Male obesity is associated with sperm telomere shortening and aberrant mRNA expression of autophagy-related genes.

BACKGROUND: Obesity is regarded a global public health crisis. It has been implicated in a variety of health problems, but when it comes to male fertility, how and to what extent obesity affects it are poorly understood. Accordingly, semen samples from 32 individuals with obesity (body mass index (BMI) ≥ 30 kg/m2) and 32 individuals with normal weight (BMI: 18.5-25 kg/m2) were obtained. Here, for the first time, we examined the association between obesity, relative sperm telomere length (STL) and autophagy-related mRNA levels such as Beclin1, AMPKa1, ULK1, BAX, and BCL2. Each group was also evaluated for conventional semen parameters, sperm apoptotic changes, DNA fragmentation index (DFI), sperm chromatin maturation, and reactive oxygen species (ROS) levels. RESULTS: Based on our findings, there was a marked reduction in relative STL in individuals with obesity as compared to the normal-weight group. We also found a significant negative correlation between relative STL and age, BMI, DFI, percentage of sperm with immature chromatin, and intracellular ROS levels in patients with obesity. In the normal-weight group, relative STL was only negatively correlated with DFI and intracellular ROS levels. Regarding mRNA expression, there was considerable upregulation of Beclin1, ULK1, and BCL2 in the group with obesity compared to the normal-weight group. Obesity was also found to be associated with a considerable decline in semen volume, total sperm count, progressive motility, and viability in comparison to normal-weight individuals. Furthermore, obesity was associated with considerably higher percentages of DFI, sperm with immature chromatin, late-stage apoptosis, and elevated ROS levels. CONCLUSION: According to our findings, obesity is associated with sperm telomere shortening and aberrant autophagy-related mRNA expression. It should be emphasized that telomere shortening in sperm may be an indirect consequence of obesity due to the oxidative stress associated with the condition. Nevertheless, further investigation is required for a more comprehensive understanding.

RéSUMé: CONTEXTE: L'obésité est considérée comme une crise mondiale de santé publique. Elle a été impliquée dans divers problèmes de santé ; mais quand il s'agit de la fertilité masculine, comment et dans quelle mesure l'obésité affecte cette fertilité restent mal compris. En conséquence, des échantillons de sperme de 32 hommes obèses (indice de masse corporelle (IMC) ≥ 30 kg/m²) et de 32 hommes ayant un poids normal (IMC : 18,5 à 25 kg/m²) ont été recueillis. A été examiné dans cette étude, pour la première fois, l'association entre l'obésité, la longueur relative des télomères des spermatozoïdes (LTS), et les taux d'ARNm liés à l'autophagie tels que Beclin1, AMPKa1, ULK1, BAX et BCL2. Chaque groupe a également été évalué pour les paramètres conventionnels du sperme, les changements apoptotiques des spermatozoïdes, l'indice de fragmentation de l'ADN (DFI), la maturation de la chromatine des spermatozoïdes et les niveaux d'espèces réactives de l'oxygène (ROS). RéSULTATS: Il y eut une réduction marquée de la LTS relative chez les hommes obèses par rapport à ceux du groupe de poids normal. Nous avons également trouvé une corrélation négative significative entre la LTS relative et l'âge, l'IMC, le DFI, le pourcentage de spermatozoïdes avec chromatine immature et les niveaux intracellulaires de ROS chez les hommes obèses. Dans le groupe d'hommes de poids normal, la LTS relative n'était corrélée négativement qu'avec les taux de DFI et de ROS intracellulaires. En ce qui concerne l'expression de l'ARNm, il y avait une régulation positive considérable de Beclin1, ULK1 et BCL2 dans le groupe d'hommes obèses par rapport à ceux du  groupe de poids normal. L'obésité s'est également avérée être associée à une baisse considérable du volume de sperme, du nombre total de spermatozoïdes, de la mobilité progressive et de la viabilité des spermatozoïdes par rapport aux hommes de poids normal. En outre, l'obésité était associée à des pourcentages considérablement plus élevés de DFI, de spermatozoïdes avec chromatine immature, d'apoptose à un stade avancé, et de niveaux élevés de ROS. CONCLUSION: Selon nos résultats, l'obésité est associée au raccourcissement des télomères des spermatozoïdes et à une expression aberrante d'ARNm liés à l'autophagie. Il convient de souligner que le raccourcissement des télomères dans les spermatozoïdes peut être une conséquence indirecte de l'obésité en raison du stress oxydatif associé à la maladie. Néanmoins, des études plus approfondies sont nécessaires pour une compréhension plus complète.

