The pollination ecology of a rare case of bee-pollinated Psittacanthus mistletoe.

The genus Psittacanthus (Loranthaceae) is widely distributed in the Neotropical region, where it is known for its large, colourful, scentless flowers. Until very recently, all Psittacanthus species were regarded as exclusively hummingbird-pollinated and the large species radiation in the genus attributed to the interactions with bird dispersers and pollinators. P. eucalyptifolius (Kunth) G.Don. is the only species reported as bee-pollinated. Here we describe the floral biology, floral visitors, and the reproductive system of P. eucalyptifolius in an Amazonian savanna, Brazil. We also compare the pollination success (reproductive performance) among different Psittacanthus species reported in previous studies. Psittacanthus eucalyptifolius produces sweet-scented flowers, and a small quantity of concentrated nectar. At least five species of scopate bees were recorded visiting and carrying pollen of P. eucalyptifolius. Xylocopa frontalis carried most pollen, visited more flowers, remained longer, and touched reproductive parts of flowers in >95% of the observed visits. Experiments indicate that P. eucalyptifolius is partially autocompatible (39% autonomous pollination) but depends on pollinators to achieve higher performance (~78% in control), indicating that bees can be as effective as birds in pollinating this group of mistletoes.

Characterization of the miRNA regulators of the human ovulatory cascade.

Ovarian follicular development and ovulation are complex and tightly regulated processes that involve regulation by microRNAs (miRNAs). We previously identified differentially expressed mRNAs between human cumulus granulosa cells (CGCs) from immature early antral follicles (germinal vesicle - GV) and mature preovulatory follicles (metaphase II - M2). In this study, we performed an integrated analysis of the transcriptome and miRNome in CGCs obtained from the GV cumulus-oocyte complex (COC) obtained from IVM and M2 COC obtained from IVF. A total of 43 differentially expressed miRNAs were identified. Using Ingenuity IPA analysis, we identified 7288 potential miRNA-regulated target genes. Two hundred thirty-four of these target genes were also found in our previously generated ovulatory gene library while exhibiting anti-correlated expression to the identified miRNAs. IPA pathway analysis suggested that miR-21 and FOXM1 cooperatively inhibit CDC25A, TOP2A and PRC1. We identified a mechanism for the temporary inhibition of VEGF during ovulation by TGFB1, miR-16-5p and miR-34a-5p. The linkage bioinformatics analysis between the libraries of the coding genes from our preliminary study with the newly generated library of regulatory miRNAs provides us a comprehensive, integrated overview of the miRNA-mRNA co-regulatory networks that may play a key role in controlling post-transcriptomic regulation of the ovulatory process.

Focused time-lapse analysis reveals novel aspects of human fertilization and suggests new parameters of embryo viability.

STUDY QUESTION: Can focused application of time-lapse microscopy (TLM) lead to a more detailed map of the morphokinetics of human fertilization, revealing novel or neglected aspects of this process? SUMMARY ANSWER: Intensive harnessing of TLM reveals novel or previously poorly characterised phenomena of fertilization, such as a cytoplasmic wave (CW) preceding pronuclear formation and kinetics of pronuclear chromatin polarization, thereby suggesting novel non-invasive biomarkers of embryo quality. WHAT IS KNOWN ALREADY: In recent years, human preimplantation development has been the object of TLM studies with the intent to develop morphokinetic algorithms able to predict blastocyst formation and implantation. Regardless, our appreciation of the morphokinetics of fertilization remains rather scarce, currently including only times of polar body II (PBII) emission, pronuclear appearance and fading, and first cleavage. This is not consistent with the complexity and importance of this process, calling for further TLM studies aimed at describing previously unrecognized or undetected morphokinetic events and identifying novel developmental biomarkers. STUDY DESIGN, SIZE, DURATION: The study involved a retrospective observation by TLM of the fertilization process in 500 oocytes utilized in consecutive ICSI cycles carried out in 2016. A maximum of five fertilized oocytes per patients were included in the analysis to reduce possible patient-specific biases. Oocytes of patients with different diagnoses of infertility where included in the analysis, while cases involving cryopreserved gametes or surgically retrieved sperm were excluded. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: Microinjected oocytes where assessed by a combined TLM-culture system (Embryoscope). Oocytes that were not amenable to TLM assessment, due to excess of residual corona cells or inadequate orientation for the observation of PBII emission, were not analysed. We identified and monitored 28 parameters relevant to meiotic resumption, pronuclear dynamics, chromatin organization, and cytoplasmic/cortical modifications. Times (T) were expressed as mean ± SD hours post-insemination (p.i.) and analysed, where appropriate, by Paired T Student or Fisher's exact tests. MAIN RESULTS AND ROLE OF CHANCE: PBII emission was occasionally followed (4.3% of cases) by the transient appearance of a protrusion of the cell surface, the fertilization cone (FC), probably resulting from interaction of the male chromatin with the oocyte cortex. Pronuclear formation was always preceded by a radial CW originating from the initial position of the male pronucleus (PN) and extending towards the oocyte periphery. The appearance of the CW followed a precise sequence, occurring always 2-3 h after PBII emission and shortly before PN appearance. Male and female PN appeared virtually simultaneously at approximately 6.2 h p.i. However, while the female PN always formed cortically and near the site of emission of the PBII, the initial position of the male PN was cortical, intermediate, or central (15.2%, 31.2% and 53.6%, respectively). PN juxtaposition involved rapid and straight movement of the female PN towards the male PN. In addition, the initial position of male PN formation was predictive of the position of PN juxtaposition. It was also observed that nucleolar precursor bodies (NPBs) aligned along the juxtaposition area and this happened considerably earlier for the female PN (8.2 ± 2.6 vs.11.2 ± 4.1 h, P = 0.0001). Although it occurred rarely, displacement of juxtaposed PN to the cortex was strongly associated (P < 0.0001) with direct cleavage into three blastomeres at the first cell division. The times of PN breakdown and first cleavage showed a very consistent trend, occurring earlier or progressively later depending on whether initial male PN positioning was central, intermediate or cortical, respectively. Finally, time intervals between discrete fertilization events were strongly associated with embryo quality on Day 3. For example, longer intervals between disappearance of the cytoplasmic halo and PN breakdown were highly predictive of reduced blastomere number and increased fragmentation (P = 0.0001). LARGE SCALE DATA: N/A. LIMITATIONS, REASON FOR CAUTION: Some of the morphokinetic parameters assessed in this study may require better definition to reduce inter-operator annotation variability. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, overall, these data represent the most detailed morphokinetic description of human fertilization. Many of the illustrated parameters are novel and may be amenable to further elaboration into algorithms able to predict embryo quality, as suggested by the findings presented in this study. STUDY FUNDING/COMPETING INTERESTS: None.

