Leukemia inhibitory factor's effect on the growth and survival of sheep's follicles of ovarian tissue during vitrification.

One of the experimental programs for fertility protection in women includes protective cryopreservation. Vitroficasion of ovarian tissue is one of the protective cryopreservation methods that use high concentrations of antifreeze and faster cooling. To reduce its complications, LIF (Leukemia inhibitory factor) was used as a pretreatment in this study. In this study, the ovaries were randomly divided into 8 groups. In NCN (without pretreatment and LIF in culture media), NCP (without pretreatment and with LIF in culture media), PCP (with pretreatment and LIF in culture media), and PCN (with pretreatment and without LIF in culture media) groups, vitrification and reversal were not performed. In the groups NVN (without pretreatment and LIF in culture media), NVP (without pretreatment and with LIF in culture media) PV, PVP (with pretreatment and LIF in culture media), and PVN (with pretreatment and without LIF in culture medium) groups, vitrification and tissue reversal were performed. All groups were cultured and histological, cellular, and molecular evaluations were performed. The results of the present study showed that LIF in the culture medium reduced the number of abnormal, primordial, primary, and secondary follicles, and DNA breakage compared to the group without LIF (P < 0.05) and increases the growth of follicles and expression of GDF9, BMP, AMH, KITLG genes (P < 0.05). The use of LIF pretreatment before vitrification and melting of sheep ovary tissue in its culture medium reduces the damage caused by it and increases the growth and development of ovarian follicles while maintaining their function.

Comparison of the Efficiency of Magnetic-Activated Cell Sorting (MACS) and Physiological Intracytoplasmic Sperm Injection (PICSI) for Sperm Selection in Cases with Unexplained Infertility.

Background: The cases with unexplained infertility may have an abnormality in their sperm chromatin structure. Sperm selection methods can be used to separate sperm with low DNA fragmentation. The purpose of this study was to compare the efficacy of physiological intracytoplasmic sperm injection (PICSI) with magnetic-activated cell sorting (MACS) in assisted reproductive techniques in cases with unexplained infertility. Methods: The semen samples were collected from couples with unexplained infertility. After semen analysis and sperm DNA fragmentation (SDF) evaluations, samples were prepared with swim-up method. The rates of SDF in different fractions including raw semen (n=20), swim-up (n=20), only motile sperm after swim-up (swim-up selection) (n=20), MACS sperm selection (n=20), only motile sperm after MACS (MACS selection) (n=20), and PICSI sperm selection (n=16) were evaluated. Also, the main sperm characteristics and fine morphology of sperm suspension after MACS were assessed. Statistical analysis was performed using GraphPad Prism. The p<0.05 was considered statistically significant. Results: DNA fragmentation index (DFI) values in PICSI and MACS groups were significantly reduced as compared to the swim-up group. The rate of this reduction was more pronounced in MACS (58.20±13.02) than PICSI (36.57±15.52) group. Also, our results showed that MACS resulted in decreased sperm motility, with no alteration in their fine morphology. Conclusion: MACS was found to be more efficient in reduction of SDF rates than PICSI. However, none of the sperm selection techniques can not totally eliminated the spermatozoa with DNA fragmentation in the final sperm sample.

Comparison of the efficacy of bone morphogenetic protein-4 on in vitro differentiation of murine adipose and bone marrow mesenchymal stem cells into primordial germ cells.

