Down-regulation of long non-coding RNAs in reproductive aging and analysis of the lncRNA-miRNA-mRNA networks in human cumulus cells.

PURPOSE: Long non-coding RNAs (lncRNAs) control gene expression at multiple levels. By interacting with microRNAs (miRNAs), they regulate their mRNA targets creating dynamic regulatory networks involved in different cellular processes. Their role in follicle development and oocyte maturation has recently emerged. lncRNA deregulation has been found associated with different pathological conditions. In this study, we identified differentially expressed lncRNAs in cumulus cells (CCs) isolated from MII oocytes of advanced maternal age women and proposed ceRNA-networks involved in signaling pathways crucial in ovarian folliculogenesis and female germ cell maturation. METHODS: We performed a high-throughput analysis of the expression profile of 68 lncRNAs from CCs of aged and young women by using NanoString technology. By miRNet, TarPmiR, miRTarBase, OKdb, and KEGG we predicted some ceRNA-networks involving the differentially expressed (DE) lncRNAs, miRNA interactors, and their mRNA target genes. RESULTS: We identified 28 lncRNAs down-regulated in CC samples from aged women. The analysis revealed that the miRNAs binding 11 of the DE lncRNAs and their mRNA targets are included in ceRNA-networks involved in the regulation of the PI3K-Akt, FOXO, and p53 signaling pathways. CONCLUSION: We proposed that the lncRNA down-regulation in CCs from aged women could influence the expression of genes encoding proteins deregulated in reproductive aging. A better understanding of the interplay of lncRNA-miRNA-mRNA networks in human CCs could increase our knowledge about the mechanisms of regulation of gene expression involved in aging, lead to the development of novel therapeutics, and improve reproductive outcomes in aged women.

Molecular profiling of follicular fluid microRNAs in young women affected by Hodgkin lymphoma.

RESEARCH QUESTION: Treatments for Hodgkin lymphoma have improved but one of their common effects is gonadal toxicity, which contributes to fertility damage of patients and induces temporary or irreversible loss of fertility. Could micro-RNA (miRNA) expression profiles in follicular fluid be influenced by Hodgkin lymphoma? Could their alteration affect molecular pathways involved in follicle growth and oocyte maturation? DESIGN: miRNA expression profile was investigated in follicular fluid samples from young women affected by Hodgkin lymphoma compared with healthy controls by NanoString technology. Bioinformatic analysis was used to verify miRNA involvement in follicle development and miRNA deregulation with Hodgkin lymphoma in a larger cohort of follicular fluid samples was confirmed by real-time quantitative polymerase chain reaction. RESULTS: Thirteen miRNAs are deregulated in Hodgkin lymphoma samples compared with controls and are involved in molecular pathways related to cancer, gametogenesis and embryogenesis. Among them, let-7b-5p, miR-423-5p, miR-503-5p, miR-574-5p and miR-1303 are implicated in biological processes related to follicle development and oocyte maturation. Let-7b-5p holds the central position in the regulatory network of miRNA-mRNA interactions, has the highest number of mRNA target genes shared with the other differentially expressed miRNAs and is significantly downregulated in Hodgkin lymphoma follicular fluid samples. CONCLUSIONS: These data led us to question the potential influence of miRNA deregulation on oocyte quality. Further studies are needed to verify the reproductive potential of young patients with Hodgkin lymphoma before starting chemotherapy protocols and an adequate protocol of fertility preservation needs to be guaranteed.

Modulating Intrafollicular Hormonal Milieu in Controlled Ovarian Stimulation: Insights From PPAR Expression in Human Granulosa Cells.

Controlled ovarian stimulation (COS) leading to ovulation of multiple follicles is a crucial aspect of biomedical infertility care. Nevertheless, biomarkers useful for COS management are still lacking. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors relevant to steroid metabolism in granulosa cells (GCs). We investigated whether PPARs and their steroidogenic targets were differentially expressed in GCs differentiated under different recombinant or urinary gonadotropin preparations. GCs from women subjected to COS with r-hFSH, r-hFSH/r-hLH, or hMG-HP were processed to assess expression of PPARα, PPARß/δ, PPARγ, and steroidogenic enzymes under PPAR modulation. As an evidence of their activation, all PPAR isotypes with their coactivators, the retinoic-X-receptors (RXRs), localized in the nucleus. When GCs from r-hFSH/r-hLH group were compared with r-hFSH, a significant reduction of PPARα protein was observed. By contrast, an increase of PPARß/δ at both protein and mRNA levels along with that of PPARγ protein were detected. The steroidogenic enzymes 17ßHSD IV, 3ßHSD II, and HMG-CoA red were downregulated in the r-hFSH/r-hLH group in comparison to r-hFSH unlike CYP19A1 that remained unchanged. In GCs from urinary FSH-LH stimulation (hMG-HP), PPARα was more expressed in comparison with r-hFSH/r-hLH group. Likewise, 3ßHSD II and 17ßHSD IV were increased suggesting that hMG-HP partially mimicked r-hFSH/r-hLH effects. In summary, transcript analysis associated to protein investigation revealed differential effects of COS protocols on PPARs and their steroidogenic targets in relation to LH and gonadotropin source. These observations candidate PPARs as new biomarkers of follicle competence opening new hypotheses on COS effects on ovarian physiology. J. Cell. Physiol. 231: 908-914, 2016. © 2015 Wiley Periodicals, Inc.

