunología , Semen/metabolismo , Implantación del Embrión/inmunologíaRESUMEN
STUDY QUESTION: Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods? SUMMARY ANSWER: The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential. WHAT IS KNOWN ALREADY: To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations. STUDY DESIGN, SIZE, DURATION: The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage. MAIN RESULTS AND THE ROLE OF CHANCE: In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05). LIMITATION, REASONS FOR CAUTION: The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients. STUDY FUNDING/COMPETING INTEREST(S): The study is funded by the Iran University of Medical Sciences and no competing interest exists. TRIAL REGISTRATION NUMBER: N/A.
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Citometría de Flujo , Análisis de Semen , Proteínas de Plasma Seminal , Espermatozoides , Masculino , Humanos , Espermatozoides/fisiología , Citometría de Flujo/métodos , Análisis de Semen/métodos , Fragmentación del ADN , Motilidad Espermática , Fosfoinositido Fosfolipasa C/metabolismo , Adulto , Microfluídica/métodos , Fertilización/fisiología , Técnicas Analíticas Microfluídicas/métodos , Separación Celular/métodos , Proteínas Portadoras/metabolismoRESUMEN
Unexpected poor ovarian response (UPOR) occurs when nine or fewer oocytes are retrieved from a young patient with normal ovarian reserve. Bone morphogenetic protein15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-specific factors with pivotal role in folliculogenesis. The aim of this study was to assess the relation between BMP15 and GDF9 variants with UPOR. Hundred women aged ≤ 39 with AMH ≥ 1.27 IU/ml participated as UPOR and normal ovarian responders (NOR) based on their oocyte number. Each group consisted of 50 patients. After genomic DNA extraction, the entire exonic regions of BMP15 and GDF9 were amplified and examined by direct sequencing. Western blotting was performed to determine the expression levels of BMP15 and GDF9 in follicular fluid. Additionally, in silico analysis was applied to predict the effect of discovered mutations. From four novel variants of BMP15 and GDF9 genes, silent mutations (c.744 T > C) and (c.99G > A) occurred in both groups, whereas missense variants: c.967-968insA and c.296A > G were found exclusively in UPORs. The latter variants caused reduction in protein expression. Moreover, the mutant allele (T) in a GDF9 polymorphism (C447T) found to be more in NOR individuals (58% NOR vs. 37% UPOR (OR = 2.3, CI 1.32-4.11, p = 0.004).The novel missense mutations which were predicted as damaging, along with other mutations that happened in UPORs might result in ovarian resistance to stimulation. The mutant allele (T) in C447T polymorphism has a protective effect. It can be concluded that there is an association between BMP15 and GDF9 variants and follicular development and ovarian response.
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Proteína Morfogenética Ósea 15 , Factor 9 de Diferenciación de Crecimiento , Humanos , Femenino , Factor 9 de Diferenciación de Crecimiento/genética , Factor 9 de Diferenciación de Crecimiento/metabolismo , Proteína Morfogenética Ósea 15/genética , Proteína Morfogenética Ósea 15/metabolismo , Ovario/metabolismo , Oocitos/metabolismo , Líquido Folicular/metabolismoRESUMEN
The cryopreservation-thawing process of spermatozoa cells has negative impacts on their structure, function, and fertility parameters, which are known as cryoinjury. Asthenozoospermia patients are more susceptible to cryoinjury. Granulocyte-macrophage colony-stimulating factor (GM-CSF) increases sperm glucose uptake via the induction of glucose transporters, resulting in increased sperm motility. This study aimed to investigate the efficiency of GM-CSF supplementation of the cryopreservation media for semen samples of asthenoteratozoospermia patients. The study was carried out on 20 semen samples from infertile men referred to diagnosing semen analysis. To avoid subjective bias, two main sperm motility parameters, including velocity along the curvilinear path and velocity along the straight-line path were considered by the computer-assisted sperm analysis system. Afterward, each semen sample was divided into three equal aliquots and randomly assigned to one of the following groups: group I (control, freezing media only), group II (+GM-CSF, freezing medium supplemented with 2 µL/mL GM-CSF), or group III (GM-CSF added after thawing and washing). Following semen thawing, standard parameters, mitochondrial membrane potential (MMP), and the DNA Fragmentation Index were analyzed. Total sperm motility (progressive and