Background and purpose: In vitro development of functional gametes from pluripotent stem cells is a promising prospect to treat infertility. Mesenchymal stem cells with a high degree of plasticity and less tumorigenicity are a reliable source of stem cells for the generation of gametes. The present study aimed to compare the differentiation potential in the mesenchymal stem cells that are derived from bone marrow (BMDMSCs) and adipose tissue derived mesenchymal stem cells (AD-MSCs) into germ cells in a culture medium containing bone morphogenic protein-4 (BMP-4). Experimental approach: In this study, MSCs were isolated from both bone marrow and adipose tissue of murine samples. To further verify the nature of the harvested stem cells, their multipotency and surface marker were examined. The identified stem cells were cultured in a medium supplemented with 0 and 25 ng/mL of BMP-4 for 4 days. Flow cytometry analysis, immunofluorescence staining, and real RT-PCR were used to assess the expression levels in germ cell-specific biomarkers (Mvh, Dazl, Stra8, and Scp3). Findings/Results: CD44+, CD45-, CD31-, BMD-MSCs, and AD-MSCs showed to be capable of differentiating to osteo-adipogenic lineages. The flow cytometry, immunofluorescence, and RT-PCR results indicated that early germ cell markers (Mvh and Dazl) were expressed in both types of cells but they were significantly higher in BMD-MSCs than AD-MSCs. Conclusion and implications: Based on our results, the addition of exogenous BMP4 to the culture medium could differentiate BMD-MSCs and AD-MSCs into primordial germ cells, but it is inadequate to further develop into late germ cells in vitro. Moreover, the results revealed that, although AD-MSCs were easier to collect and had faster growth and proliferation rates than BMD-MSCs, the BMD-MSCs were better capable of differentiation into primordial germ cells. They may serve to be considered a more suitable source of MSC for in vitro generation of gametes than AD-MSCs.

Chitosan Scaffold Containing Periostin Enhances Sternum Bone Healing and Decreases Serum Level of TNF-α and IL-6 after Sternotomy in Rat.

BACKGROUND: In the aftermath of bone injuries, such as cranium and sternum, bone wax (BW) is used to control bleeding from the bone surfaces during surgery. Made up of artificial substances, however, it is associated with many complications such as inflammation, increased risk for infection, and bone repair delay. We, therefore, in this study set out to design and evaluate a novel BW without the above-mentioned side-effects reported for other therapies. METHODS: The pastes (new BW(s)) were prepared in the laboratory and examined by MTT, MIC, MBC, and degradability tests. Then, 60 adult male Wistar rats, divided into six equal groups including chitosan (CT), CT-octacalcium phosphate (OCP), CT-periostin (Post), CT-OCP-Post, Control (Ctrl), and BW, underwent sternotomy surgery. Once the surgeries were completed, the bone repair was assessed radiologically and thereafter clinically in vivo and in vitro using CT-scan, H&E, ELISA, and qRT-PCR. RESULTS: All pastes displayed antibacterial properties and the CT-Post group had the highest cell viability compared to the control group. In contrast to the BW, CT-Post group demonstrated weight changes in the degradability test. In the CT-Post group, more number of osteocyte cells, high trabeculae percentage, and the least fibrous connective tissue were observed compared to other groups. Additionally, in comparison to the CT and Ctrl groups, higher alkaline phosphatase activity, as well as decreased level of serum tumor necrosis factor-α, interleukin-6, and OCN in the CT-Post group was evident. Finally, Runx2, OPG, and RANKL genes' expression was significantly higher in the CT-Post group than in other groups. CONCLUSION: Our results provide insights into the desirability of pastes in terms of cellular viability, degradability, antibacterial properties, and surgical site restoration compared to the BW group. Besides, Periostin could enhance the osteogenic properties of bone tissue defect site.

The effect of mitochondria-targeted antioxidant MitoQ10 on redox signaling pathway components in PCOS mouse model.

PURPOSE: Considerable evidence suggests that mitochondrial dysfunction and oxidative stress contribute to the pathogenesis of Polycystic ovary syndrome (PCOS). We aimed to evaluate the effectiveness of mitochondria-targeted antioxidant, MitoQ10, on the redox signaling pathway's component in PCOS. METHOD: We assessed TXNIP, TRX, and ASK1 expression in granulosa cells (GCs) of the DHEA-induced PCOS mouse model. Female BALB/c mice in five groups of Control, DHEA, and DHEA + MitoQ10 in three doses of 250, 500, and 750 µmol/L MitoQ10 were treated for 21 days. RESULTS: Histological investigation showed a probable improvement in folliculogenesis; besides, ASK1 and TXNIP expression were significantly increased in GCs of the PCOS mouse F4Fmodel as compared to the control groups and decreased steadily in groups treated by MitoQ10. However, TRX expression showed a drop that was restored by MitoQ10 meaningfully (P ≤ 0.05). CONCLUSION: The work presented herein suggests mitochondria-targeted antioxidant, MitoQ10, have modulating effects on folliculogenesis in the ovary and also on the redox signaling pathway in GCs of PCOS mouse model which may have potential to attenuate oxidative stress and its relative damages.

