Unveiling the impact of DNA methylation machinery: Dnmt1 and Dnmt3a in orchestrating oocyte development and cellular homeostasis.

DNA methylation can be considered the most prominent in controlling the gene expression responsible for the balance between cell proliferation and cell death. In this study, we aimed to analyze the distinct contributions of Dnmt1 and Dnmt3a enzymes in oocyte maturation, survival, autophagy, reactive oxygen species (ROS) production, and compensation capacity of Dnmt3b and Dnmt3l enzymes in mouse oocytes. Following confirming the suppression of Dnmt1or Dnmt3a through siRNA application, the assessment involved immunofluorescence staining for Dnmts, 5mC, p62, and ROS levels. Cell death rates showed a noticeable increase while oocyte maturation rates exhibited significant reduction. Global DNA methylation showed a decline, concomitant with elevated p62 and ROS levels upon Dnmt1 or Dnmt3a knockdown. Remarkably, silencing of Dnmt1 led to an upsurge in Dnmt3a expression, whereas Dnmt3a knockdown triggered an increase in Dnmt1 levels. Furthermore, Dnmt3l expression exhibited a notable decrease after silencing of either Dnmt1 or Dnmt3a, while Dnmt3b levels remained comparable between control and siRNA-treated groups. Collectively, this study underscores the pivotal roles of Dnmt1 and Dnmt3a in orchestrating various facets of oocyte development, encompassing maturation, survival, autophagy, and ROS production. These findings offer valuable insights into the intricate regulatory network governed by DNA methylation machinery within the context of oocyte physiology.

Antioxidant supplementation may effect DNA methylation patterns, apoptosis, and ROS levels in developing mouse embryos.

This study was designed to address the question: does antioxidant-containing embryo culture media affect DNA methyltransferases, global DNA methylation, inner cell mass/trophoblast differentiation, intracellular reactive oxygen species (ROS) levels, and apoptosis? Mouse zygotes were cultured in embryo culture media containing MitoQ, N-acetyl-L-cysteine (NAC), acetyl-L-carnitine (ALC), α-lipoic acid (ALA), or the mixture of NAC + ALC + ALA (AO) until the blastocyst stage, whereas in vivo-developed blastocysts were used as control. Protein expression levels of Dnmt1, 3a, 3b, and 3l enzymes were analyzed by immunofluorescence and western blot, while global DNA methylation, apoptosis, and ROS levels were evaluated by immunofluorescence. NAC, ALC, and MitoQ significantly increased the levels of all Dnmts and global methylation. ALA significantly induced all Dnmts, whereas global methylation did not show any difference. NAC and mixture AO applications significantly induced Nanog levels, ALA and MitoQ increased Cdx2 levels, while the other groups were similar. ALA and MitoQ decreased while ALC increased the levels of intracellular ROS. This study illustrates that antioxidants, operating through distinct pathways, have varying impacts on DNA methylation levels and cell differentiation in mouse embryos. Further investigations are warranted to assess the implications of these alterations on the subsequent offspring.

High-fat diet induced obesity alters Dnmt1 and Dnmt3a levels and global DNA methylation in mouse ovary and testis.

Obesity impairs reproductive capacity, and the link between imprinting disorders and obesity has been discussed in many studies. Recent studies indicate that a high-fat diet may cause epigenetic changes in maternal and paternal genes, which may be transmitted to offspring and negatively affect their development. On this basis, our study aims to reveal the changes in DNA methylation and DNA methyltransferase enzymes in the ovaries and testes of C57BL/6 mice fed a high-fat diet and created a model of obesity, by comparing them with the control group. For this purpose, we demonstrated the presence and quantitative differences of DNA methyltransferase 1 and DNA methyltransferase 3a enzymes as well as global DNA methylation in ovaries and testis of C57BL/6 mice fed a high-fat diet by using immunohistochemistry and western blot methods. We found that a high-fat diet induces the levels of Dnmt1 and Dnmt3a proteins (p < 0.05). We observed increased global DNA methylation in testes but, interestingly, decreased global DNA methylation in ovaries. We think that our outcomes have significant value to demonstrate the effects of obesity on ovarian follicle development and testicular spermatogenesis and may bring a new perspective to obesity-induced infertility treatments. Additionally, to the best of our knowledge, this is the first study to document dynamic alteration of Dnmt1 and Dnmt3a as well as global DNA methylation patterns during follicle development in healthy mouse ovaries.