non-progressive) improved significantly in group III samples after a 30-minute incubation with GM-CSF compared with the control group (26.5% ± 3.1% vs. 17.51% ± 2.59%). However, no differences in progressive motility or sperm morphology were found among the three thawed samples. The percentage of vitality was significantly higher in group III compared with the other two groups (28.38% ± 3.4% vs. 22.4% ± 3.08% and 22.14% ± 2.77%, respectively) (p < 0.05). JC-1 levels (a marker of MMP) were not significantly different between the examined groups (44.95% ± 8.26% vs. 36.61% ± 6.95% vs. 46.67% ± 7.7%, for control, group II, and group III, respectively) (p > 0.05). GM-CSF may be advantageous as an additive after freezing, improving total motility and viability after 30 minutes of post-thaw incubation; however, when supplied to the freezing media before cryopreservation, it is unable to protect against cryoinjury.
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Astenozoospermia , Preservación de Semen , Humanos , Masculino , Congelación , Motilidad Espermática , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Semen , Citocinas , Preservación de Semen/métodos , Espermatozoides , Criopreservación/métodos , Crioprotectores/farmacologíaRESUMEN
Background: Ectopic pregnancy (EP) is defined as embryo implantation in a location other than the uterine cavity. Objective: We aimed to evaluate the expression of several genes, which may play a role in EP, in the ampulla region of fallopian tubes and endometrial tissue of women with EP. Materials and Methods: In this case-control study, 5 women who underwent salpingectomy due to EP, comprised the 5 pseudo-pregnant women as a control group. These participants referred to the Royan Institute, Shariati, and Arash hospital, Tehran, Iran during 2019-2021. We evaluated the expressions of vascular endothelial growth factor A, mucin-1, colony-stimulating factor-1, heparin-binding epidermal growth factor-like growth factor (HBEGF), and fibroblast growth factor 2 genes in the fallopian tube and endometrium of EP cases by real-time polymerase chain reaction using specific primers. Results: The vascular endothelial growth factor expression was significantly higher in the ampulla region of the controls. However, no significant differences were observed in endometrial tissue. Assessments of colony-stimulating factor-1 and fibroblast growth factor 2 showed no significant differences between the case and control groups. HBEGF showed significantly higher expression in the ampulla region of EP cases, but no significant difference was observed in HBEGF expression in the endometrial tissues of the study groups. Mucin-1expression was significantly higher in both study regions of the EP cases. Conclusion: Our results have strongly suggested that these genes play important roles in proper implantation, and disruptions in their expression patterns could lead to EP. However, more studies are needed to confirm the current findings.
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OBJECTIVE: Although intracytoplasmic sperm injection (ICSI) is a way to deal with in vitro fertilization failure, 3% of couples still experience repeated fertilization failure after attempted ICSI, despite having sperm within normal parameters. These patients are a challenging group whose sperm cannot fertilize the egg during ICSI. Unfortunately, no test can predict the risk of fertilization failure. Phospholipase C zeta (PLCζ) and transition nuclear proteins (TNPs) are essential factors for chromatin packaging during sperm maturation. This study aimed to assess PLCζ1 and TNP1 expression in the sperm of patients with fertilization failure and the correlations among the DNA fragmentation index, PLCζ1 and TNP1 gene and protein expression, and the risk of fertilization failure. METHODS: In this study, 12 infertile couples with low fertilization rates (<25%) and complete failure of fertilization in their prior ICSI cycles despite normal sperm parameters were chosen as the case group. Fifteen individuals who underwent ICSI for the first time served as the control group. After sperm analysis and DNA fragmentation assays, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to compare the gene and protein expression of PLCζ and TNP1 in both groups. RESULTS: DNA fragmentation was significantly higher in the fertilization failure group. The qRT-PCR and Western blot results demonstrated significantly lower PLCζ and TNP1 gene and protein expression in these patients than in controls. CONCLUSION: The present study showed that fertilization failure in normozoospermic men was probably due to deficient DNA packaging and expression of TNP1.
