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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 (

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.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.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.The present study findings show that sperm sorting by the microfluidic method improves fertilization rate in patients with poor fertilization outcomes following ICSI.

The mechanism of Myo-Inositol, as an adjuvant, on key signaling pathways related to oocyte maturation, fertilization rate, and embryo quality as well as ovarian steroidogenesis in cumulus cells of PCOS patients, is still unclear.Infertile patients who were candidates for ART cycles were divided into three groups (n = 30 in each group), including group 1: PCOS patients only receiving folic acid, group 2: PCOS patients receiving daily Myo-Inositol combined with folic acid, and a control group (group 3): normal ovulatory women without PCOS receiving only folic acid from 1 month prior to IVF cycle until the day of ovum pick up. During the ART procedure, oocytes maturation, fertilization rate, and embryo quality were assessed. The gene expressions of FSHR, LHR, CYP11A1, CYP19A1, 3β-HSD2, and StAR were also analyzed using qRT-PCR. Western blot analysis was performed for the evaluation of AKT, ERK, CREB, and AMPK phosphorylation.Despite equal number of retrieved oocytes, the percentages of MII oocytes, fertilization rate, and embryo quality were found to be significantly higher in group 2 due to the administration of inofolic. The expressions of all the studied genes were significantly higher in the cumulus cells of group 1 compared to the group 2. Higher phosphorylation of ERK1/2 was found in the groups 2 and 3 compared to the group 1. On the other hand, p-Akt has significantly decreased in the group 2 compared to the group 1.Our study provides new insight into the molecular mechanism underlying the positive effect of Myo-Inositol on intrinsic ovarian defects in PCOS, steroidogenesis, oocyte maturation, fertilization rate, and embryo quality.

SummaryAlthough 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.

The aim of this study was to investigate the use of time-lapse morphokinetic parameters and cumulus cells transcriptomic profile to achieve a more accurate and non-invasive method in embryo evaluation. Two hundred embryos from 20 couples were evaluated based on morphokinetic characteristics using time-lapse. Embryos were divided into the high-quality, moderate-quality, and bad-quality groups. Non-fertilized oocytes were considered as the fourth group. T5 (time to five cells), S2 (time from three to four cells), and CC2 (time from two to three cells) were recorded. Also, the cumulus cells of the respective oocytes were divided into high-quality, moderate-quality, bad-quality, and non-fertilized groups based on the grading of the embryos. Then their transcriptomic profiles were analyzed by RNA-sequencing. Finally, the correlation between differentially expressed genes and embryo time-lapse parameters was investigated. T5 was the only timing that showed a statistically significant difference between high-quality group and other groups. RNA-sequencing results showed that 37 genes were downregulated and 106 genes were upregulated in moderate, bad-quality, and non-fertilized groups compared to high-quality group (q value

Background:Micro RNAs (miRNAs) are small non-coding RNAs known as essential regulators of cell-cell communication. Recent studies have revealed that miRNAs secrete by a blastocyst in culture mediums. We hypothesized that endometrial epithelial cells take up embryo-derived miRNAs and other soluble factors and regulate their receptivity-related genes expression.Methods and Results:Blastocyst culture mediums (BCM) were collected from the individually cultured embryos and, 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 decreased in the implanted BCM group compared with the non-implanted BCM group. Also, we observed decreased fold changes of miR‐29b,145, and increased miR-223 in the implanted BCM group compared with the non-implanted ones.Conclusions:miRNAs' role as potential gene expression regulators during implantation. These molecules are secreted by human blastocyst, uptake 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, and BCM transfer with the embryo can be useful as an embryo implantation trigger.

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 Ca

Polycystic ovary syndrome (PCOS) is a complex disease that causes an ovulatory infertility in approximately 10% of reproductive-age women. We searched for candidate proteins that might contribute to endometrial receptivity defects in PCOS patients, and result in adverse reproductive outcomes. Shotgun proteomics approach was used to investigate the proteome profile of the endometrium at the luteal phase in PCOS patients compared to healthy fertile individuals. Biological process and pathway analyses were conducted to categorize the proteins with differential expressions. Confirmation was performed for a number of proteins via immunoblotting in new samples. 150 proteins with higher abundance, and 46 proteins with lower abundance were identified in the endometrial tissue from PCOS patients compared to healthy fertile individuals. The proteins with higher abundance were enriched in protein degradation, cell cycle, and signaling cascades. Proteins with lower abundance in PCOS patients were enriched in extracellular matrix (ECM) composition and function, as well as the salvage pathway of purine biosynthesis. Metabolism was the most affected biological process with over 100 up-regulated, and approximately 30 down-regulated proteins. Our results indicate significant imbalances in metabolism, proteasome, cell cycle, ECM related proteins, and signaling cascades in endometrial tissue of PCOS, which may contribute to poor reproductive outcomes in these patients. We postulate that the endometria in PCOS patients may not be well-differentiated and synchronized for implantation. Possible roles of the above-mentioned pathways that underlie implantation failure in PCOS will be discussed. Our findings need to be confirmed in larger populations.