DNMT enzymes differentially alter global DNA methylation in a stage-dependent manner during spermatogenesis.

DNA methylation plays important roles during spermatogenesis. This mechanism includes maintenance and de novo methylation which are catalysed by DNA methyltransferase enzymes. DNMT1 plays role in maintenance methylation, while DNMT3A, DNMT3B and DNMT3L are primarily responsible for de novo methylation. Both maintenance and de novo methylation processes appears during primordial germ cell development and spermatogenesis. However, the function(s) of the methylation and DNMTs during spermatogenesis still remain elusive. The aim of the study was to evaluate the relationship between DNMTs levels and global DNA methylation in total testis and during spermatogenesis. For this purpose, DNMTs were analysed using Western blot and immunohistochemistry techniques. We also analysed global DNA methylation level by 5mC staining. We found that DNMTs expression and global DNA methylation levels were significantly differed in total testes and spermatogenetic cells in a stage-dependent manner. DNMT3B and DNMT3L were more abundant in testes, while DNMT1 and DNMT3A were comparatively low. Interestingly, no DNMTs signal was seen in elongated spermatid whereas global DNA methylation was at the highest level. To understand the meaning of differential expressions of DNMTs in the testes, further molecular biological studies are required.

Phospholipase C-zeta levels are not correlated with fertilisation rates in infertile couples.

In mammals, 'oocyte activation' is triggered by certain proteins, one of which is phospholipase C-zeta. Recent evidence suggests that low expression of phospholipase C-zeta might be associated with male infertility, while a limited number of studies claimed the opposite. This study was designed to test whether quantity of phospholipase C-zeta and in vitro fertilisation rates are correlated or not, assessed by flow cytometry. Semen samples from 43 infertile couples were analysed for the percentage and mean fluorescent intensity (MFI) of phospholipase C-zeta protein. Results were confirmed by immunofluorescent labelling. Patients with a fertilisation rate of 40% or lower were involved in the low fertilisation group, while the high fertilization group consisted of patients with a fertilisation rate of 60% and higher. Quantitative analyses by flow cytometry showed no significant difference among the low fertilisation and high fertilisation groups when phospholipase C-zeta ratio or MFI was considered. No correlation was found between pregnancy rates and phospholipase C-zeta quantity. None of the total fertilisation failure cases were lack of phospholipase C-zeta. In fact, fertilisation was possible even when phospholipase C-zeta levels were very low. Thus, we concluded that phospholipase C-zeta quantity cannot be considered as a diagnostic tool for male infertility.

Mitochondria-targeted therapeutics, MitoQ and BGP-15, reverse aging-associated meiotic spindle defects in mouse and human oocytes.