Metformin improves epigenetic modification involved in oocyte growth and embryo development in polycystic ovary syndrome mice model.

The possible relationship between dehydroepiandrosterone (DHEA)-induced polycystic ovary syndrome (PCOS) and epigenetic changes (ECs) leading to the impaired oocyte quality, has not been investigated yet. So, this study aimed to provide an insight into the relationship of the impaired oocyte quality with ECs in a mice DHEA-induced PCOS model and to further reveal the effect of metformin treatment. For this purpose, 80 female BALB/C mice were randomly divided into four equal groups, named as the control, sham, (DHEA) and DHEA + Metformin groups. The alterations in acetylation of H4K5 and H4K16, and in methylation of DNA (5MeC) and H3K9 were evaluated using immunocytochemical. Moreover, the expression of Hdac1, Hdac2, Dnmt1, and Dnmt3a genes involved in ECs were analyzed using reverse-transcription polymerase chain reaction. As well, the levels of mitochondrial membrane potential (MMP), oxidative stress (OS), embryo development, ovarian morphology, sexual hormone, ovulatory function, and AMPKα phosphorylation activity were compared in all the studied groups. Metformin attenuated the damages induced by DHEA as indicated by the normalized the estrous cycle, the improved ovarian morphology, the decreased sexual hormone and OS levels, and the increased MMP and AMPKα phosphorylation levels. In the metformin group, the Dnmt1, Dnmt3a, and Hdac2 genes have significantly upregulated compared to the DHEA group. However, metformin was found to have no effect on the expression level of Hdac1. In this regard, significant decrease and increase were observed in both the acetylated H4K16 and methylated H3K9 within MII oocytes in the DHEA + Metformin group compared with the DHEA group. Our results show that metformin could enhance the developmental competence of PCOS oocytes via reducing OS and ECs.

Astaxanthin Protects Human Granulosa Cells against Oxidative Stress through Activation of NRF2/ARE Pathway and Its Downstream Phase II Enzymes.

OBJECTIVE: Astaxanthin (AST) has been introduced as a radical scavenger and an anti-apoptotic factor that acts via regulating the nuclear factor-E2-related factor 2 (NRF2) and related factors. Here, we intended to examine the effect of AST on granulosa cells (GCs) against oxidative stress by examining NRF2 and downstream phase II antioxidant enzymes. MATERIALS AND METHODS: In this experimental study, we used cultured human primary GCs for the study. First, we performed the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test to evaluate cells viability after treatment with hydrogen peroxide (H2O2) and AST. The apoptosis rate and ROS levels were measured by flow cytometry. To determine NRF2 and phase II enzymes expression, we performed real-time polymerase chain reaction (PCR). Finally, we used western blot to measure the protein levels of NRF2 and Kelch-like ECsH-associated protein 1 (KEAP1). Enzyme activity analysis was also performed to detect NRF2 activity. RESULTS: This study showed that AST suppressed ROS generation (P<0.01) and cell death (P<0.05) in GCs induced by oxidative stress. AST also elevated gene and protein expression and nuclear localization of NRF2 and had an inhibitory effect on the protein levels of KEAP1 (P<0.05). Furthermore, when we used trigonelline (Trig) as a known inhibitor of NRF2, it attenuated the protective effects of AST by decreasing NRF2 activity and gene expression of phase II enzymes (P<0.05). CONCLUSION: Our results presented the protective role of AST against oxidative stress in GCs which was mediated through up-regulating the phase II enzymes as a result of NRF2 activation. Our study may help in improving in vitro fertilization (IVF) outcomes and treatment of infertility.

The Effect of Astaxanthin and Metformin on Oxidative Stress in Granulosa Cells of BALB C Mouse Model of Polycystic Ovary Syndrome.