Context: The SARS-CoV-2 virus causes dysfunction of vital organs in the body. Concerns about the destructive effect of SARS-CoV-2 on human reproductive tissues and fertility have increased. Evaluation of the possible mechanisms by which SARS-CoV-2 causes infertility is essential for effective prevention and treatment. This review aims to assess the studies that have been conducted on SARS-CoV-2 impacts on the human reproductive system. Evidence Acquisition: This review study investigated articles indexed in PubMed, Science-Direct, Scopus, and google scholar databases from 2019 to 2021. The Keywords SARS-CoV-2, COVID-19, human reproductive system, testis, and ovary were searched in the mentioned databases. Results: The present study assessed the expression of SARS-CoV-2-specific receptors, the presence of the virus in the human reproductive system, and the mechanisms by which this virus can affect human fertility. Conclusions: SARS-CoV-2, like other viruses, may indirectly influence the male reproductive system through cytokine storms, inflammation-causing oxidative stress, and its possible complications. The direct effects of SARS-CoV-2 on the male reproductive system are also reported. The testis may be a potential target for the SARS-CoV-2 virus. The impact of the SARS-CoV-2 virus on women's reproductive performance is unknown and requires further investigation.

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.

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.

Background: It is demonstrated that optimal preincubation time improves oocyte quality, fertilization potential and developmental rate. This study aimed to evaluate the effect of preincubation time in the simple and myo-inositol supplemented medium on the oocyte quality regarding oxidative stress and mitochondrial alteration. Methods: Cumulus oocyte complexes (COCs) retrieved from superovulated NMRI mice were divided in groups of 0, 4 and 8 hr preincubation time in the simple and 20 mmol/L myo-inositol supplemented media. Intracellular reactive oxygen species (H2O2), glutathione (GSH), mitochondrial membrane potential (MMP), ATP content, and mitochondrial amount were measured and analyzed in experimental groups. One-way ANOVA and Kruskal-Wallis were respectively used for parametric and nonparametric variables. Statistical significance was defined as p

Background: In about 40% of the couples, the cause of infertility problems is attributed to men because of low sperm production and disturbed motility of sperm. Pieces of evidence show that Myo-inositol has a potential role for the treatment of sperm morphology and male fertility. Objectives: This study aimed to determine the effect of Myo-inositol on the sperm parameters and fertility rate in patients with oligoasthenospermia treated by intrauterine insemination (IUI). Materials and Methods: This study was a randomized clinical trial conducted on 37 patients with oligoasthenospermia treated by IUI during 2016-2017. In this study, the patients were randomly divided into two groups of oligoasthenospermia treated with (Case group) and without Myo-inositol (Control group). The case group received 0.5 ml of Myo-inositol with a concentration of 2 mg/ml and incubated at 37°C incubator for 2 hr, but the control group had no interventions. Results: The results of this study showed that although there was no significant difference in sperm parameters including sperm motility and concentration before processing with Myo-inositol in the case group, but there was a significant increase in sperm motility during the treatment with Myo-inositol. The therapeutic effect of this method was confirmed on induction of pregnancy in 18% of the treated patients, in such a way that was about twice greater than those who did not receive the drug. Conclusion: According to the results of this study, the use of Myo-inositol is efficient enough to change sperm parameters to increase the chance of fertility. Key words: Myo-inositol, Pregnancy, Sperm, Motility, Oligoasthenospermic.

© 2019 John Wiley & Sons, Ltd. Spermatogenesis is a process in which animals generate spermatozoa from spermatogonial stem cells (SSCs). Successful in vitro differentiation of SSCs towards spermatids holds a significant promise for regeneration of impaired spermatogenesis. The present study aims to evaluate the efficiency of a 3D culture containing naringenin on proliferation and differentiation potentials of mouse SSCs. In this study, multi-walled carbon nanotubes (MWCNTs) were incorporated into poly(l-lactic acid) (PLLA) fibers via electrospinning technique. The fibrous PLLA/MWCNTs were studied by Fourier-transform infrared spectroscopy (FTIR), transmission electron microscope (TEM), water contact angle measurements, electrical conductivity, and mechanical properties. Next, the SSCs were seeded into the PLLA/MWCNTs scaffolds and exhibited preferable survival and differentiation efficiency to subsequent cell lines. To shed more light on this matter, the immunocytochemistry, reverse-transcription polymerase chain reaction (RT-PCR), and qRT-PCR results showed that the aforementioned cells on the 3D fabrics overexpressed the C-kit and SYCP3 proteins. In addition, the reactive oxygen species (ROS) measurement data demonstrated that naringenin, an effective antioxidant, plays an important role in in vitro spermatogenesis. Taken together, the results of this study revealed the synergistic effects of 3D scaffolds and naringenin for efficient spermatogenesis in laboratories.