STUDY QUESTION: Do mitochondria-targeted therapies reverse ageing- and oxidative stress-induced spindle defects in oocytes from mice and humans? SUMMARY ANSWER: Exposure to MitoQ or BGP-15 during IVM protected against spindle and chromosomal defects in mouse oocytes exposed to oxidative stress or derived from reproductively aged mice whilst MitoQ promoted nuclear maturation and protected against chromosomal misalignments in human oocytes. WHAT IS KNOWN ALREADY: Spindle and chromosomal abnormalities in oocytes are more prevalent with maternal aging, increasing the risk of aneuploidy, miscarriage and genetic disorders such as Down's syndrome. The origin of compromised oocyte function may be founded in mitochondrial dysfunction and increased reactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION: Oocytes from young and old mice were treated with MitoQ and/or BGP-15 during IVM. To directly induce mitochondrial dysfunction, oocytes were treated with H2O2, and then treated the MitoQ and/or BGP-15. Immature human oocytes were cultured with or without MitoQ. Each experiment was repeated at least three times, and data were analyzed by unpaired-sample t-test or chi-square test. PARTICIPANTS/MATERIALS, SETTING, METHODS: Immature germinal vesicle (GV) stage oocytes from 1-, 12- and 18-month-old mice were obtained from preovulatory ovarian follicles. Oocytes were treated with MitoQ and/or BGP-15 during IVM. GV-stage human oocytes were cultured with or without MitoQ. Mitochondrial membrane potential and mitochondrial ROS were measured by live-cell imaging. Meiotic spindle and chromosome alignments were visualized by immunofluorescent labeling of fixed oocytes and the 3-dimensional images were analyzed by Imaris. MAIN RESULTS AND THE ROLE OF CHANCE: MitoQ or BGP-15 during IVM protects against spindle and chromosomal defects in oocytes exposed to oxidative stress and in oocytes from aged mice (P < 0.001). In human oocytes, the presence of MitoQ during IVM promoted nuclear maturation and had a similar positive effect in protecting against chromosomal misalignments (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Our study identifies two excellent candidates that may help to improve fertility in older women. However, these potential therapies must be tested for efficacy in clinical IVM systems, and undergo thorough examination of resultant offspring in preclinical models before utilization. WIDER IMPLICATIONS OF THE FINDINGS: Our results using in-vitro systems for oocyte maturation in both mouse and human provide proof of principle that mitochondrially targeted molecules such as MitoQ and BGP-15 may represent a novel therapeutic approach against maternal aging-related spindle and chromosomal abnormalities. STUDY FUNDING/COMPETING INTEREST(S): The project was financially supported by the National Health and Medical Research Council and Australian Research Council, Australia. U.A.-Z. was supported by the Iraqi Higher Education and Scientific Research Ministry PhD scholarship and O.C. was supported by TUBITAK-1059B191601275. M.P.M. consults for MitoQ Inc. and holds patents in mitochondria-targeted therapies. R.L.R. is an inventor on patents relating to the use of BGP-15 to improve gamete quality. TRIAL REGISTRATION NUMBER: N/A.

Knockdown of Dnmt1 and Dnmt3a gene expression disrupts preimplantation embryo development through global DNA methylation.

PURPOSE: DNA methylation is one of the epigenetic mechanisms that plays critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of methylation, and Dnmt3a, for de novo methylation, are gradually erased from the zygote in succeeding stages and then reestablished in the blastocyst. This study was designed to address the vital role of Dnmt1 and Dnmt3a enzymes by silencing their gene expressions in embryonic development in mice. METHODS: Groups were (i) control, (ii) Dnmt1-siRNA, (iii) Dnmt3a-siRNA, and (iv) non-targeted (NT) siRNA. Knockdown of Dnmt genes using siRNAs was confirmed by measuring the targeted proteins using Western blot and immunofluorescence. Following knockdown of Dnmt1 and Dnmt3a in zygotes, the developmental competence and global DNA methylation levels were analyzed after 96 h in embryo cultures. RESULTS: A significant number of embryos arrested at the 2-cell stage or had undergone degeneration in the Dnmt1 and Dnmt3a knocked-down groups. By 3D observations in super-resolution microscopy, we noted that Dnmt1 was exclusively found in juxtanuclear cytoplasm, while the Dnmt3a signal was preferentially localized in the nucleus, both in trophoblasts (TBs) and embryoblasts (EBs). Interestingly, the global DNA methylation level decreased in the Dnmt1 knockdown group, while it increased in the Dnmt3a knockdown group. CONCLUSION: Precisely aligned expression of Dnmt genes is highly essential for the fate of an embryo in the early developmental period. Our data indicates that further analysis is mandatory to designate the specific targets of these methylation/demethylation processes in mouse and human preimplantation embryos.

The spatio-temporal dynamics of mitochondrial membrane potential during oocyte maturation.

