SPOM is a new approach to IVM, mimicing some characteristics of oocyte maturation in vivo and substantially improving oocyte developmental outcomes. Adaption of SPOM for clinical application should have significant implications for infertility management and bring important benefits to patients.

The phosphodiesterase (PDE) family is a group of enzymes that catalyzes the transformation of cyclic nucleotides into 5' nucleotides. Based on rodents, the current mammalian model of PDE distribution in the ovarian follicle predicts Pde3a in the oocyte and Pde4d in the somatic cells. Using bovine as an experimental model, the present results showed that PDE3 was the predominant PDE activity in oocytes. However, cumulus cell cAMP-PDE activity was predominantly resistant to inhibition by 3-isobutyl-methylxantine, indicating PDE8 activity (60% of total PDE activity) and a minor role for PDE4 (<5%). A total of 20% of total oocyte PDE activity was also attributed to PDE8. The PDE activity measurements in mural granulosa cells from 2 to 6 mm in diameter suggest the presence of PDE4 and PDE8. In granulosa cells from follicles >10 mm, total PDE and PDE8 activities along with PDE8A protein level were increased compared with smaller follicles. The RT-PCR experiments showed that cumulus cells expressed PDE8A, PDE8B, and PDE10A. Western blot experiments showed PDE8A, PDE8B, and PDE4D proteins in mural granulosa cells and cumulus-oocyte complexes. PDE8 inhibition using dipyridamole in a dose-dependent manner increased cAMP levels in the cumulus-oocyte complexes and delayed oocyte nuclear maturation. These results are the first to demonstrate the functional presence of PDE8 in the mammalian ovarian follicle. This challenges the recently described cell-specific expression of cAMP-PDEs in the ovarian follicle and the notion that PDE4 is the predominant granulosa/cumulus cell PDE. These findings have implications for our understanding of hormonal regulation of folliculogenesis and the potential application of PDE inhibitors as novel contraceptives.

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Within the context of an oocyte in vitro maturation (IVM) program for reproductive treatment, oocyte cumulus complexes (COCs) derived from follicles <6 mm in patients with PCOS were matured in vitro. Key transcripts related to meiotic maturation (FSHR, LHCGR, EGFR, PGR) and oocyte competence (AREG, ADAMTS, HAS2, PTGS2) were quantified in cumulus cells (CCs) before and after maturation. Control CC samples were collected from PCOS and normo-ovulatory patients who had undergone conventional gonadotrophin stimulation for IVF/ICSI. Additional control samples from a non-stimulated condition were obtained ex vivo from patients undergoing ovariectomy for fertility preservation. Expression data from CCs from follicles with a diameter of <6 mm before (IVM-CCs) and after in vitro maturation (IVM-CCs) were obtained after pooling CCs into four groups in relation to the percentage of matured (MII) oocytes obtained after 40 h of IVM (0; 40-60; 61-80; 100% MII) and values were compared with in vivo matured controls (IVO-CCs). Genes encoding key receptors mediating meiotic resumption are expressed in human antral follicles of <6 mm before and after IVM. The expression levels of FSHR, EGFR and PGR in CCs were significantly down-regulated in the IVO-CCs groups and in the 100% MII IVM group compared with the BM groups; all the receptors studied in the 100% MII IVM group reached an expression profile similar to that of IVO-CCs. However, after maturation in a conventional IVF/ICSI cycle, IVO-CCs from large follicles contained significantly increased levels of ADAMTS1, AREG, HAS2 and PTGS2 compared with IVM-CCs and IVM-CCs; the expression patterns for these genes in all IVM-CCs were unchanged compared with IVM-CCs. In conclusion, genes encoding receptors involved in oocyte meiotic resumption appeared to be expressed in CCs of small human antral follicles. Expression levels of genes-encoding factors reflecting oocyte competence were significantly altered in IVM-CCs compared with in vivo matured oocytes from large follicles. Observed differences might be explained by the different stimulation protocols, doses of gonadotrophin or by the intrinsic differences between in vivo and in vitro maturation.

Purpose The in-vitro environment influences oocyte competence and gene expression in cumulus cells and oocytes. Effects of culturing under non-attachment conditions and varying follicle exposure to FSH were investigated at the mRNA level and on oocyte developmental capacity. Methods Quantitative PCR analysis of Gdf9, Mater, Nmp2 (in oocytes), Lhcgr and Amh (in cumulus cells), and oocyte developmental competence after in vitro follicle culture were evaluated. Results Follicle survival (98.7%) and polar body rate (94%) were similar for all conditions. Estradiol and progesterone production were significantly lower in non-attachment follicles (10-fold and 3-fold, respectively). Under nonattachment conditions, a higher two-cell rate (69.9%) and total blastocyst yield (48.5%) were obtained and, by decreasing FSH levels during culture, Lhcgr transcripts were significantly reduced to levels similar to in-vivo. Levels of oocyte-specific transcripts were not significantly influenced by in-vitro conditions. Conclusion Non-attachment conditions influence follicle steroid secretory capacity and, together with dynamic FSH doses, positively influence cumulus cell gene expression and oocyte developmental competence.

Semen parameters of varicocele men have been usually suspected to exhibit higher levels of abnormalities including DNA fragmentation, reactive oxygen species (ROS) and apoptotic markers. Negative correlation between increased level of DNA fragmentation and assisted reproductive techniques (ART) outcome has been studied by several authors. In the current study, we aim to evaluate the possible value of magnetic activated cell sorting (MACs) technology in reduction of DNA fragmentation in infertile varicocele patients prior to ART. Semen samples, collected from 36 varicocele patients, were prepared by density gradient centrifugation (DGC). Every sample was subsequently divided into two aliquots. One aliquot was kept untouched as pre-MACs control while the other aliquot was subjected to MACs technique, for depletion of apoptotic spermatozoa, and serves as post-MACs test. Sperm count, motility and DNA fragmentations were evaluated for both control and test samples. Post-MACs samples showed no deleterious reduction in total sperm motility (80.64 ± 6.97%) compared with control samples (80.97 ± 7.74%) while sperm DNA fragmentations were significantly reduced in post-MACs samples (9.61 ± 5.62%) compared with pre-MACs controls (12.43 ± 6.29%) (P < 0.05). It can be concluded that MACs technique is a simple, noninvasive, technique that can efficiently reduce DNA fragmentation in infertile varicocele patients prior to ART.

This is the first study reporting preclinical validation and accuracy assessment of the Ion Torrent Personal Genome Machine (PGM) NGS-based comprehensive chromosome screening method using blastomeres and blastocysts. The NGS proved to be a robust methodology and is ready for clinical application in reproductive medicine, with the major advantage of low cost and enhanced precision when compared with other technologies used for comprehensive chromosome screening.