Study question Can GM-CSF as a sperm medium supplement improve sperm quality in OAT men? Summary answer GM-CSF can be used to improve sperm quality in OAT men by activating the PI3K/Akt pathway. What is known already OAT patients have very low sperm parameters, including morphology, count, and motility, which can adversely affect the results of assisted reproduction technologies. It seems the development of sperm media is necessary to improve the sperm parameters of these patients. GM-CSF is a natural growth factor produced by the reproductive organs, culture media supplemented with GM-CSF is widely commercially available and enhances the embryo development and implantation rate. Studies show that this growth factor in the semen of infertile men is lower than that of fertile men. However, there is no study to assess the effect of GM-CSF on sperm quality. Study design, size, duration In the present study, Semen specimens were collected from 20 OAT patients who have male infertility factors, according to WHO criteria. After the swim-up washing procedure, each of the samples is divided into two groups; experiment, and control. In the experimental group, samples are incubated with a medium containing 2 ng/ml GM-CSF for one hour, yet, in the control group, the sperms are incubated without GM-CSF for the same time. Participants/materials, setting, methods The sperm motility was examined with phase-contrast microscopy, Eosin-nigrosin staining method was used to assess sperm viability. The expression of sperm glucose transporters (GLUT 1, 3) was determined using immunofluorescent staining, the ratio of pAkt to total Akt was assessed by the Western blotting method. Image J software was used to quantify results; the data was analyzed by SPSS software. P-value Main results and the role of chance As compared to the control group, supplementation with GM-CSF improved sperm progressive motility, enhanced GLUT 1 and 3, and p-AKT/AKT expression (P Limitations, reasons for caution This study needs further investigations, including Real-time PCR for the genes of this signaling pathway which is ongoing. Wider implications of the findings: We showed for the first time that GM-CSF can improve sperm quality by influencing motility and energy metabolism in spermatozoa which can be affected by increasing the phosphorylation of AKT. This growth factor could be an appropriate supplement in sperm media for OAT patients. Trial registration number IRCT20200519047508N1

Study question Which sperm preparation technique separate the best quality sperm? Summary answer Microfluidic method improved the sperm parameters and decreased sperm DNA damage. What is known already About 40% of infertility issues are due to male factor. One of the known causes of male infertility is associated with low sperm parameters and high level DNA fragmentation. Sperm preparation techniques in ICSI procedures is used in order to obtain the best-quality sperm. Study design, size, duration The present study was designed to compare Microfluidic, Zeta potential, Magnetic Activated Cell Sorting (MACS) and Swim-up methods for sperm preparation and the effect of these methods on semen parameters and sperm DNA integrity in infertile men (n = 25) with a mean age of 38. Participants/materials, setting, methods In this study, each sample was divided into 4 groups, one part for preparing by Microfluidic method, one of them for preparing by Swim -up method, the other one was prepared by MACS and the last one was prepared by zeta potential. Then sperm count, viability, motility and morphology were assessed according to WHO 2010. DNA damage were assessed by Sperm DNA Fragmentation assay and sperm chromatin packaging assessed by CMA3 staining test Main results and the role of chance Sperm parameters including viability, motility, and morphology in the Microfluidic method were significantly improved and sperm DNA damage were significantly lower than three other methods (P-value Limitations, reasons for caution The fertilization and pregnancy rate of the resulting embryos are not available. Wider implications of the findings: Our results showed that Microfluidic can be an effective way to improve sperm quality of infertile male compared to conventional preparation methods. We also found instead of the MACS method, we can use the Zeta potential method according to their costs, for sperm preparation during ART cycle. Trial registration number *