Mitochondria are highly dynamic organelles and their distribution, structure and activity affect a wide range of cellular functions. Mitochondrial membrane potential (∆Ψm) is an indicator of mitochondrial activity and plays a major role in ATP production, redox balance, signaling and metabolism. Despite the absolute reliance of oocyte and early embryo development on mitochondrial function, there is little known about the spatial and temporal aspects of ΔΨm during oocyte maturation. The one exception is that previous findings using a ΔΨm indicator, JC-1, report that mitochondria in the cortex show a preferentially increased ΔΨm, relative to the rest of the cytoplasm. Using live-cell imaging and a new ratiometric approach for measuring ΔΨm in mouse oocytes, we find that ΔΨm increases through the time course of oocyte maturation and that mitochondria in the vicinity of the first meiotic spindle show an increase in ΔΨm, compared to other regions of the cytoplasm. We find no evidence for an elevated ΔΨm in the oocyte cortex. These findings suggest that mitochondrial activity is adaptive and responsive to the events of oocyte maturation at both a global and local level. In conclusion, we have provided a new approach to reliably measure ΔΨm that has shed new light onto the spatio-temporal regulation of mitochondrial function in oocytes and early embryos.

Optimizing the transport and storage conditions of current Good Manufacturing Practice -grade human umbilical cord mesenchymal stromal cells for transplantation (HUC-HEART Trial).

BACKGROUND: The HUC-HEART Trial is a clinical study of intramyocardial delivery of current Good Manufacturing Practice (cGMP)-grade human umbilical cord multipotent stromal cells (HUC-MSCs) in ischemic cardiomyopathy where 2 × 107 cells are administered to peri-infarcted myocardium. Prior to the onset of the trial, we aimed to optimize the transport/storage conditions for obtaining the highest cell viability and proliferation rate of cells to be transplanted. METHODS: Cells were tested after being transported in phosphate-buffered saline (PBS) or Ringer's lactate-based (RL) transport media supplemented with human serum albumin (HSA) and/or hydroxyethyl starch (HES) at two temperatures (2-10°C or 22-24°C). RESULTS: The effects of transport conditions on cell viability following 6 h were found highest (93.4 ± 1.5) in RL-based media at 2-10°C. Karyotypes were found normal upon transportation in any of the formulations and temperatures. However, the highest proliferation rate was noted (3.1-fold increase) in RL (1% HSA) media at 2-10°C over 6 days in culture. From that point, RL (1% HSA) media at 2-10°C was used for further experiments. The maximum cell storage time was detected around 24 h at 2-10°C. Extended storage periods resulted in a decrease in cell viability but not in MSC marker expression. An increase in actin quantity was detected in hypoxia (5% O2) groups in early culture days; no difference was noted between hypoxic versus normoxic (21% O2) conditions in later days. DISCUSSION: The overall results suggest that non-commercial, simple media formulations with extended storage intervals at 2-10°C temperatures are capable of retaining the characteristics of clinical-grade HUC-MSCs. The above findings led us to use RL (1% HSA) media at 2-10°C for transport and storage in the HUC-HEART Trial; 23 patients received HUC-MSCs by August 2018; no adverse effects were noted related to cell processing and transplantation.

Is leptin receptor expression triggered in the case of embryo transfer to endometrium coculture?

Background/aim: A synchronized dialogue between maternal and embryonic tissues is required for successful implantation. Low uterine receptivity is responsible for two-thirds of implantation failures and leptin is effective in the physiology of reproduction by binding to specific receptors. In this study, we investigateleptin receptor expression in cases of embryo transfer to endometrial coculture. Materials and methods: Biopsy materials were taken from 20 females with indication for coculture application and were cultured in an appropriate medium after the epithelial cells were isolated. The grown cells were cultured in chamber slides as the first group. For the second group, day 3 embryo was added to chamber slides and the development was observed. The embryo was transferred 1 or 2 days later and other cells (after the transfer process) were used to form the second group. After fixation, immunohistochemical staining was performed with anti-leptin primary antibody. Results: Regarding the coculture without embryo transfer, moderate leptin receptor immunoreactivity was seen in the perinuclear region and the cell membrane. Also, regarding the coculture with embryo transfer, moderate leptin receptor immunoreactivity was seen in the cytoplasm and strong leptin receptor immunoreactivity was seen in the cell membrane. Conclusion: Embryo transfer to endometrium coculture triggers leptin receptor expression

Umbilical cord mesenchymal stromal cell transplantations: A systemic analysis of clinical trials.