Derivation of hormone-responsive human endometrial organoids and stromal cells from cryopreserved biopsies.

The human endometrium is a dynamic tissue that undergoes cyclic changes in response to sex steroid hormones to provide a receptive status for embryo implantation. Disruptions in this behavior may lead to implantation failure and infertility; therefore, it is essential to develop an appropriate in vitro model to study endometrial changes in response to sex hormones. In this regard, the first choice would be human endometrial cells isolated from biopsies that could be used as monolayer cell sheets or to generate endometrial organoids. However, the need for fresh samples and short-time viability of harvested endometrial biopsy limits these approaches. In order to overcome these limitations, we sought to develop an efficient, simple, robust and reproducible method to cryopreserve human endometrial biopsies that could be stored and/or shipped frozen and later thawed to generate endometrial organoids and endometrial stromal cells (EnSCs). These cryopreserved biopsies could be thawed and used to generate simple endometrial organoids or organoids for co-culture with matched stromal cells that are functionally responsive to sex hormones as similar as the organoids generated from fresh biopsy. An optimal endometrial tissue cryopreservation method would allow the possibility for endometrial tissue biobanking to enable future organoid generation from both healthy tissues and pathological conditions, and open new venues for generate endometrial assembloids, consisting of epithelial organoids and primary stromal cells.

A decade of molecular preimplantation genetic diagnosis of 350 blastomeres for beta-thalassemia combined with HLA typing, aneuploidy screening and sex selection in Iran.

BACKGROUND: Preimplantation genetic diagnosis (PGD) has been developed to detect genetic disorders before pregnancy which is usually done on blastomeres biopsied from 8-cell stage embryos obtained from in vitro fertilization method (IVF). Here we report molecular PGD results for diagnosing of beta thalassemia (beta-thal) which are usually accompanied with evaluating chromosomal aneuploidies, HLA typing and sex selection. METHODS: In this study, haplotype analysis was performed using short tandem repeats (STRs) in a multiplex nested PCR and the causative mutation was detected by Sanger sequencing. RESULTS: We have performed PGDs on 350 blastomeres from 55 carrier couples; 142 blastomeres for beta-thal only, 75 for beta-thal and HLA typing, 76 for beta-thal in combination with sex selection, and 57 for beta-thal and aneuploidy screening. 150 blastomeres were transferable, 15 pregnancies were happened, and 11 babies born. We used 6 markers for beta-thal, 36 for aneuploidy screening, 32 for sex selection, and 35 for HLA typing. To our knowledge combining all these markers together and the number of STR markers are much more than any other studies which have ever done. CONCLUSIONS: PGD is a powerful diagnostic tool for carrier couples who desire to have a healthy child and wish to avoid medical abortion.

Conditioned medium derived from seminal extracellular vesicles-exposed endometrial stromal cells induces inflammatory cytokine secretion by macrophages.

OBJECTIVE: Seminal plasma (SP) contains large numbers of sub-cellular structures called extracellular vesicles (EV) which have been postulated to have immunological functions due to their bioactive contents including proteins and small non-coding RNAs. Although the response of endometrial cells to seminal EV (SEV) is recently being elucidated, the impact of these signaling vesicles on stroma-immune crosstalk is still unknown. Herein, we aimed to investigate the effect of conditioned medium (CM) derived from SEV-exposed endometrial stromal cells (eSC) on cytokine secretion by macrophages. STUDY DESIGN: SEV were isolated from SP samples of healthy donors and characterized by common methods needed for EV characterization, including size determination by dynamic light scattering (DLS), transmission electron microscopy (TEM), and western blot analysis of EV markers. Endometrial biopsies were obtained from healthy individuals and eSC were isolated and characterized. EV internalization assay was performed by labeling the SEV with PKH67 green fluorescent dye. Then, the eSC were exposed to SEV and the CM was collected. Finally, the CM from SEV-exposed eSC was added to the macrophage culture and the level of inflammatory (interleukin (IL)-1α and IL-6) and anti-inflammatory (IL-10) cytokines were measured in the culture supernatant of macrophages. RESULTS: The results demonstrated that the CM derived from SEV-exposed eSC induce IL-1α and IL-6 secretion by the macrophages, while the secretion of IL-10 was reduced. CONCLUSION: Our results support the idea that the stroma-immune interaction is affected by SEV. This effect may be a part of immunoregulatory function of SP inside upper female genital tract and have an obvious impact during peri-implantation period.

