Is the phosphoinositol 1,3-kinase/protein kinase B (PI3K/AKT) pathway expression profile in cumulus cells (CCs) a potential marker of oocyte competence and predictive of pregnancy outcome?
Eleven ...genes (AKT1, ARHGEF7, BCL2L1, CCND1, E2F1, HRAS, KCNH2, PIK3C2A, SHC1, SOS1 and SPP1) in the PI3K/AKT pathway were significantly down-regulated in CCs from oocytes that went on to produce a pregnancy compared to CCs associated with a negative outcome.
The PI3K/AKT pathway plays a pivotal role in the interdependence and continuous feedback between the oocyte and CCs.
The expression analysis of 92 transcripts in the PI3K/AKT pathway in CCs from patients with negative or positive pregnancy outcome, after single embryo transfer, was performed. Mouse CCs target gene expression was conducted to associate the expression profile of PI3K/AKT pathway to oocyte developmental profile.
Fifty-five good prognosis IVF patients who had been referred to IVF or intracytoplasmic sperm injection treatment for male-factor infertility or tubal disease were enroled. CCs from single cumulus-oocyte complexes (COCs) from 16 patients who underwent a single embryo transfer were analyzed. Twenty-five CD-1 mice were used to assess gene expression in CCs associated with oocytes with different competence in relation to hCG priming. A total 220 human COCs were collected. The RNA extracted from CCs of 16 selected patients was used to analyze PI3K/AKT pathway gene expression employing a 96-well custom TaqMan Array. Expression data of CCs associated to positive IVF outcome were compared to data from negative outcome samples. Mice were sacrificed after 9, 12, 15, 21 and 24 h post-hCG administration to obtain CCs from MII oocytes with different developmental competence. Akt1, Bcl2l2 and Shc1 expression were tested in the collected mouse CCs. In addition, the expression of upstream regulator ESR1, the gene encoding for the oestrogen receptor ERβ, and the downstream effectors of the pathway FOXO1, FOXO3 and FOXO4 was evaluated in human and mouse samples.
Transcripts involved in the PI3K Signaling Pathway were selectively modulated according to the IVF/ICSI outcome of the oocyte. Eleven transcripts in this pathway were significantly down-regulated in all samples of CCs from oocytes with positive when compared those with a negative outcome. These outcomes were confirmed in mouse CCs associated with oocytes at different maturation stages. Expression data revealed that the down-regulation of ESR1 could be related to oocyte competence and is likely to be the driver of expression changes highlighted in the PI3K/AKT pathway.
Small sample size and retrospective design.
The CCs expression profile of PI3K/AKT signaling genes, disclosed a specific CCs gene signature related to oocyte competence. It could be speculated that CCs associated with competent oocytes have completed their role in sustaining oocyte development and are influencing their fate in response to metabolic and hormonal changes by de-activating anti-apoptotic signals.
Supported by Merck Serono an affiliate of Merck KGaA, Darmstadt, Germany (research grant for the laboratory session; Merck KGaA reviewed the manuscript for medical accuracy only before journal submission. The authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors). The authors declare no conflict of interest.
Recently, studies on inositol supplementation during in vitro fertilization program (IVF) have gained particular importance due to the effect of this molecule on reducing insulin resistance improving ...ovarian function, oocyte quality, and embryo and pregnancy rates and reducing gonadotropin amount during stimulation. Inositol and its isoforms, especially myoinositol (MYO), are often used as prestimulation therapy in infertile patients undergoing IVF cycle. Inositol supplementation started three months before ovarian stimulation, resulting in significant improvements in hormonal responses, reducing the amount of FSH necessary for optimal follicle development and serum levels of 17beta-estradiol measured the day of hCG injection. As shown by growing number of trials, MYO supplementation improves oocyte quality by reducing the number of degenerated and immature oocytes, in this way increasing the quality of embryos produced. Inositol can also improve the quality of sperm parameters in those patients affected by oligoasthenoteratozoospermia.
Abstract
Objective: To evaluate the effects the administration of myo-inositol (MYO) on hormonal parameters in a group of polycystic ovary syndrome (PCOS) patients.
Design: Controlled clinical study.
...Setting: PCOS patients in a clinical research environment.
Patients: 50 overweight PCOS patients were enrolled after informed consent.
Interventions: All patients underwent hormonal evaluations and an oral glucose tolerance test (OGTT) before and after 12 weeks of therapy (Group A (n¼10): MYO 2 g plus folic acid 200 mg every day; Group B (n¼10): folic acid 200 mg every day). Ultrasound examinations and Ferriman-Gallwey score were also performed.
Main outcome measures: Plasma LH, FSH, PRL, E2, 17OHP, A, T, glucose, insulin, C peptide concentrations, BMI, HOMA index and glucose-to-insulin ratio.