The advances and success of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in experimental disease animal models have fueled the development of targeted therapies in humans. The therapeutic potential of allogeneic transplantation of UC-MSCs has been under examination since 2009. The purpose of this systematic analysis was to review the published results, limitations and obstacles for UC-MSC transplantation. An extensive search strategy was applied to the published literature, 93 peer-reviewed full-text articles and abstracts were found published by early August 2017 that investigated the safety, efficacy and feasibility of UC-MSCs in 2001 patients with 53 distinct pathologies including many systemic/local, acute/chronic conditions. Few data were extracted from the abstracts and/or Chinese-written articles (n = 7, 8%). Importantly, no long-term adverse effects, tumor formation or cell rejection were reported. All studies noted certain degrees of therapeutic benefit as evidenced by clinical symptoms and/or laboratory findings. Thirty-seven percent (n = 34) of studies were found published as a single case (n = 10; 11%) or 2-10 case reports (n = 24; 26%) with no control group. Due to the nature of many stem cell-based studies, the majority of patients also received conventional therapy regimens, which obscured the pure efficacy of the cells transplanted. Randomized, blind, phase 1/2 trials with control groups (placebo-controlled) showed more plausible results. Given that most UC-MSC trials are early phase, the internationally recognized cell isolation and preparation standards should be extended to future phase 2/3 trials to reach more convincing conclusions regarding the safety and efficacy of UC-MSC therapies.

Can dicoumarol be used as a gonad-safe anticancer agent: an in vitro and in vivo experimental study.

STUDY HYPOTHESIS: Dicoumarol (DC) has potential for use as a gonad-safe anticancer agent. STUDY FINDING: DC altered cell proliferation, decreased viability and increased apoptosis in Vero and MCF-7 cell lines but did not show any toxic effect on mouse ovarian tissues and developing oocytes in vitro and in vivo. WHAT IS KNOWN ALREADY: DC suppresses cell proliferation and increases apoptosis in various cancer cells such as breast, urogenital and melanoma. DC has also been reported to alter the anticancer effects of several chemotherapeutics, including cisplatin, gemcitabine and doxorubicin in prostate, liver and uroepithelial cancer cells, respectively. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Vero (African green monkey kidney epithelial cells) and MCF-7 (human cancerous breast epithelial cells) cell lines and mouse granulosa cells isolated from 21-day-old female BALB/c mice (n = 21) were used to assess the effects of DC (10, 50, 100 and 200 µm) for 24 and 48 h on cell proliferation, viability and apoptotic cell death. In vivo experiments were performed with a single i.p. injection of 32 mg/kg DC in 21-day-old female BALB/c mice (n = 12). Following 48 h, animals were sacrificed by cervical dislocation and histological sections of isolated ovaries were evaluated for apoptosis. Viability assays were based on the trypan blue dye exclusion method and an automated cell counter device was used. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and Annexin-V immunofluorescence were assessed by 3D confocal microscopy to address apoptotic cell death. We also assessed whether DC inhibits cell proliferation and viability through NQO1 [NAD(P)H Quinone Oxidoreductase 1], an intracellular inhibitor of reactive oxygen species (ROS). The meiotic spindle and chromosomes were studied in mouse oocytes by α-ß-tubulin and 7-aminoactinomycine D (7-AAD) immunostaining in vitro and in vivo. MAIN RESULTS AND THE ROLE OF CHANCE: DC does not block oocyte maturation and no significant alteration was noted in meiotic spindle or chromosome morphology in metaphase-II (M-II) stage oocytes following DC treatment in vitro or in vivo. In contrast, exposure to DC for 24 h suppressed cell proliferation (P = 0.026 at 200 µm), decreased viability (P = 0.002 at 200 µm) and increased apoptosis (P = 0.048 at 100 µm) in Vero and MCF-7 cell lines, compared with controls. These changes were not related to intracellular NQO1 levels. Mouse granulosa cells were unaffected by 50 or 100 µm DC treatment for 24 and 48 h in vitro. DC treatment in vivo did not alter the number of primordial follicles or the ratio of apoptosis in primordial, primary and secondary follicles, as well as in antral follicles, compared with the controls. LIMITATIONS, REASONS FOR CAUTION: DC was tested for ovarian toxicity only in isolated mouse oocytes/ovaries and healthy BALB/c mice. No cancer formation was used as an in vivo test model. The possibility that DC may potentiate ovarian toxicity when combined with traditional chemotherapeutic agents, such as mitomycin-C, cisplatin, gemcitabine and doxorubicin, must be taken into account, as DC is known to alter their effects in some cancer cells. WIDER IMPLICATIONS OF THE FINDINGS: The present study evaluated, for the first time, the effect of DC on ovarian tissue. The results suggested that DC is not toxic to ovarian tissues and developing oocytes; therefore, DC should be assessed further as a potential anticancer agent when female fertility preservation is a concern. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This work includes data from dissertation thesis entitled 'Effects of dicoumarol on mitotic and meiotic cells as an anticancer agent' by DA, 2014 and was partly supported by The National Scientific and Technological Research Council of Turkey (SBAG-109S415) to AC, OC and SO. The authors confirm that this article content presents no conflicts of interest.