Culture strategy as a modulator of target assessments: Functionality of suspension versus hanging drop-derived choriocarcinoma spheroids as in vitro model of embryo implantation.

The choriocarcinoma spheroid model has been amply applied to study the underlying molecular mechanism of implantation. Reproducibility and functionality of spheroid tumor models were addressed precisely. To mimic embryo-endometrium crosstalk, no functional characteristics of spheroids have been provided based on culture strategies. In this study, choriocarcinoma spheroids were provided as suspension culture (SC) or hanging drop culture (HDC). Primary assessments were performed based on morphology, cellular density, and hormonal secretion. Spheroid-endometrial cross talk was assessed as coculture procedures. Further, alkaline phosphatase (ALP) activity and expression of genes involved in attachment, invasion, and inducing migration were quantified. We found HDC spheroids provided a homogenous-shaped aggregate with a high grade of viability, cellular integration, hormonal secretion, and the dominant role of WNTs expression in their microarchitecture. SC spheroids showed a higher level of ALP activity and the expression of integrated genes in modulating attachment, invasion, and migration abilities. Spheroid confrontation assays clearly clarified the superiority of SC spheroids to crosstalk with epithelial and stromal cells of endometrium in addition to motivating an ideal endometrial response. Conclusively, culture strategies by affecting various molecular signaling pathways should be chosen precisely according to specific target assessments. Specifically, SC assumed as an ideal model in spheroid-endometrial cross talk.

Design and Microfabrication of An On-Chip Oocyte Maturation System for Reduction of Apoptosis.

OBJECTIVE: In customary assisted reproductive technology (ART), oocyte culture occurs in static micro drops of Petri dishes with vast media volume; while, the in vivo condition is dynamic. In this study, we aimed to improve the maturation efficiency of mammalian oocytes by designing an optimal microchamber array to obtain the integration of oocyte trapping and maturation within a microfluidic device and evaluate the role of microfluidic culture condition in lipid peroxidation level of the culture medium, in vitro matured oocytes apoptosis, and its comparison with the conventional static system. MATERIALS AND METHODS: In this experimental research, immature oocytes were collected from ovaries of the Naval Medical Research Institute (NMRI) mice. Oocytes were randomly laid in static and dynamic (passive and active) in vitro maturation culture medium for 24 hours. The lipid peroxidation level in oocyte culture media was assessed by measuring the concentration of malondialdehyde (MDA), and the rate of apoptosis in in vitro matured oocytes was assessed by the TUNEL assay after a-24 hour maturation period. RESULTS: The MDA concentration in both dynamic oocyte maturation media were significantly lower than the static medium (0.003 and 0.002 vs. 0.13 µmol/L, P<0.01). Moreover, the rate of apoptosis in matured oocytes after a-24 hour maturation period was significantly lower in passive dynamic and active dynamic groups compared with the static group (16%, 15% vs. 35%, P<0.01). CONCLUSION: The dynamic culture for in vitro oocyte maturation (IVM) improves the viability of IVM oocytes in comparison with the static culture condition.

Meiosis Resumption of Immature Human Oocytes following Treatment with Calcium Ionophore In Vitro.

OBJECTIVE: In vitro maturation (IVM) of human oocytes is used to induce meiosis progression in immature retrieved oocytes. Calcium (Ca2+) has a central role in oocyte physiology. Passage through meiosis phase to another phase is controlled by increasing intracellular Ca2+. Therefore, the current research was conducted to evaluate the role of calcium ionophore (CI) on human oocyte IVM. MATERIALS AND METHODS: In this clinical trial study, immature human oocytes were obtained from 216 intracytoplasmic sperm injection (ICSI) cycles. After ovarian stimulation, germinal vesicle (GV) stage oocytes were collected and categorized into two groups: with and without 10 µM CI treatment. Next, oocyte nuclear maturation was assessed after 24-28 hours of culture. Real-time reverse transcription polymerase chain reaction (RT-PCR) was used to assess the transcript profile of several oocyte maturation-related genes (MAPK3, CCNB1, CDK1, and cyclin D1 [CCND1]) and apoptotic-related genes (BCL-2, BAX, and Caspase-3). Oocyte glutathione (GSH) and reactive oxygen species (ROS) levels were assessed using Cell Tracker Blue and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent dye staining. Oocyte spindle configuration and chromosome alignment were analysed by immunocytochemistry. RESULTS: The metaphase II (MII) oocyte rate was higher in CI-treated oocytes (73.53%) compared to the control (67.43%) group, but this difference was not statistically significant (P=0.13). The mRNA expression profile of oocyte maturation-related genes (MAPK3, CCNB1, CDK1, and CCND1) (P<0.05) and the anti-apoptotic BCL-2 gene was remarkably up-regulated after treatment with CI (P=0.001). The pro-apoptotic BAX and Caspase-3 relative expression levels did not change significantly. The CI-treated oocyte cytoplasm had significantly higher GSH and lower ROS (P<0.05). There was no statistically significant difference in meiotic spindle assembly and chromosome alignment between CI treatment and the control group oocytes. CONCLUSION: The finding of the current study supports the role of CI in meiosis resumption of human oocytes. (Registration Number: IRCT20140707018381N4).