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There is a correlation between teratozoospermia and production of reactive oxygen species leading to poor assisted reproductive techniques outcomes. This study aimed to examine the effect of plasma-rich in growth factors (PRGF) on teratozoospermic samples. Twenty-five teratozoospermic samples were included in this study. After sperm preparation, it was divided into four groups, including 0 (control), 1, 5, and 10% PRGF. Sperm motility, viability (eosin-nigrosin staining), morphology (Papanicolaou staining), DNA fragmentation (sperm chromatin dispersion test), mitochondrial membrane potential (JC-1 staining by flow cytometry), and lipid peroxidation (measurement of malondialdehyde, MDA) were evaluated before and after 1 h of incubation with or without PRGF. Our results showed that after 1 h of incubation, the addition of 1% PRGF improved sperm progressive motility (47.72 ± 13.76%) compared to the control group (17.36 ± 8.50%) (p < 0.001). Also, 1% PRGF preserved the sperm's total motility (77.50 ± 13.28% vs. 65.63 ± 19.03%, for 1% PRGF and control, respectively) and viability after incubation. The rate of normal sperm morphology was the same between different groups. Higher mitochondrial membrane potential and lower DNA fragmentation were also observed in sperm treated with different concentrations of PRGF compared to the control group, but the differences were non-significant. The MDA levels were significantly decreased in PRGF-treated groups compared to the control group (0.99 ± 0.62, 0.95 ± 0.33, 0.95 ± 0.79, and 1.49 ± 0.27 for 1% PRGF, 5% PRGF, 10% PRGF and control, respectively). Based on our results, it seems that PRGF incubation can improve sperm parameters and especially decrease the level of malondialdehyde as an indicator of oxidative stress, which is one of the main problems of teratozoospermic samples.
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Teratozoospermia , Humanos , Masculino , Semen , Motilidad Espermática , Espermatozoides/metabolismo , Malondialdehído/metabolismo , Malondialdehído/farmacologíaRESUMEN
OBJECTIVE: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. METHODS: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. RESULTS: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1ß, IL-6, IL-12, interferon α (IFN-α), IFN-ß, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. CONCLUSION: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.
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RESEARCH QUESTION: Can time-lapse parameters and the transcriptional profile of cumulus cells be used to achieve a more stringent and non-invasive method of embryo assessment and to identify possible factors affecting the embryo's ability to implant in repeated implantation failure (RIF) patients? DESIGN: A total of 190 embryos from 18 oocyte donors and 145 embryos from 15 RIF patients were evaluated based on time-lapse parameters. Three morphokinetic parameters including T5 (time to reach five cells), T3 (time to reach three cells) and CC2 (time to two to three cells) were recorded for all embryos. Embryos that had all three parameters in the normal range were graded as high quality and comparison between these parameters were compared in high-quality embryos between two groups. The transcriptional profile of cumulus cells related to high-quality embryos of both groups were analysed by RNA sequencing and compared. Finally, the possible relationship between differentially expressed genes and time-lapse parameters was examined. RESULTS: T5 was significantly lower in the RIF group than the donor group (P = 0.011). The cumulus cell transcriptome analysis showed 193 genes were down-regulated and 222 genes up-regulated. The mammalian target of rapamycin and the transforming growth factor beta pathways were significantly increased in the RIF group compared to the donor group (P = 0.007 and 0.01, respectively). Vitamin B12 and fatty acid beta-oxidation pathways were also significantly reduced in the RIF group compared to the donor group (P = 0.006 and 0.01, respectively). CONCLUSIONS: Differences in the transcriptomic profiles of cumulus cells and some morphokinetic parameters may be one of the main factors contributing to unexplained RIF.