Study question Is implantation different in euploid and aneuploid embryos? Summary answer By simulating the human endometrium using a three-dimensional scaffold, aneuploid embryos were unable to attach to the endometrial cells, while euploid embryos attached. What is known already Although embryo selection for transfer is usually based on morphology, 70% of embryos with high morphological quality have chromosomal abnormalities. The results of implantation and pregnancy rate assessments following Preimplantation Genetic Screening (PGS) are controversial. There is still no in vitro study to compare the implantation of human euploid and aneuploid embryos. Study design, size, duration After informed consent, 10 endometrial biopsies were taken from fertile women. For scaffolding, the stromal cells were resided within the matrix, after 24 hours, the epithelial cells were seeded on the scaffold. Cell culture continued for 5 days to reach the appropriate confluence. The embryos were also examined by performing PGS following CGH Array. 10 euploid and 10 aneuploid blastocysts were selected and co-cultured for 72 hours with the 3D structure of human endometrial cells. Participants/materials, setting, methods Endometrial cells were isolated and expanded in 2D cultures to achieve enough cells. The fibrin-agarose scaffold was made and stromal and epithelial cells were cultured into and on the scaffold, respectively. Then, cell proliferation was assessed by MTT assay. The simulated endometrial construct was confirmed by H&E and IHC. Partial hatching of blastocysts was performed using a laser system. The blastocyst’s attachment to the endometrial-like structure was examined under a phase-contrast microscope and SEM. Main results and the role of chance The MTT OD of scaffolds increased during 5 days of cell culture (P In the 3D culture, the origins and locations of epithelial and stromal cells were defined by cytokeratin and vimentin immunostaining, respectively. IHC for cytokeratin was only positive for epithelial cells in the surface epithelium. IHC for the vimentin was positive for the stromal cells located in the 3D matrix. These results showed that fibrin-agarose scaffold could simulate the human endometrial structure. Using SEM and phase-contrast microscopy, it was found that only euploid embryos were able to attach to the endometrial construct while aneuploid embryos weren’t. Limitations, reasons for caution Since the co-culture does not contain a unique cell type, and the MTT OD standard curve against cell number is specified for cell type, the number of growing cells in the co-culture cannot be calculated; therefore it is reported as OD. Wider implications of the findings: Our findings determined that PGS allows us to transfer top-quality embryos with higher implantation potential. It improves implantation and pregnancy rate during ART cycles, especially in patients with recurrent implantation failure. Trial registration number Not applicable

BACKGROUND Endometrial scratching (ES) has been proposed as a potential treatment for implantation improvement in unexplained repeated implantation failure (uRIF) patients, however, little is known about its exact molecular mechanisms. OBJECTIVE This randomized controlled trial (RCT) was conducted on twenty uRIF patients to investigate the expression of innate and adaptive immune signaling genes after ES. METHODS Ten uRIF patients in the intervention (twice endometrial sampling in follicular and luteal phases) and 10 uRIF patients in the control group (only luteal phase sampling) were randomly enrolled. Gene expression analysis with innate and adaptive immune response PCR-array kit between intervention and control groups were performed. RESULTS Among innate immune-associated genes, a significant decrease was observed in the expression of APCS, CPR, CCL2, NLRP3, HLA-A, TLR3 and TLR4 in the intervention group. In adaptive immune-related genes, the expression level of CD80, CD86, CXCR3, IFNγ, IFNα1, IFNβ, MBL2, CCR6, CCR8 and IL17A were decreased and CSF2, GATA3, and IL4 increased significantly in the intervention group (P < 0.05). Of 14 uRIF patients, five live birth (35.71 %) was achieved. CONCLUSION ES in uRIF patients may exert positive effects on the endometrial preparation which increases its receptivity for embryo implantation by modulating the expression of an array of immune signaling pathway genes.

Objective The aim of our study was to detect a biomarker for selection of competent oocytes with acceptable fertilization potential. Calcium ion fluctuation play the most critical role of modulating intercellular signaling pathways in oocyte maturation, egg activation and the egg-to-embryo transition. Since, the stimulatory action of calcium ion is mediated by binding to certain proteins, the calcium/calmodulin-binding genes (CBGs), as the main calcium binding group, was analyzed in detail. Methods In this work, bioinformatics analysis was conducted on the CBGs of human cumulus cells (CCs) to elucidate a reliable biomarker for fertile oocyte selection. Calcineurin (CaN) or protein phosphatase 3 (PPP3) was selected which consists of a catalytic subunit A with PPP3CA (Aα), PPP3CB (Aβ), and PPP3CC (Aγ) isoforms and a regulatory subunit B. Whereas CaN A regulates calcium ion function, our study gives insights to probable role of related isoforms within human oogenesis process. The presence of CaN A in CCs surrounding growing and mature oocytes was confirmed by western blotting and the expression patterns of related isoforms were examined by reverse transcription-quantitative PCR (RT-qPCR). Results Our results indicated the increased expression of the catalytic subunit of CaN protein in the CCs of metaphase (M) II oocytes. The expression level of PPP3CB was significantly elevated in CCs of fertile MII compared with those in the germinal vesicle (GV), MI and unfertilized MII oocytes (P ≤ 0.05). Conclusion Elevated level of PPP3CB isoform in the CCs of fertile MII oocyte could be a reliable indication of oocyte fertilization potential. However, further researches are required to introduce CaN Aβ as an appropriate biomarker for oocyte selection in assisted reproduction technique (ART) programs.