Fertility preservation issues in pediatric hematopoietic stem cell transplantation: practical approaches from the consensus of the Pediatric Diseases Working Party of the EBMT and the International BFM Study Group.

Fertility preservation is an urgent challenge in the transplant setting. A panel of transplanters and fertility specialists within the Pediatric Diseases Working Party of the European Society for Blood and Marrow Transplantation (EBMT) and the International BFM Study Group provides specific guidelines. Patients and families should be informed of possible gender- and age-specific cryopreservation strategies that should be tailored according to the underlying disease, clinical condition and previous exposure to chemotherapy. Semen collection should be routinely offered to all postpubertal boys at the diagnosis of any disease requiring therapy that could potentially impair fertility. Testicular tissue collection might be offered to postpubertal boys; nevertheless, its use has been unsuccessful to date. Oocyte collection after hormonal hyperstimulation should be offered to postpubertal girls facing gonadotoxic therapies that could be delayed for the 2 weeks required for the procedure. Ovarian tissue collection could be offered to pre-/post-pubertal girls. Pregnancies have been reported after postpubertal ovarian tissue reimplantation; however, to date, no pregnancy has been reported after the reimplantation of prepubertal ovarian tissue or in vitro maturation of pre-/post-pubertal ovarian tissue. Possible future advances in reproductive medicine could change this scenario. Health authorities should prioritize fertility preservation projects in pediatric transplantation to improve patient care and quality of life.

Characterization of the human cumulus cell transcriptome during final follicular maturation and ovulation.

Cumulus expansion and oocyte maturation are central processes in ovulation. Knowledge gained from rodent and other mammalian models has revealed some of the molecular pathways associated with these processes. However, the equivalent pathways in humans have not been thoroughly studied and remain unidentified. Compact cumulus cells (CCs) from germinal vesicle cumulus oocyte complexes (COCs) were obtained from patients undergoing in vitro maturation (IVM) procedures. Expanded CCs from metaphase 2 COC were obtained from patients undergoing IVF/ICSI. Global transcriptome profiles of the samples were obtained using state-of-the-art RNA sequencing techniques. We identified 1746 differentially expressed (DE) genes between compact and expanded CCs. Most of these genes were involved in cellular growth and proliferation, cellular movement, cell cycle, cell-to-cell signaling and interaction, extracellular matrix and steroidogenesis. Out of the DE genes, we found 89 long noncoding RNAs, of which 12 are encoded within introns of genes known to be involved in granulosa cell processes. This suggests that unique noncoding RNA transcripts may contribute to the regulation of cumulus expansion and oocyte maturation. Using global transcriptome sequencing, we were able to generate a library of genes regulated during cumulus expansion and oocyte maturation processes. Analysis of these genes allowed us to identify important new genes and noncoding RNAs potentially involved in COC maturation and cumulus expansion. These results may increase our understanding of the process of oocyte maturation and could ultimately improve the efficacy of IVM treatment.