Reactive oxygen species (ROS), involved in the pathogenesis of the polycystic ovary syndrome (PCOS), play a key role in the onset of apoptosis in follicles and granulosa cells (GCs). We aimed to investigate the antioxidant effects of AST and metformin separately and in combination on GCs using a PCOS mouse model. Forty-eight prepubertal female BALB C mice aged 25-30 days and weighing 12-14 g were studied. The PCOS model was created by subcutaneous injection of the dehydroepiandrosterone (DHEA) hormone in 8 mice of BALB C for 20 consecutive days. Apoptosis and the amount of ROS were evaluated in GCs of the ovaries via flow cytometry. The activity of AKT protein was measured by western blot, and the viability of GCs was investigated using spectrophotometry. Ovarian tissue sections were prepared, stained with H&E, and the morphology of the sections was examined. Statistical analysis was performed by SPSS v22.0 software using one-way ANOVA. We found that AST administration leads to a significant reduction in oxidative stress (P<0.01) and consequently a significant decrease in the rate of apoptosis (P<0.01). While the expression of AKT in the AST group revealed a significant increase (P<0.05), it decreased in the metformin group. However, it was still significantly higher than the control and PCOS groups. Ovulation was confirmed in both metformin and AST groups. Further studies are warranted to prove the efficacy of AST and to introduce it as a complementary therapeutic agent in PCOS.

Effect of miR-30a-5p on Apoptosis, Colonization, and Oxidative Stress Variables in Frozen-Thawed Neonatal Mice Spermatogonial Stem Cells.

Cryopreservation of spermatogonial stem cells (SSCs) is a useful method for fertility preservation in preadolescent children suffering from cancer. However, SSCs may become damaged during cryopreservation due to the generation of reactive oxygen species (ROS). For this reason, various antioxidant agents have been used to protect SSCs from cryopreservation-induced damages. Recently, it has been reported that miR-30a-5p has antiapoptotic and antioxidant activity. The aim of this study was to assess the antiapoptotic and antioxidant effects of miR-30a-5p mimics in frozen-thawed SSCs. To this end, SSCs were isolated from male BALB/C mice (3-6 days old) and cultivated for 14 days. After the detection of optimum concentration, a miR-30a-5p mimic or miR-30a-5p inhibitor with Lipofectamine was transfected into SSCs and, finally, the cell groups were frozen for 1 week. After thawing, different properties, including cell viability (using MTT), colonization of SSCs (number and diameter of colonies), ROS generation (using DCFH-DA assay), levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and gene expression of Bcl-2 and BAXBax (using quantitative real-time PCR), were investigated. The transfection of SSCs with miR-30a-5p mimics before the freezing-thawing process significantly increased the viability, number, and diameter of SSCs colonies. Also, the miR-30a-5p mimic decreased the levels of ROS production and MDA, but it increased the SOD levels. Moreover, the miR-30a-5p mimic decreased BAX and increased Bcl-2 expression in frozen-thawed SSCs. The transfection of SSCs with the miR-30a-5p mimic can increase cell viability and antioxidant defense, and it can decrease apoptosis during the freezing-thawing process. If SSC is able to produce spermatozoa after the transfection of miR-30a-5p and the freezing-thawing process, it can be suggested as a promising strategy for the cryopreservation of SSCs in prepubertal boys suffering from cancer.

A randomized exploratory trial to assess the effects of resveratrol on VEGF and TNF-α 2 expression in endometriosis women.

Resveratrol, a naturally synthesized polyphenolic compound found in some fruits, has anti neoplastic, anti-inflammatory, anti-oxidative, and anti-angiogenic properties. Angiogenesis is an important process in endometriosis which provides blood supply for implantation, proliferation and survival of endometriotic lesions. In this study, we assessed the effects of resveratrol on vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-α) expression in the eutopic endometrium of infertile patients with endometriosis within the window of implantation as a randomized exploratory trial. Subjects, who confirmed their endometriosis (stage III-IV) by a pathologist after laparoscopic surgery, were recruited to the present trial. A total of 34 patients were randomly divided into treatment (n = 17) and control (n = 17) groups, beside the routine protocol for treatment of endometriosis, they received resveratrol and placebo (400 mg) for 12-14 weeks, respectively. Endometrial tissue was collected from both groups before and after the intervention in the mid-secretory phase. Gene and protein expression levels of VEGF and TNF-α in the eutopic endometrium were assessed by Real-Time PCR and Western blotting, respectively. VEGF and TNF-α gene and protein levels in the treatment group showed significant decrease following intervention. It seems resveratrol may improve the endometrium of endometriosis patients in window of implantation period by modifying the expression of VEGF and TNF-α but further investigations are needed to reveal the potential role of this compound.