Odorant or olfactory receptors are mainly localized in the olfactory epithelium for the perception of different odors. Interestingly, many ectopic olfactory receptors with low expression levels have recently been found in nonolfactory tissues to involve in local functions. Therefore, we investigated the probable role of the olfactory signaling pathway in the surrounding microenvironment of oocyte. This study included 22 women in intracytoplasmic sperm injection cycle. The expression of olfactory target molecules in cumulus cells surrounding the growing and mature oocytes was evaluated by Western blotting and real-time polymerase chain reaction. Additionally, integrated bioinformatics analyses were carried out and 6 ectopic olfactory receptors were selected for further evaluation. The initiation of olfactory transduction cascade in cumulus cells of competent oocytes was confirmed by analyzing the expression of adenylyl cyclase type 3 and olfactory market protein. Moreover, the expression pattern of the selected olfactory receptors was evaluated and OR10H2 was selected due to a high level of expression in mature fertile oocytes. We suggested that OR10H2 could be considered as a reliable biomarker for oocyte selection in assisted reproduction technique programs. However, further studies are required to elucidate the role of olfactory transduction cascade in embryo quality and implantation.

Objective: The fallopian tubes play a critical role in the early events of fertilization. The rapid innate immune defense is an important part of the fallopian tubes. Toll-like receptor 3 (TLR3), as a part of the innate immune system, plays an important role in detecting viral infections. In this basic and experimental study, the effect of sex hormones on the function of TLR3 in the OE-E6/E7 cell line was investigated. Methods: The functionality of TLR3 in this cell line was evaluated by cytokine measurements (interleukin [IL]-6 and IL-1b) and the effects of sex hormones on TLR3 were tested by an enzyme-linked immunosorbent assay kit. Additionally, TLR3 small interfering RNA (siRNA) and a TLR3 function-blocking antibody were used to confirm our findings. Results: The production of IL-6 significantly increased in the presence of polyinosinic-polycytidylic acid (poly(I:C)) as the TLR3 ligand. Using a TLR3-siRNA-ransfected OE-E6/E7 cell line and function-blocking antibody confirmed that cytokine production was due to TLR3. In addition, 17-β estradiol and progesterone suppressed the production of IL-6 in the presence and absence of poly(I:C). Conclusion: These results imply that sex hormones exerted a suppressive effect on the function of TLR3 in the fallopian tube cell line when different concentrations of sex hormones were present. The current results also suggest that estrogen receptor beta and nuclear progesterone receptor B are likely to mediate the hormonal regulation of TLR3, as these two receptors are the main estrogen and progesterone receptors in OE-E6/E7 cell line.

The tight junction between epithelial cells helps making connections in the fallopian tube and contributes to successful fertilization. Breaking the tight junction complex induces various diseases such as the EP. Previous studies have shown that glucocorticoids are effective in repairing and maintaining intercellular tight junctions in epithelial cells of the fallopian tube, although their mechanism is still unknown. This research is a genomic study of hydrocortisone’s effect on epithelial cells of the fallopian tube. Using the human fallopian tube, epithelial cell line (OE-E6/E7) was cultured in four concentrations of hydrocortisone (0 nM, 50 nM, 100 nM and 200 nM) for three durations (24 h, 48 h and 72 h). Glucocorticoids are effective on the expression of Zona occluding-1(ZO-1), Claudin 4, Claudin3, Desmoglein and E-cadherin genes involved in the tight junctions of the fallopian tube. The expression of all genes was up-regulated in the concentrations of 100 nM after 48 h treatment, as compared with the control (0 nM). However, their expression was down-regulated significantly after 72 h treatment (P The present study showed that treatment of epithelial cells of the fallopian tube with glucocorticoid increased the expression of genes involved in tight junctions, including claudin-3, claudin-4, E-cadherin, zona occludin-1 and Desmoglein-1. The obtained data suggests that a new mechanism is developed for glucocorticoid induction of tight junctions by increasing the expression of claudin-3, claudin-4, E-cadherin, zona occludin-1 and Desmoglein-1 genes.

Objective To determine the localization, expression, and function of Toll-like receptors (TLRs) in fallopian tube epithelial cells. Methods The localization of TLRs in fallopian tube epithelial cells was investigated by immunostaining. Surprisingly, the intensity of staining was not equal in the secretory and ciliated cells. After primary cell culture of fallopian tube epithelial cells, ring cloning was used to isolate colonies of ciliated epithelial cells, distinct from non-ciliated epithelial cells. The expression of TLRs 1-10 was examined by quantitative real-time polymerase chain reaction, and protein localization was confirmed by immunostaining. The function of the TLRs was determined by interleukin (IL)-6 and IL-8 production in response to TLR2, TLR3, TLR5, TLR7, and TLR9 ligands. Results Fallopian tube epithelial cells expressed TLRs 1-10 in a cell-type-specific manner. Exposing fallopian tube epithelial cells to TLR2, TLR3, TLR5, TLR7, and TLR9 agonists induced the secretion of proinflammatory cytokines such as IL-6 and IL-8. Conclusion Our findings suggest that TLR expression in the fallopian tubes is cell-type-specific. According to our results, ciliated cells may play more effective role than non-ciliated cells in the innate immune defense of the fallopian tubes, and in interactions with gametes and embryos.