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Implantación del Embrión , Embrión de Mamíferos , Imagen de Lapso de Tiempo/métodos , BlastocistoRESUMEN
Although the role of myo-inositol (MYO) in promoting the oocyte quality of PCOS patients has been documented in human studies; the cellular effects of this supplement on oocytes have not been directly examined due to ethical limitations. In the first phase of this study, MYO dosimetry was carried out simultaneously with the PCOS model development. An effective dose was obtained following the assessment of fasting insulin and testosterone levels using ELISA and ovarian morphology appraisal by histopathology. In the second phase, following the continuous administration of the effective dose of MYO and dehydroepiandrosterone (DHEA), cellular evaluation was performed. The quality of oocytes from superovulation was analyzed by examining maturity and normal morphology percentage using a stereomicroscope, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels using fluorometry, and ATP count evaluation using ELISA. The results revealed that, among the four different MYO concentrations, the 0.36 mg/g dose compared with the DHEA group reduced testosterone levels and large atretic antral follicles (LAtAnF) diameter. This dose also increased the corpus luteum count and the granulosa:theca (G/T)layer thickness ratio in antral follicles. Furthermore, this dose increased mature oocytes and normal morphology percentage, ATP count, and GSH levels; however, it decreased ROS levels in mature oocytes. Our findings provide the grounds for further cellular and molecular studies on the PCOS mouse model, suggesting that the improvement in mitochondrial function and its antioxidant properties is probably one of the mechanisms by which MYO increases oocyte quality.
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Ácido Fólico , Síndrome del Ovario Poliquístico , Femenino , Animales , Ratones , Humanos , Ácido Fólico/farmacología , Especies Reactivas de Oxígeno , Inositol/farmacología , Oocitos , Glutatión , Testosterona/farmacología , Deshidroepiandrosterona/farmacología , Adenosina Trifosfato/farmacologíaRESUMEN
BACKGROUND: Micro RNAs (miRNAs) are small non-coding RNAs known as essential regulators of cell-cell communication. Recent studies have revealed that miRNAs are secreted by a blastocyst in culture media. We hypothesized that endometrial epithelial cells take up embryo-derived miRNAs as well as other soluble factors and regulate their receptivity-related gene expression. METHODS AND RESULTS: Blastocyst culture media (BCM) were collected from the individually cultured embryos, while human endometrial epithelial cells (HEECs) were collected from healthy fertile volunteers. To evaluate the effect of BCM on the endometrial receptivity gene expression, HEECs were co-cultured with implanted BCM, non-implanted BCM, and a control culture medium. After determining altered gene expression in the HEECs, the miRNAs-related genes through bioinformatics databases were identified and evaluated in the BCM. Co-culture of primary HEECs with BCM significantly stimulated the expression levels of VEGFA, HBEGF, HOXA10, and LIF in the implanted group compared with non-implanted and control groups. The fold changes of miR-195 significantly diminished in the implanted BCM group compared with the non-implanted BCM group. Reduced fold changes of miR-29b, 145 and increased miR-223 were also observed in the implanted BCM group compared with the non-implanted ones. CONCLUSION: miRNAs could function as potential gene expression regulators during implantation. These molecules are secreted by human blastocyst, taken up by endometrial epithelial cells, and cause a change in the endometrial function. We found that BCMs can be effective in implantation process by stimulating related receptivity gene expression.