Results: After 12 weeks of MYO administration plasma LH, PRL, T, insulin levels and LH/FSH resulted significantly reduced. Insulin sensitivity, expressed as glucose-to-insulin ratio and HOMA index resulted significantly improved after 12 weeks of treatment. Menstrual cyclicity was restored in all amenorrheic and oligomenorrheic subjects. No changes occurred in the patients treated with folic acid.
Conclusions: MYO administration improves reproductive axis functioning in PCOS patients reducing the hyperinsulinemic state that affects LH secretion.
It is a known fact that abnormal seminal liquid specimens contain abnormal amounts of oxygen free radicals and reactive oxygen species (ROS), and that the use of antioxidant molecules both in vivo ...and in vitro leads to improvement of semen quality in terms of motility, reduction in DNA damage, with obvious consequences on the fertilization potential. Myo-inositol has been observed to have anti-oxidant properties and be present in much greater concentrations specifically in seminal liquid than in the blood. Moreover, there seems to be a direct relationship between myo-inositol and mitochondrial membrane potential (MMP) and sperm motility. Studies performed in vivo have demonstrated that a dietary supplementation with myo-inositol in men undergoing assisted reproduction techniques may improve sperm quality and motility in oligoasthenospermia (OAT) patients. In the following study we utilized myo-inositol in vitro to verify its effect on semen quality in both normal and OAT patients undergoing in vitro fertilization (IVF) with respect to standard sperm medium. In vitro incubation of seminal liquid carried out using myo-inositol (Andrositol-Lab, Lo.Li. Pharma-Roma, Italy) at a concentration of 15 μl/ml improved progressive motility in both normospermia and OAT subjects. In our opinion, myo-inositol may prove to be a useful strategy to improve sperm preparation for clinical use in IVF.
Polycystic ovary syndrome (PCOS) is an endo-crine disorder that occurs in 8-10% of women of reproduc-tive age. It is characterized by oligo or anovulation, hyperandrogenism and/or polycystic ovaries, ...but also by an increased insulin plasma level especially in overweight/obese women or in those with familial diabetes. In the last years, among the insulin sensitizers, the use of the two active isoforms of inositols (myo-inositol and d-chiro-inositol) has been spreading for the treatment of PCOS insulin resistance. Several studies have shown a positive role of inositols both on the metabolic profile of PCOS patients, but also on hormonal parameters. Hence, inositols can positively affect the infertility that characterizes many PCOS patients, acting both on ovarian function and spontaneous ovulation and during IVF procedures, in terms of oocyte quality and pregnancy rate.
Maternal RNAs are synthesized by the oocyte during its growth; some of them are utilized for oocyte-specific processes and metabolism, others are stored and used during early development before ...embryonic genome activation. The appropriate expression of complex sets of genes is needed for oocyte maturation and early embryo development. In spite of the basic role of noncoding RNAs in the regulation of gene expression, few studies have analyzed their role in human oocytes. In this study, we identified the microRNAs (miRNAs) expressed in human metaphase II stage oocytes, and found that some of them are able to control pluripotency, chromatin remodeling, and early embryo development. We demonstrated that 12 miRNAs are differentially expressed in women of advanced reproductive age and, by bioinformatics analysis, we identified their mRNA targets, expressed in human oocytes and involved in the regulation of pathways altered in reproductive aging. Finally, we found the upregulation of miR-29a-3p, miR-203a-3p, and miR-494-3p, evolutionarily conserved miRNAs, also in aged mouse oocytes, and demonstrated that their overexpression is antithetically correlated with the downregulation of DNA methyltransferase 3A (Dnmt3a), DNA methyltransferase 3B (Dnmt3b), phosphatase and tensin homolog (Pten), and mitochondrial transcription factor A (Tfam). We propose that oocyte miRNAs perform an important regulatory function in human female germ cells, and their altered regulation could explain the changes occurring in oocyte aging.
Abstract
Study question
Does reproductive aging alter the expression of genes involved in NAD+ metabolism in the mammalian oocyte?
Summary answer
We found that aging alters the expression of thirty ...genes encoding for NAD+-producing and NAD+-consuming enzymes pathway in mouse MII oocytes.
What is known already
NAD, a multifunctional metabolite in living cells, is known to convert between its oxidized NAD+ and reduced NADH forms during nutrients breakdown; the intracellular NAD+/NADH redox state reflects cell ability in generating ATP energy. NAD+ is utilized by proteins that control gene expression, DNA repair, apoptosis, mitochondrial biogenesis (i.e. sirtuins). Raising NAD+ by inducing its biosynthesis leads to sirtuins activation, so directly linking the cellular redox state with signalling events. The NAD+ pool is set by a critical balance between NAD+ biosynthetic and NAD+ consuming pathways. NAD(P)H levels declined in aged oocytes and NAD+ precursors seem to counteract ovarian aging.