Photobiomodulation preconditioned human semen protects sperm cells against detrimental effects of cryopreservation.

The biological consequences of semen samples preconditioning with photobiomodulation (PBM) were studied on human sperm cells post cryopreservation. Donated semen samples were collected from 22 married men with normal sperm parameters according to World Health Organization (WHO) criteria. Included samples were divided into control and PBM-preconditioning (one session, 810 nm, diode laser, and 0.6 J/cm2) groups before cryopreservation procedure. Progressive sperm motility (PSM), morphology, viability, sperm mitochondrial membrane potential(MMP), intracellular reactive oxygen species (ROS) and lipid peroxidation of sperm cells were assessed post thawing. PBM preconditioning of cryopreserved semen samples most prominently increased the PSM percentage 30 min post thawing (p = 0.000).Application of PBM before cryopreservation significantly increased the number of viable spermatozoa (p = 0.000), increased significantly the number of spermatozoa with high MMP (p = 0.004) and decreased significantly the number of spermatozoa with low MMP post-thawing(P = 0. 007)compared to control group. Cryopreserved human sperm cells with PBM preconditioning showed significant decrease in the levels of intracellular ROS (47.66 ± 2.14 versus 60.42 ± 3.16, p = 0.002) and lipid peroxidation (3.06 ± 0.13 versus 3.68 ± 0.27, p = 0.05)compared to control group. Our findings, as the first evidence, indicated that PBM-preconditioning of human semen before cryopreservation provides a real and substantial advantage. This might lead to a novel strategy in improving PBM application in the procedures of assisted reproductive technologies.

Effect of vitrification on biogenesis pathway and expression of development-related microRNAs in preimplantation mouse embryos.

Vitrification of embryos has been known as the most efficient cryopreservation method in assisted reproductive technology clinics. Vitrification of preimplantation embryo might be associated with altered gene expression profile and biochemical changes of vitrified embryos. Stringent regulation of gene expression in early embryonic stages is very critical for normal development. In the present study, we investigated the effect of vitrification on the canonical miRNA biogenesis pathway, and also the expression of developmental related miRNAs, in 8-cell and blastocyst mouse embryos. Although the expression pattern of the miRNA biogenesis pathway genes differed between 8-cell and blastocyst mouse embryos, vitrification did not affect the expression level of these genes in preimplantation embryos. The expression levels of miR-21 and let-7a were significantly decreased in vitrified 8-cell embryos and fresh blastocysts when compared with fresh 8-cell embryos. The expression of Stat3 was significantly reduced in blastocysts after vitrification. The alteration in the expression pattern of miRNAs, due to their mode of action, can affect broad downstream key developmental signaling pathways. Therefore, the blastocyst stage is the preferred point for embryo vitrification as they are less susceptible to cryo-damages regarding the stability of miRNAs related to the developmental and implantation competence of embryo.

Gelatin Electrospun Mat as a Potential Co-culture System for In Vitro Production of Sperm Cells from Embryonic Stem Cells.