A significant part of couples in IVF-ICSI cycles experience Repeated Implantation Failure (RIF). Screening of the embryos with new methods like Next Generation Sequencing and arrays showed that even euploid embryos fail to implant. Immunology is a potent window maybe resolve the RIF problem. In this investigation we employed innate and adaptive immune system PCR array to compare the transcriptome profiles of endometrium in unexplained RIF and healthy fertile women. A total of 21 women were enrolled in the present study, 11women with unexplained RIF and 10 healthy fertile women. After RNA extraction and cDNA synthesis PCR array was performed using RT2 profiler PCR array human innate and adaptive immune responses kit (Qiagen, Cat.No: PAHS-052A). PCR Array data analysis identified significantly greater expression of IL6, IFNG, IL17A, IL23A, IFNA1, IFNB1, CD40 L, CCR4, CCR5, CCR6, CXR3, CCL2, IL2, TLR4, IRF3, STAT3, RAG1, IFNAR1 in unexplained RIF women than in controls (P < 0.05). However, expression of IL1B, IL8, NFKB, HLA-A, HLA-E, CD80, CD40 was significantly lower in unexplained RIF group than in controls (P < 0.05). Our results showed that modulation of immune system in RIF patient is shifted to inflammatory responses as pNK cells, Th17 signaling pathway and TLR signaling pathway are activated. So, by stimulation of immune system and initiation of humoral immune responses the panel of immunity and immunotolerance is completely changed in RIF patients comparing normal. It seems that attention to these alterations individually help physician to manage RIF patients better.

Stimulation of ovulation with clomiphene citrate can cause side effects on endometrial receptivity. Formulation with nano-size may be an alternative therapy for women with ovulatory disorders. In this study, we investigated sustained-release clomiphene citrate by using Phosal-based formulation (PBF) and evaluate its decreased side effect on the endometrial receptivity.In the in-vitro study, CC loaded PBF was analyzed using Zetasizer, Fourier-transform infrared spectroscopy (FTIR), and Transmission electron microscopy (TEM). In the in-vivo study, 24 female mice were randomly divided into three groups: CC (5 mg/kg), CC/PBF (5 mg/kg) and SS (1 ml) daily administered and injected with 5 IU HCG and mated after two days. At day 4.5, pregnant mice were euthanized and endometrial tissue was extracted for quantitative polymerase chain reaction (Q-PCR) analysis.The optimized PBF contained Phosal 50PG/glycerol in a 2:8 ratios (w/w) and the particle size of optimum formulation was 67 ± 0.30551 nm and the release of CC from CC-containing PBF was slightly faster in the first 24 h; wherein, 29% of CC was released, and 76% of CC was released up to 120 h. The mRNA levels of leukemia inhibitory factor (LIF), leukemia inhibitory factor receptor alpha (LIFR), HOXA10, Heparin-binding epidermal growth factor (HB-EGF), and epidermal growth factor (EGF) were significantly upregulated and MUC1 and PGR mRNA levels were significantly downregulated in the CC-containing PBF-treated animals compared with only CC group (P 0.05).Sustained release formulation of clomiphene citrate increased its targeting efficiency and improved the impact of the CC on implantation. Graphical abstract A new Phosal Based Formulation (PBF) was designed to decrease the side effects of Clomiphene citrate (CC) on endometrium. This drug formulation could react better during implantation by increasing the expression of genes involved in implantation. The in vivo study demonstrated that the CC-containing PBF in mice has a significantly higher endometrial receptivity, compared with the suspension.

Background Spermatozoa interactions with fallopian tubes may influence fertilization. The purpose was to investigate cytokines, chemokines and growth factors expression from human fallopian tube epithelial cells (OE-E6/E7) exposed to spermatozoa. Methods Fresh semen samples were obtained from 10 healthy normozoospermic men. Sperms were prepared and co-cultured with OE-E6/E7. The cell line without spermatozoa was considered as the control group. Afterwards, Expression of 84 cytokines from OE-E6/E7 cell line in the presence and absence of spermatozoa were measured using PCR-array. Quantitative PCR was performed on seven genes to confirm the results of PCR-array analysis. Differentially expressed genes were subjected to www.geneontology.org and www.pantherdb.org to perform GO enrichment and panther pathway analysis. The concentration of IL-8, IL-10, IL-1B and BMP-4 in culture medium were analyzed by ELISA. Results Sperm interaction with the epithelial cells resulted in a significant increase in expression of TGF-β2, BMP-4, IL-10, IL-9, and CD40LG markers. Moreover, expression of IL-16, IL-17F, SPP-1, CXCL-13, MSTN, IL-1A, IL-1B, IL-8, BMP-7, CSF-2, CSF-3, VEGF-A, OSM, LTA, TNF, TNFRSF11B, TNFSF11, CCL-11, CCL-20, CCL-24, CCL-3, CCL-8, CX3CL1 and CXCL-9 were considerably reduced in presence of spermatozoa. Panther pathway analysis discovered 3 pathways for upregulated genes including gonadotropin-releasing hormone receptor, TGF-beta and interleukin signaling pathways. Furthermore, 9 pathways were detected for down-regulated genes. Inflammation signaling pathway which is mediated by chemokine and cytokine contains the most number of genes. Conclusion This study indicates that sperm modifies expression of cytokines, chemokines and growth factors from OE-E6/E7. Moreover, altered genes expression are toward higher survival chance of the spermatozoa.