The modulating effects of vitamin D on the activity of ß-catenin in the endometrium of women with endometriosis: a randomized exploratory trial.

AIMS: The aim of this prospective study was to investigate the effects of vitamin D on the expression and activity of ß-catenin, as the key molecule of the Wnt/ß-catenin signaling pathway, in endometriosis women. MATERIALS AND METHODS: Thirty four infertile women with stage III or IV endometriosis were randomly divided to two groups. The control group received the routine treatment and the treatment group, beside the routine protocol, received 50000 IU vitamin D weekly for 12-14 weeks. Blood and endometrial tissue were collected from both groups before and after the intervention. Protein and Gene expression levels of ß-catenin were assessed by Western blotting and Real-Time PCR, respectively. RESULTS: Compared to before intervention, the expression of active form of ß-catenin reduced significantly within treatment group (p = .000), in addition, the difference between control and treatment groups (p = .012) was significant after intervention, too. Also, the ratio of active/total form of ß-catenin protein expression was significantly decreased within the treatment group at the end of intervention period (p = .000). CONCLUSIONS: It seems vitamin D can change the activity of ß-catenin protein in the endometrial cells of endometriosis patients. Further studies on the therapeutic potential of vitamin D in modifying the ß-catenin activity in endometriosis patients are warranted. CLINICAL TRIAL REGISTRATION NUMBER: IRCT2015081823678N1. TRIAL REGISTRATION DATE: 29 September 2015.

Assessing the effect of MitoQ10 and Vitamin D3 on ovarian oxidative stress, steroidogenesis and histomorphology in DHEA induced PCOS mouse model.

Polycystic ovary syndrome (PCOS) continues to be one of the most complex reproductive and endocrine disorder among women of reproductive age. Recent reports have identified close interaction of Vitamin D deficiency and oxidative stress (OS) in exacerbating the pathophysiology of PCOS. This current study aims at assessing the combine effect of MitoQ10 and Vitamin D3 on dehydroepiandrosterone (DHEA) induced PCOS. Following successful induction of PCOS using DHEA, mice were organized into five groups (n = 8) namely: Negative Control (NC), Vitamin D3 Vehicle (VDV), Vitamin D3 (VD), MitoQ10 (MQ), Vitamin D3 plus MitoQ10 (V+M) and DHEA, ethanol and distilled water, Vitamin D3, MitoQ10 and Vitamin D3 plus MitoQ10 were respectively administered for 20 consecutive days. The study also included positive control (PC) group (n = 8) in which no treatment was applied. Treatment effects were assessed using hormonal assays, biochemical assays, Real-Time PCR, western blotting and histological analysis. Combination of Vitamin D3 and MitoQ10 significantly reduced levels of estradiol, progesterone, FSH, LH, LH/FSH, SOD and MDA. The expression rate of mRNAs of 3ß-HSD, Cyp19a1, Cyp11a1, StAR, Keap1, HO-1 and Nrf2 were also significantly low in V+M group. Moreover, the histomorphological inspection of ovaries from this group revealed many healthy follicles at various stages of development including few atretic follicles, pre-antral and antral follicles and many corpora lutea. The characteristics observed in this group were in many ways similar to that of the PC group. The combination of MitoQ10 and Vitamin D3 may be potential candidate to ameliorate PCOS.

Altered TH1, TH2, TH17 balance in assisted reproductive technology conceived mice.