Engineering of 3D substrates with maximum similarity to seminiferous tubules would help to produce functional sperm cells in vitro from stem cells. Here, we present a 3D electrospun gelatin (EG) substrate seeded with Sertoli cells and determine its potential for guided differentiation of embryonic stem cells (ESCs) toward germline cells. The EG was fabricated by electrospinning, and its morphology under SEM, as well as cytobiocompatibility for Sertoli cells and ESCs, was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and cell attachment assay. Embryoid bodies (EBs) were formed from ESCs and co-cultured with Sertoli cells, induced with BMP4 for 3 and 7 consecutive days to induce the differentiation of EBs toward germline cells. The differentiation was investigated by immunocytochemistry (ICC), flow cytometry, and RT-PCR in four experimental groups of EBs (EBs cultured in gelatin-coated cell culture plates); Scaffold/EB (EBs cultured on EG); ESCs/Ser (EBs and Sertoli cells co-cultured on gelatin-coated cell culture plates without EG); and Scaffold/EB/Ser (EBs and Sertoli cells co-cultured on EG). All experimental groups exhibited a significantly increased MVH (germline-specific marker) and decreased c-KIT (stemness marker) expression when compared with the EB group. ICC and flow cytometry revealed that Scaffold/EB/Ser had the highest level of MVH and the lowest c-KIT expression at both 3 and 7 days postdifferentiation compared with other groups. RT-PCR results showed a significant increase in the germline marker (Dazl) and a significant decrease in the ESC stemness marker (Nanog) in Scaffold/EB compared to the EB group. The germline markers Gcna, Stella, Mvh, Stra8, Piwil2, and Dazl were significantly increased in Scaffold/EB/Ser compared to the Scaffold/EB group. Our findings revealed that the EG scaffold can provide an excellent substrate biomimicking the micro/nanostructure of native seminiferous tubules and a platform for Sertoli cell-EB communication required for growth and differentiation of ESCs into germline cells.

Effects of treatment with hydroxychloroquine on the modulation of Th17/Treg ratio and pregnancy outcomes in women with recurrent implantation failure: clinical trial.

AIM: The imbalance of Th17/Treg cells has been recently suggested as a new risk factors for recurrent implantation failure (RIF). Furthermore Th17/Treg cells are involved in immune regulation in peripheral blood and endometrial tissue of patients with RIF. In this research, we investigated the effects of Hydroxychloroquine (HCQ) on the level and function of Th17 and Treg cells in women with RIF. It may be possible to improve pregnancy outcomes by modulating high cytokine levels. METHODS: Women with RIF received oral HCQ (n = 60) on day 4 of the menstrual cycle and continued until day 20 of the menstrual cycle and 2 days before embryo transfer and continued until the day of the pregnancy test, for a total of 16 days in another cycle. The serum levels of IL-17 and IL-10, the expression of transcription factors related to Th17 and Treg cells and the immune-reactivity of IL-17, IL-21 as Th17 related cytokines and IL-10, TGF- ß as Treg related cytokines in endometrial tissues were evaluated by ELISA, real-time PCR, and fluorescent immunohistochemistry respectively.Results: Treatment with HCQ down-regulated Th17 related cytokines and function and up-regulated Treg related cytokines and function significantly (p < .001). RORγt, the Th17 transcription factor, expression was down-regulated and FOXP-3, the T-reg transcription factor, expression was up-regulated. The biochemical pregnancy rate was not significantly different in RIF patients before and after treatment. CONCLUSION: Our results demonstrated that the administration of HCQ in RIF women with immune cell disorders during pregnancy could affect the Th17/Treg ratio and enhance Treg and diminish Th17 responses which may be associated with successful pregnancy outcomes. However, significant difference in pregnancy outcomes was not observed in the present study.

The effect of PEGylated iron oxide nanoparticles on sheep ovarian tissue: An ex-vivo nanosafety study.

Today, nanotechnology plays an important role in our ever-continuous quest to improve the quality of human life. Because of their infinitesimal size, nanostructures can actively interact and alter cellular functions. Therefore, while the clinical benefits of nanotechnology may outweigh most of the associated risks, assessment of the cytotoxicity of nanostructures in respect to cells and tissues early in product development processes is of great significance. To the best of our knowledge, no such assessment has been performed for nanomaterials on the ovarian cortex before. Herein, silica-coated, PEGylated silica-coated, and uncoated iron oxide nanoparticles (IONP) with core diameter of 11 nm (±4.2 nm) were synthesized. The oxidative stress in cultured ovarian tissue exposed to the various IONP was subsequently assessed. The results indicate that among the four groups, uncoated IONP induce the most oxidative stress on the ovarian cortex while tissues treated with PEGylated IONP exhibit no significant change in oxidative stress.

Photobiomodulation with 810 nm Wavelengths Improves Human Sperms' Motility and Viability In Vitro.