The aim of the present study was to evaluate the effect of myo-Inositol administration on oocyte quality, fertilization rate and embryo quality in patients with PCOS during assisted reproductive technology (ART) cycles. Fifty infertile PCOS patients were randomly designated in two groups. In the study group, patients received daily doses of 4 g myo-Inositol combined with 400 mg folic acid and in the control group patients received only 400 mg folic acid from 1 month before starting the antagonist cycle until the day of ovum pick up. Oocyte and embryo qualities were assessed according to European Society of Human Reproduction and Embryology (ESHRE) guidelines. The gene expression of PGK1, RGS2 and CDC42 as a factor of oocyte quality in granulosa cells was analyzed using real-time RT-PCR. Levels of total antioxidant capacity (TAC) and reactive oxygen species (ROS) were evaluated by chemiluminescence assay in follicular fluid. The percentage of metaphase II oocyte, fertilization rate and embryo quality significantly improved in the study group (p

The aim of this study is to evaluate the beneficial effect of Myoinositol (MYO) supplement in freezing media on the post thaw sperm quality. Semen samples from 40 normozoospermic men were divided into two aliquots and frozen with simple or 2 mg/mL MYO supplemented freezing medium. Post thaw process including, computer-assissted sperm analysis was used to analyze sperm motility and morphology. Reactive oxygen species was evaluated by the fluorometry of DCFH-DA, as well as total antioxidant capacity and lipid peroxidation were measured based on colorimetric assay by ELISA reader. Eventually, DNA fragmentation was assessed using TUNEL staining. MYO significantly improved progressive motility and normal morphology in treated samples (p 0.05). Lipid peroxidation (malondialdehyde level) can be diminished in samples were frozen by MYO supplemented freezing media (p 0.05). While MYO did not affect the amount of ROS (p 0.05), it was associated with high values of total antioxidant capacity (p 0.05). DNA integrity was significantly affected by MYO, as in MYO treated samples, DNA fragmentation was decreased compared to control ones (p 0.001). The findings support the use of 2 mg/mL myoinositol supplemented freezing media in sperm cryopreservation to increase sperm quality after freezing-thawing procedures.

Background: Women with polycystic ovary syndrome have lower pregnancy rates, possibly due to the decreased uterine receptivity.Successful implantation depends on protein networks that are essential for cross-talk between the embryo and endometrium.Apolipoprotein A1 has been proposed as a putative anti-implantation factor. In this study, we evaluated apolipoprotein A1 expression in human endometrial tissues. Materials and Methods: Endometrial apolipoprotein A1 messenger RNA (mRNA) and protein expression were investigated using quantitative real-time polymerase chain reaction (PCR) and Western blot. The distribution of apolipoprotein A1 was also detected by immunostaining. Samples were obtained from 10 patients with polycystic ovary syndrome and 15 healthy fertile women in the proliferative (on day 2 or day 3 before ovulation, n = 7) and secretory (on days 3-5 after ovulation,n = 8) phases. Results: Endometrial apolipoprotein A1 expression was upregulated in patients with polycystic ovary syndrome compared to normal subjects. However, apolipoprotein A1 expression in the proliferative phase was signifi cantly higher than in the luteal phase (P value < 0.05). Conclusion: It seems that diff erentially expressed apolipoprotein A1 negatively aff ects endometrial receptivity in patients with polycystic ovary syndrome. The results showed that apolipoprotein A1 level signifi cantly changes in the human endometrium during the menstrual cycle with minimum expression in the secretory phase, coincident with the receptive phase (window of implantation). Further studies are required to clarify the clinical application of this protein. Key words: Apolipoprotein A1, endometrium , polycystic ovary syndrome, proliferative phase, secretory phase