AIM AND PURPOSE: The higher prevalence rate of different diseases may accentuate the possible alteration of the immune system in individuals conceived through the assisted reproductive technologies (ART). The present study was conducted to evaluate the TH1, TH2, TH17 balance in intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) - conceived mice in comparison to naturally conceived offspring. METHODS: Mice (6-8 weeks) were divided into three groups (IVF- conceived, ICSI- conceived and naturally conceived). They were subjected to subcutaneous immunization witMycobacterium bovis Bacille Calmette-Guérin (BCG). The blood samples were taken and the sera were separated. Then the spleens were surgically removed at the time the mice were sacrificed. Serum levels of IFN-γ, IL-17A and IL-4 were detected by ELISA. Then the proportion of TCD4 cells possessing the T-bet+, GATA3+, and ROR-γt + were measured using FACS caliber flow cytometer. RESULTS: In comparison with naturally conceived mice, intracellular expression of T-bet and serum levels of IFN-γ were significantly decreased in ART- conceived mice. Moreover, ART- conceived offspring showed marked increase in IL-4 and IL-17A. CONCLUSION: It is concluded that compared to naturally conceived male mice, ART- conceived counterparts exhibit less efficient immune responses against BCG through further promotion of humoral and inflammatory related immune response characteristics.

The modulating effects of Resveratrol on the expression of MMP-2 and MMP-9 in endometriosis women: a randomized exploratory trial.

Endometriosis is an inflammatory disease; the hallmark of inflammation is over-activation of matrix metalloproteinases (MMPs). The regulatory effects of Resveratrol on MMPs were formerly depicted in other cell lines. This study aimed at investigating the effects of Resveratrol on expression of MMP-2 and -9 in endometriosis patients. This trial was carried out on endometriosis patients (n = 34) who were randomly divided into treatment (i = 17) and control (n = 17) groups. Alongside the routine protocol, the control and treatment groups took placebo and Resveratrol (400 mg), respectively, for 12-14 weeks. Endometrial tissue and fluid as well as blood sampling from both groups were done before and after the intervention. The level of mRNA and protein of both MMP-2 and -9 reduced in the endometrium of treatment group following intervention. Also, the serum and the endometrial fluid concentration of them lowered within the treatment group. Moreover, the serum and endometrial fluid levels of MMP-2 as well as MMP-9 were also diminished following the surgical removal of endometritic lesions. We showed that Resveratrol can modify the inflammation process in the endometrium of women with endometriosis at least in the level of MMP-2 and -9 expressions. The therapeutic potency of Resveratrol in endometriosis needs more clinical studies.

Effects of letrozole and clomiphene citrate on Wnt signaling pathway in endometrium of polycystic ovarian syndrome and healthy women†.

Polycystic ovary syndrome (PCOS) is an endocrine disorder in women of reproductive age. In addition to anovulation, endometrial dysfunction can reduce fertility in PCOS. The cyclical changes of endometrium are controlled by estrogen and progesterone via modulating the Wnt/B-catenin pathway. Clomiphene citrate (CC) and letrozole are used to induce ovulation; unlike letrozole, there is a discrepancy between ovulation and pregnancy rates in CC-treated cycles. Because of the anti-estrogenic effects of CC on endometrium, we compared the expression of the key molecules of the Wnt/B-catenin pathway in the endometrium of women taking CC and letrozole. This study included PCOS and healthy women divided into the groups stimulated with letrozole (5 mg) or CC (100 mg) as well as NO-treatment groups. The endometrial thickness and hormonal profile were measured on day 12 of the menses. Using real-time polymerase chain reaction and western blot, we evaluated mRNA and protein expression of B-catenin, glycogen synthase kinase 3 beta (GSK3B), dickkopf Wnt signaling pathway inhibitor 1 (DKK1), and estrogen receptor 1 (ESR1) in the endometrial samples. Significantly, the mean serum estrogen and progesterone were lower and higher, respectively, in letrozole than CC groups. The endometrial thickness was significantly reduced in CC. The proteins expression of active B-catenin, inactive GSK3B, and ESR1 were significantly decreased in CC-treated groups. The mRNA and protein assessment of DKK1 showed significantly higher expression in CC. Our results indicate that letrozole can provide an acceptable activation of the Wnt/B-catenin pathway, resulting in adequate proliferation of endometrium in the women receiving letrozole compared to CC.