Does combining magnetic-activated cell sorting with density gradient or swim-up improve sperm selection?

PURPOSE: The present study aimed to evaluate whether combining the magnetic-activated cell sorting (MACS) with density-gradient (DG) or swim-up (SU) sperm separation techniques can improve sperm selection to obtain higher quality spermatozoa. METHODS: Two commonly used sperm selection techniques, SU and DG, were compared to MACS combined with either SU or DG. Spermatozoa obtained from normozoospermic (n = 10) and oligozoospermic (n = 10) cases were grouped as SU, DG, SU+MACS, and DG+MACS followed by the analysis of sperm morphology, motility, DNA integrity, and the levels of Izumo-1 and PLCZ proteins. RESULTS: Although spermatozoa obtained by SU or DG when combined with MACS have improved aspects when compared to SU or DG alone, results did not reach a statistically significant level. Moreover, separation with MACS caused a significant loss in the numbers of total and rapid progressive spermatozoa. CONCLUSIONS: Considering the cost/benefit ratio, MACS application together with traditional techniques may only be preferred in certain cases having higher concentrations of spermatozoa, but it does not seem to be an ideal and practical sperm selection technique for routine use.

Total fertilization failure: is it the end of the story?

PURPOSE: To study parameters that could predict in-vitro fertilization (IVF) success in patients who experienced total fertilization failure (TFF) with intracytoplasmic sperm injection (ICSI) in their previous cycles. METHODS: Cycle characteristics of patients with TFF (Group I, n = 136 cycles), cycles resulting in embryo transfer (ET) following TFF (Group II, n = 36 cycles) and recurrent TFF (Group III, n = 25 cycles) and were studied retrospectively. Demographic features, cycle characteristics of three groups were compared. RESULTS: Follicle count measuring 15-17 mm was significantly higher in group II when compared to group I (p = 0.02). Total number of retrieved oocytes and mature oocytes were significantly higher in group II when compared to groups I and III (p = 0.001). Estradiol level at oocyte pick up (OPU) day was significantly higher in group II when compared to group I (p = 0.02). When the characteristics of ET cycles and preceding TFF cycles of the same patient were compared, total number of retrieved oocytes (5.11 ± 0.72 (95% CI 3.69-6.52) vs. 11.44 ± 1.60 (95% CI 5.29-17.59)) and mature oocytes (3.26 ± 3.66 (95% CI 2.04-4.47) vs. 6.92 ± 5.61 (95% CI 5.09-8.75)) were found to be significantly lower in TFF cycles (p = 0.001). Five biochemical and 5 clinical pregnancies occurred while only 2 healthy babies were born, corresponding to a live birth rate 5.5%. CONCLUSIONS: Increasing the number of retrieved and mature oocytes may increase the success of fertilization in patients with a history of previous failed fertilization. However, live birth rate is still low in embryo transfer cycles.

Altered DNA methylation and Dnmt expression in obese uterus may cause implantation failure.