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MicroARNs , Humanos , Femenino , MicroARNs/metabolismo , Implantación del Embrión/genética , Blastocisto/metabolismo , Medios de Cultivo/farmacología , Expresión Génica , Endometrio/metabolismoRESUMEN
OBJECTIVE: Animal-free scaffolds have emerged as a potential foundation for consistent, chemically defined, and low-cost materials. Because of its good potential for high biocompatibility with reproductive tissues and well-characterized scaffold design, we investigated whether polyglycolic acid (PGA) could be used as an animal-free scaffold instead of natural fibrin-agarose, which has been used successfully for three-dimensional human endometrial cell culture. METHODS: Isolated primary endometrial cells was cultured on fibrin-agarose and PGA polymers and evaluated various design parameters, such as scaffold porosity and mean fiber diameter. Cytotoxicity, scanning electron microscopy (SEM), and immunostaining experiments were conducted to examine cell activity on fabricated scaffolds. RESULTS: The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay and SEM results showed that endometrial cells grew and proliferated on both scaffolds. Immunostaining showed cytokeratin and vimentin expression in seeded cells after 7 days of culture. On both scaffolds, an epithelial arrangement of cultured cells was found on the top layer and stromal arrangement matrix on the bottom layer of the scaffolds. Therefore, fibrin-agarose and PGA scaffolds successfully mimicked the human endometrium in a way suitable for in vitro analysis. CONCLUSION: Both fibrin-agarose and PGA scaffolds could be used to simulate endometrial structures. However, because of environmental and ethical concerns and the low cost of synthetic polymers, we recommend using PGA as a synthetic polymer for scaffolding in research instead of natural biomaterials.
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PURPOSE: Sperm quality plays a vital role in successful fertilization and pregnancy. Patients with fertilization failure (total failure or low-fertilization rate) despite having normal semen parameters are a challenging group whose sperm cannot fertilize the oocyte via the intracytoplasmic sperm injection (ICSI) technique. Microfluidics is offered as a new method for proper sperm sorting. METHODS: This study aimed to evaluate sperm parameters, DNA fragmentation index (DFI), expression of phospholipase C zeta 1 (PLCZ1), and transition nuclear proteins 1 (TNP1) mRNAs in sperm selected by microfluidic sperm sorting (MSS) chip compared with conventional density gradient centrifugation technique in patients with fertilization failure following ICSI. Subsequence fertilization rate and embryo quality were assayed. RESULTS: Normal morphology and total motility were significantly higher, and DFI was significantly lower in sperm selected by the MSS chip in fertilization failure and control groups. The RT-PCR results demonstrated a significant increase in the expression of PLCZ1 and TNP1 genes in sperm of both groups selected by MSS chips compared to the DGC method. In addition, with the selected sperm by MSS chip, an increase in fertilization rate and improvement of embryo quality was obtained. CONCLUSION: The present study findings show that sperm sorting by the microfluidic method improves fertilization rate in patients with poor fertilization outcomes following ICSI.
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Microfluídica , Semen , Fragmentación del ADN , Femenino , Fertilización , Fertilización In Vitro , Humanos , Masculino , Embarazo , Índice de Embarazo , Espermatozoides/metabolismoRESUMEN
Assisted reproductive techniques can help many infertile couples conceive. Therefore, there is a need for an effective method to overcome the widespread problems of infertile men and women. Oocyte and sperm quality can increase the chances of successful in vitro fertilisation. The maturation environment in which gametes are present can affect their competency for fertilisation. It is well established that myo-inositol (MI) plays a pivotal role in reproductive physiology. It participates in cell membrane formation, lipid synthesis, cell proliferation, cardiac regulation, metabolic alterations, and fertility. This molecule also acts as a direct messenger of insulin and improves glucose uptake in various reproductive tissues. Evidence suggests that MI regulates events such as gamete maturation, fertilisation, and embryo growth through intracellular Ca2 + release and various signalling pathways. In addition to the in-vivo production of MI from glucose in the reproductive organs, its synthesis by in vitro-cultured sperm and follicles has also been reported. Therefore, MI is suggested as a therapeutic approach to maintain sperm and oocyte health in men and women with reproductive disorders and individuals of reproductive age.