Background: Enhanced sperm motility is necessary for the successful journey of sperm inside the female genital tract, successful fertilization, and the increased chance of pregnancy. Objective: We investigated the impact of red and near-infrared (NIR) ranges of photobiomodulation (PBM) alone and together on fresh human sperm to validate an optimized PBM protocol that would maximize sperm motility and viability in vitro. Methods: We randomly divided 30 normal human semen samples into 3 different PBM protocols (red, NIR, and red+NIR lasers). Each sample was divided into four subparts, one control group sample and three experimental group samples. Each experimental group received one of the PBM protocols (red, NIR, or red+NIR). Each protocol was adjusted to three energy densities (0.6, 1.2, and 2.4 J/cm2). After exposure to the selected protocol, we determined the percentage of either viable or progressive sperm motility (PSM) and measured the DNA Fragmentation Index (DFI). Results: The NIR and red+NIR lasers at 2.4 J/cm2 energy density significantly increased PSM after 60 min compared with the control groups [least significant difference (LSD) test, p = 0.023 and p = 0.04, respectively]. Samples treated with the red laser at 0.6 J/cm2 had significantly decreased viability compared with the control group (LSD test, p = 0.003). Samples treated with the red+NIR lasers had significantly decreased viability at 0.6 J/cm2 (p = 0.003), 1.2 J/cm2 (p = 0.001), and 2.4 J/cm2 (p = 0.04) energy densities when compared with the control groups. The NIR laser resulted in no significant difference in sperm viability between the control and experimental groups. At 120 min after exposure, treatment with the red+NIR and red lasers at 2.4 J/cm2 density significantly increased DFI compared to the control groups (LSD test, p = 0.000, p = 0.007). Conclusions: In this study, sperm motility, viability, and DFI data confirmed the superiority of the NIR laser at 0.6 J/cm2 energy density compared with the red and red+NIR PBM protocols.

Exposing Mouse Oocytes to MitoQ During In Vitro Maturation Improves Maturation and Developmental Competence.

BACKGROUND AND PURPOSE: Mitochondrion is the main indicator of oocyte quality and one of the components of oocyte, which is sensitive to oxidative damage during the maturation process. Mitoquinone mesylate (MitoQ) is a strong antioxidant targeting mitochondria as well as anti-apoptotic agent. However, the effect of MitoQ on the quality of oocytes during in vitro maturation (IVM) is still unknown. OBJECTIVES: This study investigated the possible effects of MitoQ on maturation and developmental competency in mice oocytes. MATERIALS AND METHODS: The oocytes were collected at germinal vesicle stage from 6-8-week old female NMRI mice and then cultured in TCM-199 medium supplemented with 0, 0.01, 0.02 and 0.04 µM MitoQ. The sham group was treated with DMSO (0.01% v.v). Then intracellular Glutathione (GSH), reactive oxygen species (ROS) levels, mitochondria membrane potential (ΔΨm), as well as in vitro fertilization (IVF) rate in the 18-20 h matured oocytes and metaphase II (MII) oocytes (in vivo-control), were assessed. RESULTS: The results showed that between three dose of MitoQ, the 0.02 µM significantly increased nuclear maturation rate, GSH level, fertilization rate and blastulation (92.6, 231.7, 90.19 and 81.66%, respectively) than the in vitro-control (71.14, 152, 78.84 and 73.50%, respectively) and more comparable to that of the in vivo matured oocytes (100, 243.5, 92.10 and 83%, respectively). Also, the mitochondria membrane potential in the 0.02 µM MitoQ was significantly higher compared with those in the other groups (4.4). However, the intracellular ROS level in 0.02 µM MitoQ was significantly decreased (38.72%) compared to in vitro-control (82.2%) and was similar to the in vivo-control (33.5%). CONCLUSION: The results indicated that supplementation of IVM medium with MitoQ (specially 0.02 µM) enhance maturation and fertilization rate. In conclusion, MitoQ might be considered as a novel component that could be added to IVM media.

Protective Effect of Gallic Acid on Testicular Tissue, Sperm Parameters, and DNA Fragmentation against Toxicity Induced by Cyclophosphamide in Adult NMRI Mice.