Obesity is defined by increased adipose tissue volume and has become a major risk factor for reproduction. Recent studies have revealed a substantial link between obesity and epigenetics. The epigenome is dynamically regulated mainly by DNA methylation. DNA methylation, which is controlled by DNA methyltransferases (Dnmts), has been widely studied because it is essential for imprinting and regulation of gene expression. In our previous study, we showed that the levels of Dnmt1, Dnmt3a and global DNA methylation was dramatically altered in the testis and ovary of high-fat diet (HFD)-induced obese mice. However, the effect of HFD on Dnmts and global DNA methylation in mouse uterus has not yet been demonstrated. Therefore, in the present study, we aimed to evaluate the effect of HFD on the level of Dnmt1, Dnmt3a, Dnmt3b, Dnmt3l and global DNA methylation in uterus. Our results showed that HFD significantly altered the levels of Dnmts and global DNA methylation in the uterus. The total expression of Dnmt1, Dnmt3a and Dnmt3b was significantly upregulated, while level of Dnmt3l and global DNA methylation were dramatically decreased (p < 0.05). Furthermore, we observed that the expression of Dnmt3b and Dnmt3l was significantly increased in endometrium including gland and epithelium (p < 0.05). Although Dnmt3b was the only protein whose expression significantly increased, the level of global DNA methylation and Dnmt3l significantly decreased in stroma and myometrium (p < 0.05). In conclusion, our results show for the first time that obesity dramatically alters global DNA methylation and expression of Dnmts, and decreased DNA methylation and Dnmt expression may cause abnormal gene expression, especially in the endometrium.

Estradiol supplementation in intracytoplasmic sperm injection cycles with thin endometrium.

OBJECTIVE: To evaluate the efficacy of estradiol supplementation starting on the day of human chorionic gonadotrophin (hCG) in patients with thin endometrium in intracytoplasmic sperm injection (ICSI) cycles. METHODS: A total of 117 consecutive patients with the endometrial thickness on the hCG day ≤ 8 mm were reviewed. Estradiol supplementation was given in 57 patients and the remaining 60 patients were accepted as control group. Estradiol supplemented (ES) group received estradiol hemihydrate 4 mg/day started on the day of hCG. Luteal phase was supported using the vaginal progesterone gel in both groups. Clinical pregnancy rate, implantation rate, miscarriage rate, endometrial thickness on the day of oocyte pick-up and on the day of embryo transferred were accepted as main outcome measures. RESULTS: There were no statistical differences in terms of clinical pregnancy rate (28.1% vs. 23.3%), implantation rate (16% vs. 10.4%), miscarriage rate (21% vs. 31.6%), endometrial thickness on the oocyte pick-up day (8.5 ± 1.8 vs. 8.4 ± 1.4, mm) and embryo transferred day (9.6 ± 2.9 vs. 10.3 ± 2.4, mm) in the ES group vs. control group. CONCLUSION: Estradiol supplementation starting on the hCG day for the patients with thin endometrium does not provide any benefit on the pregnancy outcome in ICSI cycles.

The effect of cabergoline on folicular microenviroment profile in patients with high risk of OHSS.

The aim of this study to evaluate the effect of cabergoline on follicular microenvironment by measuring follicular fluid (FF) insulin like growth hormone -I (IGF-I), antimullerian hormone (AMH), inhibin B and hepatocyte growth factor (HGF) levels in women with PCOS and high risk of ovarian hyperstimulation syndrome (OHSS). In this prospective cohort study, 41 women with PCOS undergoing controlled ovarian hyperstimulation for assisted reproduction and having the high risk factors for OHSS are included. The women in the study group (n = 15) received cabergoline for OHSS prevention while the women in the control did not received any medications for OHSS prevention. FF samples were collected during oocyte pick-up procedure for all women were determined using commercially available ELISA kits. Concentrations of FF IGF-I, AMH, inhibin B and HGF were assessed. In the study group FF AMH (2.96 ± 1.27 versus 1.91 ± 0.64 ng/mL), Inhibin B (1339.47 ± 198.56 versus 1200.09 ± 133.64 pg/mL), HGF (5623.21 ± 2411.09 versus 3787.42 ± 2269.89 pg/mL) and IGF-I (298.60 ± 37.80 versus 219.90 ± 71.40 pg/mL) concentrations were significantly decreased compared with control group. Cabergolin prevents OHSS in high risk patients by disrupting FF hormone microenvironment.

DNA methyltransferase (Dnmt) silencing causes increased Cdx2 and Nanog levels in surviving embryos.