MicroRNAs Are Stored in Human MII Oocyte and Their Expression Profile Changes in Reproductive Aging.

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.

Azoospermic patient's treatment: An experience of a PMA hospital unit and role of ultrasonography.

INTRODUCTION: Azoospermia causes about 10% of male infertility and the best therapeutic option is the retrieval of sperm from testis or epididymis. MATERIAL AND METHODS: From Juanary 2008 to June 2016, 92 men (median 36 years; range: 25-54 years) were submitted in 47 cases to TESE (testicular sperm extraction) and in 45 cases to PESA (percutaneous epididymal sperm aspiration) for secretory and obstructive azoospermia, respectively; moreover, all the patients previously underwent color Doppler ultrasound of the testis and transrectal ultrasound of the prostate. RESULTS: Serum FSH values were 9.4 ml/UI and 36.4 ml/UI (median 18.2 ml/UI) with an estimated volume of the testis equal to 5 ml; 40 men had the mutation for cystic fibrosis with bilateral agenesis of the deferentia vasa, 4 men had a cyst of the prostatic utricle, 1 man had retrograde ejaculation, 7 had an epididymis cyst and 2 had anejaculation secondary to traumatic neurologic spinal cord injury. The retrieval of sperm was performed in 39 (83%) and 36 (80%) of the patients submitted to TESE and PESA, respectively. The pregnancy rate was equal to 28% and 33% in men with secretory and obstructive azoospermia, respectively. DISCUSSION: Assisted reproduction technology with a multidisciplinary team is provided of a pregnancy rate equal about 30% in men with azoospermia; ultrasound allows to evaluate abnormalities of the testis and prostate improving the percentage of pregnancy.

A Circular RNA Derived from the Pumilio 1 Gene Could Regulate PTEN in Human Cumulus Cells.

CircRNAs are a class of non-coding RNAs able to regulate gene expression at multiple levels. Their involvement in physiological processes, as well as their altered regulation in different human diseases, both tumoral and non-tumoral, is well documented. However, little is known about their involvement in female reproduction. This study aims to identify circRNAs potentially involved in reproductive women's health. Candidate circRNAs expressed in ovary and sponging miRNAs, already known to be expressed in the ovary, were selected by a computational approach. Using real time PCR, we verified their expression and identified circPUM1 as the most interesting candidate circRNA for further analyses. We assessed the expression of circPUM1 and its linear counterpart in all the follicle compartments and, using a computational and experimental approach, identified circPUM1 direct and indirect targets, miRNAs and mRNAs, respectively, in cumulus cells. We found that both circPUM1 and its mRNA host gene are co-expressed in all the follicle compartments and proposed circPUM1 as a potential regulator of PTEN, finding a strong positive correlation between circPUM1 and PTEN mRNA. These results suggest a possible regulation of PTEN by circPUM1 in cumulus cells and point out the important role of circRNA inside the pathways related to follicle growth and oocyte maturation.

Live Birth from Cryopreserved Oocyte After Uterus Transplantation: A Case Report.

BACKGROUND Important legal and ethical issues must be addressed in the practice of uterus transplantation, because it is a non-life-saving intervention. In all cases reported in the literature so far, uterus transplantation is preceded by oocyte retrieval, fertilization of the collected oocytes, and subsequent freezing of the embryos produced. This element should be considered because of the potential ethical, legal, and moral implications related to the existence and fate of supernumerary embryos in the event of transplantation failure. CASE REPORT The Italian Research Project for Uterus Transplantation from a brain-dead donor was approved in 2018 (No. 1438/CNT2018). A 28-year-old patient with Mayer-Rokitansky-Küster-Hauser syndrome, ectopic ovaries, and good ovarian reserve received uterus transplantation in 2020 after oocyte retrieval with laparoscopic assistance. Metaphase oocytes were cryopreserved and thawed after the successful transplantation to perform in vitro fertilization followed by embryo transfer. The pregnancy course was regular, without symptoms until week 30, when PCR positivity for SARS-CoV-2 was recorded. The patient underwent an emergency cesarean delivery at 34 weeks' gestation because of fever and the appearance of regular uterine contractions. An infant was born alive and vital at 34 weeks of pregnancy and weighed 1725 g. The newborn was discharged in good condition and with a body weight of 2740 g. CONCLUSIONS This case report shows that cryopreservation of oocytes can overcome the ethical issue related to embryo retrieval before a successful uterus transplantation can be demonstrated. Our result supports the possibility of bypassing embryo freezing before ascertaining the success of uterus transplantation.