Evaluation of prooxidant-antioxidant balance in in vitro fertilization-conceived mice.

OBJECTIVE: Concerns about the safety of assisted reproductive technology (ART) have been raised, as some studies have shown elevated incidence rates of childhood cancer, asthma, allergies, and other diseases in ART-conceived babies. Findings regarding the health of ART-conceived babies are controversial. The present study was conducted to evaluate the prooxidant-antioxidant balance (PAB) in in vitro fertilization (IVF)-conceived mice in comparison to naturally conceived offspring. METHODS: Mice (6-8 weeks) were divided into two groups (IVF-conceived and naturally conceived) matched by sex, age, weight, and litter size. A 1-mL blood sample was taken and the sera were separated. The oxidant-antioxidant balance was evaluated using a fast and reliable PAB assay. The results were expressed as mean±standard deviation. RESULTS: The mean PAB values (HK units) in the IVF-conceived and naturally conceived groups were 59.70±22.30 and 54.70±18.22, respectively (p=0.82). CONCLUSION: Since free radicals contribute to several pathological conditions and antioxidants play an important protective role against oxidative stress, evaluating the oxidant-antioxidant balance is very important. Although the results of this study showed that the quality of the defense mechanism against free radicals was not significantly different between the IVF-conceived and naturally conceived mice, other parameters of metabolic dysfunction need to be measured.

The effects of letrozole and clomiphene citrate on ligands expression of Wnt3, Wnt7a, and Wnt8b in proliferative endometrium of women with Polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is a common endocrinologic disorder in women of reproductive age characterized by polycystic ovaries, oligo/anovulation, and hyperandrogenism. Not only anovulation but also endometrial dysfunction can reduce fertility in PCOS patients. Wnt pathway is responsible for endometrial proliferation which be strongly regulated by estradiol. To determine the effects of clomiphene citrate (CC) and letrozole, we measured the expression of some main ligands of Wnt/ß-catenin signaling including Wnt7a, Wnt3, and Wnt8b in the endometrial samples taken from PCOS women on day 12 of the menses who received 100 mg CC or 5 mg letrozole as well as from women without treatment. Significantly, the mean estrogen and progesterone concentration were lower and higher, respectively, in letrozole than CC. The mean endometrial thickness (ET) was significantly greater in letrozole compared to CC. Assessment of the mRNA and protein expression of Wnt7a, Wnt3, and Wnt8b showed significantly lower expression in CC than the letrozole and control groups. Collectively, letrozole provided a better molecular response in the endometrium of PCOS patients during the proliferative phase, similar to natural cycles, compared to CC. CC decreased the ligands expression of Wnt3, Wnt7a, and Wnt8b, resulting in endometrial dysfunction.

Effect of brain-derived neurotrophic factor (BDNF) on sperm quality of normozoospermic men.

The neurotrophin family of proteins and their receptors act as important proliferative and pro-survival factors in differentiation of nerve cells and are thought to play key roles in the development of reproductive tissues and normal function of spermatozoa. The objective of the present study was to evaluate the effect of Brain-Derived Neurotrophic Factor (BDNF) on the sperm viability and motility, lipid peroxidation (LPO), mitochondrial activity and concentration of leptin, nitric oxide (NO) and insulin in normozoospermic men. Semen samples from 20 normozoospermic men were divided into three groups: (i) control, (ii) BDNF and (iii) BDNF + K252a. BDNF and K252a were added in the dose of 0.133 and 0.1 nM, respectively. Viability was assessed by eosin-nigrosin staining technique, and motility was observed by microscopy. NO concentration and mitochondrial activity were measured with flow cytometry, and LPO was analyzed using enzyme-linked immunosorbent assay (ELISA) kits. Results showed that exogenous BDNF at 0.133 nM could significantly (p < 0.05) influence viability, motility, NO concentration, mitochondrial activity and LPO content. Secretions of insulin and leptin by human sperm were increased in cells exposed to the exogenous BDNF, whereas viability, mitochondrial activity and insulin and leptin secretions were decreased in cells exposed to the K252.