Study design, size, duration
Pools of 25 oocytes at MII stage were obtained from young (4–8 weeks) and reproductively aged (48–52 weeks) CD–1 mice and processed for the analysis of 41 genes participating in NAD+ biosynthetic and NAD+ consuming pathways NAD+ pathways. Each experiment was performed three times and data were subjected to bioinformatic analysis to unravel potential age-related effects on NAD metabolism.
Participants/materials, setting, methods
Mice were superovulated by intraperitoneal injection of PMSG followed by hCG 48h apart. MII oocytes were isolated by 0.3 mg/ml hyaluronidase. RNA was obtained from each sample by Arcturus PicoPure Kit, and reverse transcribed. Each cDNA was analysed in triplicate by employing a NAD Metabolism H41 Predesigned panel for use with SYBR® Green, containing 41 genes of the NAD pathway, 2 housekeeping genes and 6 control probes. Raw data were analysed by DataAssist software.
Main results and the role of chance
The comparison between aged and young oocytes were focused on genes showing an absolute fold change (FC) <0.7 or > 1.4, a present call in at least the 50% of experiments and a p-Value <0.05 (ANOVA). Excluding transcripts showing a concordant value < 80%, n.30 differentially expressed genes (DEGs) were found: n.26 transcripts down-expressed and n.4 genes up-regulated. Data obtained by Ingenuity Pathways Analysis (IPA) software (Ingenuity Systems) provide evidence that NAD+ biosynthesis in aged oocytes is severely compromised.
Limitations, reasons for caution
Our results on animal model must be taken with caution. Validation of NAD+ precursor or activators of NAD+ biosynthesis in vivo administration is required.
Wider implications of the findings: Present results demonstrate that aging affect oocyte genes involved in the regulation of NAD+ availability and supports the hypothesis that modulation of NAD+ metabolism may be an “anti-aging” strategy. Overall, these data laid the foundation for a central role of NAD+ metabolism in the maintenance of oocyte competenc
Trial registration number
Not applicable
Patients with endometriosis are characterized by the ability of the endometrium to implant and by the peritoneal response to the tissue; angiogenic factors may play a significant role in the ...aetiology of endometriosis supporting the implantation of ectopic endometrial cells. Vascular endothelial growth factor (VEGF) is a mitogen, morphogen and chemoactractant for endothelial cells and, in vivo, it is a powerful mediator for vessel permeability. Interleukin-8 (IL-8) is a chemoatractant for neutrophils and is a potent angiogenic factor. Women (n = 20) with ovarian endometriomata and 10 women with follicular cysts were enrolled in this study to investigate the role of VEGF and IL-8 in the development and maintenance of ovarian endometriomata. Cystic fluids were collected by laparoscopy, immediately centrifuged and stored until the enzyme-linked immunosorbent assays were performed. The VEGF and IL-8 concentrations were found to be significantly higher in the fluids of the ovarian endometriomata than in those of the follicular cysts of controls (P < 0.001 and P < 0.001 respectively); in addition, a significant inverse correlation between the VEGF cystic fluid concentrations and the diameter of the endometriotic adnexal masses was found (r = –0.56, P = 0.01). The evidence that the high concentrations of VEGF and IL-8 are present in the ovarian endometriomata indicates that angiogenesis could be a specific event both for the progression and maintenance of such cysts.
Introduction: Laparoscopic myomectomy has recently gained wide acceptance but this procedure remains technically highly demanding and concerns have been raised about the increased blood loss and an ...higher risk of postoperative uterine rupture of the pregnant uterus. Objective: The aim of the present study is to evaluate the fertility and endocrine outcome in women underwent robot-assisted laparoscopic myomectomy (RALM). Methods: Data from 48 RALM performed in our department between the years 2007 and 2011 have been collected. Conception rate, abortion rate, incidence of feto-maternal morbidity or severe pregnancy and labor-related complications were reported; FSH and AMH levels and ultrasound valuation of AFC has been made before and 6 months after operation. Number of cesarean sections and vaginal deliveries were described. Results: The average age of the patients was 35 years and median Body Mass Index was 23 kg/m2 (range 18-35 kg/m2). Seven women (13%) became pregnant after RALM with eight pregnancies. One pregnancy is actually on going; there were six deliveries with caesarian section and one spontaneous delivery. No spontaneous abortions. No uterine ruptures occurred. No significant modification of ovarian function was found after myomectomy. Conclusion: RALM seems to have a favorable impact on the reproductive outcome of young patients with no impact on the ovarian function.
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