Anti-Müllerian hormone remains highly expressed in human cumulus cells during the final stages of folliculogenesis.

This study evaluated whether anti-Müllerian hormone (AMH) was differentially expressed in cumulus (CC) and granulosa (GC) cells from large antral and pre-ovulatory follicles collected from individual follicles in women undergoing in-vitro maturation (IVM) or IVF treatment. Expression studies of AMH, AMH receptor 2, FSH receptor, aromatase and androgen receptor were performed in CC in IVM patients where cumulus-oocyte-complex had expanded, CC in IVM patients where cumulus-oocyte-complex remained compacted, GC from immature follicles and CC and GC from IVF patients. Microarray data on corresponding GC and CC from follicles from IVF patients was included. AMH expression was significantly higher in CC than in GC from both mature and immature follicles and in CC from immature follicles than in CC from pre-ovulatory follicles from IVF patients (P < 0.05). AMH expression was significantly higher in CC that remained compacted compared with those that had expanded (P < 0.008). AMH was correlated to the expression of FSH receptor, androgen receptor and AMH receptor 2 but not to aromatase expression. The expression pattern of AMH receptor 2 reflected that of AMH. AMH may exert intrafollicular functions even in human large antral and pre-ovulatory follicles and may be related to follicular health.

Effect of different gonadotrophin priming on IVM of oocytes from women with normal ovaries: a prospective randomized study.

This study was designed to determine if the efficiency of in-vitro maturation (IVM) in women with normal ovaries can be improved by gonadotrophin administration. 400 women were randomly allocated in four groups: group A, non-primed cycles; group B, human chorionic gonadotrophin (HCG)-primed cycles; group C, FSH-primed cycles; and group D, FSH- plus HCG-primed cycles. There were significant differences in the IVM rate among the groups. In groups where HCG was used, the overall maturation rate was higher (57.9% in group B and 77.4% in group D; 48.4% in group A and 50.8% in group C) and the percentage of total available metaphase II-stage oocytes was higher (60.4% in group B and 82.1% in group D; 48.4% in group A and 50.8% in group C). The overall clinical pregnancy rate per transfer (CPR) was 18.3% and the implantation rate (IR) was 10.6%. There was a difference in CPR among the groups: group D (29.9%) versus group A (15.3%), P = 0.023; group D versus group B (7.6%), P < 0.0001; group D versus group C (17.3%), P = 0.046. The results of this study are clearly in favour of FSH plus HCG priming. FSH priming and HCG priming alone showed no significant effects on clinical outcome.

Human oocyte cryopreservation: comparison between slow and ultrarapid methods.

The success of reproductive technologies is facilitated by the cryopreservation of embryos and gametes. In Italy, where legislation prohibits zygote and embryo cryopreservation, clinics have extensively introduced oocyte cryopreservation. Two different strategies of oocyte cryopreservation are available: slow freezing or ultrarapid cooling (vitrification). Although the results are very encouraging with both methods, there is still controversy regarding both the procedure itself and the most suitable method to use. This study reports the routine application of the two different oocyte cryopreservation methods in programmes running in two consecutive periods. The study centre carried out 286 thawing cycles for a total of 1348 thawed oocytes cryopreserved by the slow-freezing method and 59 warming cycles for a total of 285 warmed oocytes cryopreserved by vitrification. Comparison of the outcomes obtained with the slow-freezing method versus vitrification in women who underwent IVF for infertility showed survival, fertilization, pregnancy and implantation rates of 57.9% versus 78.9% (P < 0.0001), 64.6% versus 72.8% (P = 0.027), 7.6% versus 18.2% (P = 0.021) and 4.3% versus 9.3% (P = 0.043) respectively. These results suggest that oocyte vitrification is associated with a better outcome than the slow-freezing method.

Predictive factors in in-vitro maturation in unstimulated women with normal ovaries.

The in-vitro maturation protocol (IVM) is an intriguing tool in assisted reproduction since it omits the side-effects of drug stimulation and reduces the cost of the entire procedure, both in terms of time and patient/society costs. In the Biogenesi Reproductive Medicine Centre, the IVM technique has been applied for more than 3 years, obtaining successful results in terms of maturation and fertilization rates, number of pregnancies and healthy babies born. At present, IVM is widely accepted in polycystic ovary and polycystic ovarian syndrome patients but its application in other women is still controversial. This study has been carried out in order to determine the efficiency of unstimulated IVM in women with morphologically and endocrinologically normal ovaries. Body mass index, basal FSH and oestradiol concentrations, antral follicle count, endometrial thickness and lead follicle size were correlated with the outcome of the procedure so as to obtain useful criteria to select women with regular cycles for an IVM technique. It was found that basal oestradiol concentration, FSH concentration and antral follicle count are useful criteria in deciding whether to start and continue the procedure, while lead follicle size and endometrial thickness are important criteria in deciding the timing of oocyte retrieval.