Expression and shedding of CD44 in the endometrium of women with endometriosis and modulating effects of vitamin D: A randomized exploratory trial.

Endometriosis is an estrogen-dependent disease. The impaired estrogen and progesterone signaling over-activates the Wnt/ß-catenin pathway in endometriosis patients, which can explain the increased invasion potency of endometrial cells derived from the endometrium of women with endometriosis. The regulatory effects of vitamin D on Wnt/ß-catenin pathway were demonstrated by previous studies. According to gene prioritization method, among Wnt target genes, CD44 was in high ranking in relation to endometriosis. The aim of this study is to investigate the expression of CD44 in the endometrium of women with endometriosis and to study the effects of vitamin D on its expression. This prospective study was performed, during a 12 months period from December 2015 to November 2016, on healthy women as the control group (n = 14) and endometriosis patients (n = 34). The endometriosis patients randomly divided into two groups: One group treated according to the routine protocol and the other group, alongside the routine protocol, took 50,000 IU vitamin D weekly for 12-14 weeks. Blood, endometrial fluid, and endometrial tissue samples were obtained from the control group and endometriosis groups before and after the intervention. We used in silico gene prioritization to study the relevance of CD44. The expression of CD44 was evaluated using the techniques of Western blot, real-time polymerase chain reaction, and ELISA. The eutopic endometrium of women with endometriosis in mid-secretory phase expressed significantly higher levels of CD44s, CD44V, and CD44v6. The concentration of soluble CD44 in the serum and endometrial fluid of endometriosis patients was higher than of healthy women. The expression level of CD44s, CD44V, and CD44v6 in the eutopic endometrium as well as the concentration of soluble CD44 in the endometrial fluid was decreased after modification of the circulating levels of 25(OH)D. It seems that the increased expression and extensive shedding of CD44 in eutopic endometrium play a role in the pathogenesis of endometriosis. Vitamin D can control and modify this process at least in part. We suggest more in vivo investigations on the therapeutic potency of vitamin D in endometriosis.

The Wnt/ß-catenin signaling in endometriosis, the expression of total and active forms of ß-catenin, total and inactive forms of glycogen synthase kinase-3ß, WNT7a and DICKKOPF-1.

OBJECTIVES: The cyclical changes in proliferation and differentiation of endometrial cells are regulated by estrogen and progesterone via modulating Wnt/ß-catenin signaling. Imbalance in the expression of estrogen and progesterone receptors causes progesterone resistance in endometriosis patients. The aim of this study was to investigate the expression of some main components of Wnt/ß-catenin signaling including WNT7a, DKK-1, ß-catenin, and GSK-3ß in eutopic endometrium and peritoneal endometriotic lesions of endometriosis patients compared to healthy endometrium in the mid-secretory phase of menstrual cycle. STUDY DESIGN: This prospective study was performed, during a 12 months period from December 2015 to November 2016, on healthy women as the control group (n=14) and endometriosis patients (n=34). We used real-time polymerase chain reaction and Western blot techniques. RESULTS: Protein and mRNA expression of DKK-1 were significantly down-regulated in both endometriotic lesions and eutopic endometrium of endometriosis group. We also demonstrated that the expression of non-phosphorylated ß-catenin (active form) and phosphorylated GSK-3ß (inactive form) were up-regulated in endometriosis patients. The mRNA levels of ß-catenin, GSK-3ß, and WNT7a, as well as the protein levels of total ß-catenin, total GSK-3ß, and WNT7a in endometriosis group, were not significantly different with those in control group. The patterns of mRNA and protein expression of all interested factors in the lesions were similar to those in the eutopic endometrium of same patients. CONCLUSIONS: It seems that the aberrant activation of Wnt/ß-catenin signaling in the secretory phase of the menstrual cycle in endometriosis has two essential elements: excessive inactivation of GSK-3ß and suppression of the expression of Wnt signaling inhibitor DKK-1. Interventions in this signaling pathway may allow for the exploration of potential new targets for the control of development and progression of endometriosis.