PURPOSE: This study aimed to investigate the protective effect of Gallic acid (GA) on the Cyclophosphamide (CP) toxicity induced in the reproductive system. MATERIALS AND METHODS: After a pilot study for dose responses of Gallic acid ,Forty adult male NMRI mice were divided into 5 groups (n=8): control, sham (NaCl Serum: 0.2mL per day), CP (15 mg kg-1 per week; IP), GA (12.5 mg kg-1 per day ; IP) and GA (12.5 mg kg-1 per day ; IP) +CP(15 mg kg-1 per week; IP). After treatment, the left testis was detached and used for Histological examination and right testis used for Malondialdehyde (MDA) measures. Left caudal epididymis was placed in the Ham's F10 medium and released spermatozoa were used in order to analyze sperm parameters. Sperm DNA fragmentation was assessed by Sperm Chromatin Dispersion (SCD) method. RESULTS: In the CP group, there was a significant increase in the sperm DNA fragmentation (% 57.89 ± 23.91) compared with control group (% 24.52 ± 10.27). That was significantly improved by GA  (12.5 mg kg-1 per day ; IP)  in GA+CP group (% 28.4 ± 8.85) compared to CP group (p< .001).A significant increase was reported about MDA levels in CP group (6.26 ± 2.59) in compared with the control group (4.30 ± 2.05), But GA (3.24 ± 1.33) decreased it in GA+ CP group (p< .01).  The histopathological investigation revealed marked testicular atrophy in CP group, whereas GA diminished these deviations (P< .05). CONCLUSION: Gallic acid can modify the reproductive toxicity of cyclophosphamide in NMRI mice and increase the antioxidant capacity of testis tissue.

Does in vitro fertilization affect the expression of miRNAs and their biogenesis pathway in preimplantation mouse embryos?

BACKGROUND: In vitro fertilization (IVF) is a well-accepted procedure which has been utilized for the treatment of infertile patients. As embryos at early stages of development are very vulnerable, the IVF conditions may influence genetic and epigenetic regulation of preimplantation mouse embryo. METHODS: We assessed the effect of IVF on the expression of developmental and implantation related miRNAs (miR-21, miR-93, miR-24, and let-7a), their common presumptive target (Stat3), and miRNA biogenesis pathway genes (Drosha, Dgcr8, Exportin-5, Dicer, and Ago2). in vivo 8-cell and blastocysts were compared to IVF embryos. Expression levels of miRNAs, Stat3, and miRNA biogenesis pathway genes were evaluated by qRT-PCR in in vivo (n = 8) and IVF (n = 4) embryos. RESULTS: The expression levels of let-7a and Stat3 were significantly reduced in IVF blastocyst when compared with in vivo (p = .004 and p = .009, respectively). Nevertheless, the IVF procedure did not influence the expression levels of miRNA biogenesis pathway components in 8-cell and blastocyst embryos. CONCLUSIONS: Downregulation of let-7a and developmental related transcription factor, Stat3, in IVF mouse blastocysts may affect preimplantation development and implantation of embryos. Moreover, the genes of the miRNA biogenesis pathway were not changed in preimplantation mouse embryos through the IVF procedure.

MicroRNA-based regulatory circuit involved in sperm infertility.

miRNAs (MicroRNAs), known as noncoding and important endogenous factors regulating the expression protein-coding genes, are vital regulators in each biological process. Thus, this study aims to explore the key role of four microRNAs in regulating the spermatogenesis. To conduct this experiment, 55 infertile and fertile men provided the study with the sperm and testicular tissue samples. To study the spermatozoa in terms of the morphology, Diff-Quick was applied. Then, quantitative real-time polymerase chain reaction (RT-PCR) was conducted on samples. Our data indicated that in contrast to the miR-15b, significant increasing of miR-383 and miR-122 occurred in both severe oligoasthenoteratozoospermia (SOAT) and moderate oligoasthenoteratozoospermia (MOAT) compared to normal sperm group (N). In addition, it was observed that miR-15b and miR-122 increased in patients with nonobstructive azoospermia (NOA) compared with obstructive azoospermia (OA) group. Expression levels of target genes including P53, CASPASE-9 and CYCLIN D1 underwent principle changes according to miRNAs expression level. Our finding indicated that miRNAs had essential role in the regulation of spermatogenesis, and their expression altering was associated with sperm abnormalities. Thus, microRNAs can be introduced as useful biomarkers to determine male infertility reasons to choose the effective treatment.

Folliculogenesis-Associated Genes Expression in Human Vitrified Ovarian Tissue after Xenotransplantation in γ-Irradiated Mice.