Epigenetic mechanisms are one of the essential regulators of gene expression which do not involve altering the primary nucleotide sequence. DNA methylation is considered among the most prominent epigenetic mechanisms in controlling the functions of genes related to cell differentiation, cell cycle, cell survival, autophagy, and embryo development. DNA methyl transferases (Dnmts) control DNA methylation, the levels of which are differentially altered during embryonic development, and may determine cell differentiation fate as in the case of pluripotent inner cell mass (ICM) or trophectoderm (TE). In this study, we aimed to analyze the role of Dnmt1 and Dnmt3a enzymes in ICM (using the Nanog marker) and TE (using the Cdx2 marker) differentiation, autophagy (using p62 marker), reactive oxygen species (ROS) production, and apoptosis (using TUNEL) during mouse preimplantation embryo development. Following knockdown of Dnmt1 and Dnmt3a in zygotes, expression levels of Cdx2 in the trophectoderm and Nanog in the inner cell mass were measured, as well as p62 levels, reactive oxygen species (ROS) production, and apoptosis levels after 96 hours in embryo culture. We found that knockdown of Dnmt1 or Dnmt3a significantly induced Cdx2 and Nanog expression. Similarly, p62 expression, ROS levels and apoptosis significantly increased after silencing. This study shows that Dnmt genes are highly crucial for embryonic fate determination and survival. Further studies are required to reveal the specific targets of these methylation processes related to cell differentiation, survival, autophagy, and ROS production in mouse and human preimplantation embryos.

Potential role of decidual apoptosis in the pathogenesis of miscarriages.

To investigate the existence and the distribution of decidual apoptosis in normal pregnancies and miscarriages (spontaneous and recurrent), a comparative immunofluorescent tissue labelling of normal control (n = 12) and miscarried pregnancies (n = 24) was designed. Evaluation of the existence and distribution of decidual apoptosis in normal pregnancies and miscarriages, characterization of the apoptotic cell types and the involvement of caspase-dependent pathways was analyzed with TUNEL, anti-active caspase-3, anti-pancytokeratin and anti-CD45 antibodies. Normal decidua showed few apoptotic cells, whereas decidua from recurrent miscarriages had a significantly higher number of apoptotic cells preferentially localized to the sub-epithelial and periarteriolar regions, where the onset of decidualization occurs. Apoptosis occurred via a caspase-dependent pathway. Neither immune nor epithelial cells were positively stained for any apoptotic markers. The increased number of apoptotic cells, which are strictly restricted to the periarteriolar stroma particularly in recurrent miscarriages leads us to suggest that decidual apoptosis could result a series of cellular dysfunctions that may threaten the course of pregnancy.

Does empty zona pellucida indicate poor ovarian response on intra cytoplasmic sperm injection cycles?

The factors behind the empty zona pellucida (EZP) formation and its relationship with in vitro fertilization (IVF) outcomes were analyzed. A total of 104 patients who underwent IVF treatment and collected oocytes including EZP were enrolled in this study. EZP index (EZPI = the ratio of number of EZP to number of cumulus-oocyte complex (COC)) was used for the statistical analysis. Patients were grouped as Group 1 when EZPI ≤ 0.17 (n = 57) and Group 2 when EZPI > 0.17 (n = 47). Type-2 EZP, a variation or an advanced type of oocyte degeneration, is tested. Woman age, basal hormone levels, and total gonadotropin dose were significantly higher in Group 2 compared to Group 1. Total antral follicle count was significantly low in Group 2. Total number of mature oocytes, oocyte quality index, the number of fertilized oocytes, and the numbers of Grade 1 embryos were significantly low in Group 2. On the linear regression analysis, using gonadotropin releasing hormone antagonist protocol (B = 0.086, p = 0.030), the number of ≥17 mm follicle (B = 0.015, p = 0.047), peak serum level of estradiol (B = -3.625; p = 0.014), number of fertilized oocytes (B = -0.02, p = 0.0001) and the day-2 embryo score (B = -0,044, p = 0.001) significantly affected EZPI. An increment of the EZPI may be revealed decreased oocyte quality, and it is also related to the poor ovarian response.