Research question What is the molecular basis of infertility related to uterine dysfunction in women with polycystic ovary syndrome (PCOS)? Design In this study, differences in protein expression between PCOS and normal endometrium were identified using a proteomic approach based on two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS). The proteome of endometrium were analysed during the proliferative (on day 2 or 3 before ovulation, n = 6) and luteal phases (on day 3–5 after ovulation, n = 6) from healthy women and PCOS patients (12–14 days after spontaneous bleeding, n = 12). The differentially expressed proteins were categorized based on the biological process using the DAVID bioinformatics resources. Results Over 803 reproducible protein spots were detected on gels, and 150 protein spots showed different intensities between PCOS and normal women during the proliferative and luteal phases. MS analysis detected 70 proteins out of 150 spots. For four of the 70 proteins, 14-3-3 protein, annexin A5, SERPINA1 and cathepsin D, 2-DE results were validated and localized by Western blot and immunohistochemistry, respectively, and their gene expression profiles were confirmed by real-time quantitative PCR. The obtained results corresponded to the proteomic analysis. The differentially expressed proteins identified are known to be involved in apoptosis, oxidative stress, inflammation and the cytoskeleton. Conclusions The processes related to the differentially expressed proteins play important roles in fecundity and fecundability. The present study may reveal the cause of various endometrial aberrations as a limiting factor for achieving pregnancy in PCOS women.

Background: Tumor development is angiogenesis dependent. There is evidence that leptin contributes to tumor growth. However, all the mechanisms by which leptin does this has not been clearly established. The objective of the present study was to test the hypothesis that leptin enhances melanoma tumor growth through inducing angiogenesis and cell proliferation. Materials and Methods: We injected 2 × 106 B16F10 melanoma cells subcutaneously to 32 C57BL6 mice. The mice were randomly divided into four groups of eight animals, on day 8. Two groups received twice daily intraperitoneal (i.p.) injections of either phosphate buffered saline or recombinant murine leptin (1 μg/g initial body weight). Two groups received i.p. injections of either 9F8 an anti leptin receptor antibody or the control mouse IgG at 50 μg/injection every 3 consecutive days. By the end of the 2nd week, the animals were euthanized and blood samples and tumors were analyzed. Angiogenesis and proliferation were assessed by immunohistochemical staining for CD31 and Ki-67 respectively. Results: Tumors size, capillary density, plasma levels of vascular endothelial growth factor, and the number of Ki-67-positive stained cells were significantly more in the leptin than 9F8 and both control groups (P < 0.05). Conclusion: Taken together, our findings reinforce the idea that leptin acts as an angiogenic and mitogenic factor to promote melanoma growth.

To evaluate expression of glucocorticoid-induced tumor necrosis factor receptor (GITR), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and IL-10 in peripheral blood mononuclear cells (PBMCs) of 20 women with unexplained recurrent spontaneous abortion (URSA) compared to 20 normal non-pregnant women (NNP) during luteal phase in the window of implantation.Quantitative real-time PCR (qRT-PCR) was performed using the Taqman method for expression of GITR and SYBR Green method for expression of CTLA-4 and IL-10.Expression of CTLA-4 in the NNPs (median; interquartile range; 3; 1.8-10) was significantly higher than the URSAs (0.72; 0.26-3.81, p = 0.015). Expression of GITR in the NNPs (53; 10-139) was significantly higher than the URSAs (6; 3-27, p = 0.005). However, IL-10 expression in the URSAs was significantly higher than the NNPs, did not meet a significant value. A significant correlation was found between CTLA-4 and GITR expression in the study population (p = 0.0001).Expression of CTLA-4 and GITR were significantly down-regulated in the URSAs compared to NNPs at the window of implantation, which shows the essential role of Treg cells in creating an immunological privileged site for fetus as an allograft at the maternal-fetal interface by high expression levels of CTLA-4 and GITR during a normal pregnancy.

Background: Prenatal drug exposure, as a common public health concern, is associated with an increased risk of adverse effects on early embryo development. Objective: To investigate the in vitro development of - embryo from experimentally Kerack-addicted mice. Materials and Methods: Twenty-five female mice were studied in five groups: control, vehicle, and three experimental groups of Kerack-dependent mice (I, II, and III) which received different doses of Kerack for 14 days. After the establishment of addiction model (7 days), experimental groups I, II, and III were given Kerack intraperitoneally at the doses of 5, 35, and 70 mg/kg, twice a day for a period of 7 days, respectively. The vehicle group received normal saline and lemon juice whilst the control group just received water and food. Morulae were obtained through oviduct flashing. The survived embryos were cultured in T6+ 5mg/ml bovine serum albumin. The developmental rates up to hatched stage daily and embryo quality (differential staining and Tunnel staining) were also assessed Results: The developmental potential of embryos obtained from the addicted mother was significantly decreased in comparison with control group. There was a significant reduction in the rate of blastocyst formation in the high dose Kerack dependent group. However, in addicted mice there was reduction in the total cell number (40.92% vs. 65.08% in control) and, inner cell mass percentage (17.17% vs. 26.15% in control) while apoptotic cells numbers were increased (7.17 vs. 1.46 in control) (p