OBJECTIVE: Autograft transplantation of vitrified cortical ovarian tissue is an acceptable clinical technique for fertility preservation in women. Xenograft transplantation into animal models could be useful for evaluating the safety of human vitrified ovarian tissue. This study targeted to evaluate impact of vitrification on expression of the genes associated with folliculogenesis after xenograft transplantation of human vitrified ovarian tissue to γ-irradiated mice. MATERIALS AND METHODS: In this experimental study, ovarian biopsies were gathered from six transsexual persons. The cortical section of ovarian biopsies was separated and chopped into small pieces. These pieces were randomly divided into vitrified and non-vitrified groups. In each group some pieces were considered as non-transplanted tissues and the others were transplanted to γ-irradiated female National Medical Research Institute (NMRI) mice. Before and after two weeks of xenograft transplantation, histological assessment and evaluation of the expression of folliculogenesisassociated genes (FIGLA, GDF-9, KL and FSHR) were performed in both vitrified and non-vitrified groups. RESULTS: Percentage of the normal follicles and expression of the all examined genes from transplanted and nontransplanted tissue were similar in both vitrified and non-vitrified groups (P>0.05). After transplantation, the normal follicle rate was significantly decreased and among the folliculogenesis-associated genes, expression of GDF-9 gene was significantly increased, rather than before transplantation in vitrified and non-vitrified tissues (P<0.05). CONCLUSION: The vitrification method using dimethyl solphoxide and ethylene glycol (EG) had no remarkable effect on the normal follicular rate and expression of folliculogenesis-associated genes after two weeks human ovarian tissue xenografting. In addition, transplantation process can cause a significant decrease in normal follicular rate and expression of GDF-9 gene.

Expression of miR-302 in human embryo derived from in-vitro matured oocyte.

BACKGROUND: The expression of miR-302 over the period of early embryogenesis could possibly regulate the maternal transcript clearance. Zygotic transcription activation is mostly related to maternal messages degradation. OBJECTIVE: In this study, the effects of in-vitro maturation technique (IVM) on the expression of miR-302 in human embryo produced from immature and mature human oocytes (matured in vitro and in vivo, before sperm exposure) obtained from females under gonadotrophin therapy were evaluated for assisted reproduction. MATERIALS AND METHODS: Immature oocytes were cultured in vitro. The injection of oocytes-producing polar bodies was given using fresh sperm. Then, the embryo quality score was assessed in the IVM group compared with the control group. In both the groups, embryos with normal morphology were included in the molecular study. Only one blastomere was removed from three-day embryos and then the embryos were frozen. The expression of miR-302 in embryos was measured through quantitative real-time polymerase chain reaction. RESULTS: Our data showed a significant reduction of miR-302 expression in the IVM group vs. the control group (p = 0.02). The embryo quality score showed a significant difference between the two groups (p = 0.01). CONCLUSION: The present study demonstrated that the IVM process had a negative effect on the expression level of miR-302 in human pre-implantation embryos. Considering the major role of expression miR-302, a reduced potential in miR-302 expression could be related to a decrease in the early embryonic development.

Serum and Peritoneal Fluid Cytokine Profiles in Infertile Women with Endometriosis.

BACKGROUND: Endometriosis is a chronic inflammatory disease with the growth of endometrial cells out of uterus and in the peritoneal cavity. T cell subsets participate in the establishment and progress of the disease by producing different cytokines. OBJECTIVE: To investigate a group of cytokines related to Th1/Th2/Th17/Treg subsets within both peripheral blood and peritoneal fluid (PF) samples from infertile endometriosis women. METHODS: Peripheral blood and PF samples were collected from 30 infertile endometriosis and 30 non-endometriosis fertile women during laparoscopy. Concentration of cytokines, including TNF-α, IFN-γ, TGF-ß1, IL-4, IL-10, IL-17 and IL-23 were evaluated using ELISA method. RESULTS: Results indicated that the concentration of IFN-γ within serum was significantly reduced in endometriosis group (p=0.001). Regarding PF cytokines, TGF-ß1 was increased in endometriosis group (p=0.030). Furthermore, the ratios of IFN-γ/TGF-ß1 and IL-17/IL-23 were significantly different between endometriosis and non-endometriosis women in serum samples (p<0.001 and p<0.01 respectively). The ratios of TNF-α/IL-10 and IL-17/IL-10 were also significantly different regarding PF samples between the two studied groups (p<0.04 and p<0.03 respectively). Finally, significant correlations were observed between the levels of IL-17 and IL-23, inflammatory and anti-inflammatory cytokines, in both samples and serum to PF inflammatory cytokines. CONCLUSION: Based on the results of the present study, in women with endometriosis, the disturbance of cytokines network might gradually activate the inflammatory responses and tissue repair, resulting in endometriosis development after several years.