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ABSTRACT Objective: Despite the treatment of anovulation, infertility is still one of the main complications in PCOS women during reproductive age, which appears to be mainly due to impaired uterine receptivity. This study investigated the transcriptome profiles of endometrium in PCOS patients and healthy fertile individuals as the control group. Material and methods: Total mRNA was extracted from endometrial tissues of PCOS patients (n = 12) and healthy fertile individuals (n = 10) during the luteal phase. After cDNA synthesis, PCR array was performed using Human Female Infertility RT² Profiler PCR Array kit (Qiagen, Cat. No: PAHS-164Z) for evaluating expression of 84 genes contributing to the female infertility. Results: PCR Array data analysis identified significantly greater expression of CSF, IL11, IL15, IL1r1, IL1b, TNF, LIF, TNFRSF10B, TGFβ, C3, ITGA4 (Cd49d), SPP1, and Calca in PCOS women than in controls (P < 0.05). However, the expression of LIFR, C2, CD55, CFD, CALCA, LAM1, LAMC2, MMP2, MMP7, MMP9, ESR, SELL, ITGB3, and VCAM1 was significantly lower in PCOS group than in controls (P < 0.05). The results revealed dysregulation of immune-inflammatory molecules, complement activation and downregulation of IGF-I as well as adhesion molecules in PCOS group. Conclusion: The findings of this study indicated some potential causes of reduced receptivity of endometrium thus compromising the fertility in PCOS patients.
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Objective: Despite the treatment of anovulation, infertility is still one of the main complications in PCOS women during reproductive age, which appears to be mainly due to impaired uterine receptivity. This study investigated the transcriptome profiles of endometrium in PCOS patients and healthy fertile individuals as the control group. Methods: Total mRNA was extracted from endometrial tissues of PCOS patients (n = 12) and healthy fertile individuals (n = 10) during the luteal phase. After cDNA synthesis, PCR array was performed using Human Female Infertility RT2 Profiler PCR Array kit (Qiagen, Cat.No: PAHS-164Z) for evaluating expression of 84 genes contributing to the female infertility. Results: PCR Array data analysis identified significantly greater expression of CSF, IL11, IL15, IL1r1, IL1b, TNF, LIF, TNFRSF10B, TGFß, C3, ITGA4 (Cd49d), SPP1, and Calca in PCOS women than in controls (P < 0.05). However, the expression of LIFR, C2, CD55, CFD, CALCA, LAM1, LAMC2, MMP2, MMP7, MMP9, ESR, SELL, ITGB3, and VCAM1 was significantly lower in PCOS group than in controls (P < 0.05). The results revealed dysregulation of immune-inflammatory molecules, complement activation and downregulation of IGF-I as well as adhesion molecules in PCOS group. Conclusion: The findings of this study indicated some potential causes of reduced receptivity of endometrium thus compromising the fertility in PCOS patients.
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The present study investigated the effects of DNA fragmentation of spermatozoa on the growth factors expression by a human oviduct epithelial cell line (OE-E6/E7). Two separate groups were examined in this study. The cell line was cultured in the presence of spermatozoa with normal DNA fragmentation index (DFI) or abnormal DFI. Total RNA from the cell line in each group was isolated, and relative expression of objective genes was analysed using PCR array. Also, the concentration of VEGF, BMP-2, BMP-7 and MSTN in the supernatant of cell culture was analysed by the ELISA method. The PCR array analysis revealed that most of the growth factors had been upregulated in the abnormal group. However, the differences between groups were statistically significant (p < 0.05) for five genes, including VEGF-A, BMP-2, BMP-6, BMP-7 and OSM. Furthermore, MSTN was the only gene that down-regulated significantly under the influence of the spermatozoa with abnormal DFI. Moreover, the results of ELISA analysis were in agreement with the data of the PCR array. It has been concluded that DNA fragmentation in human spermatozoa can probably change regular events throughout the oviducts. Consequently, the genes of interest may change sperm function and probably its fate in the female reproductive tract.