Should we worry about the clock? Relationship between time to ICSI and reproductive outcomes in cycles with fresh and vitrified oocytes.

STUDY QUESTION: Is there an optimal time to perform ICSI with respect to the times of oocyte pick-up (OPU), in order to maximize the reproductive outcomes in cycles with fresh and vitrified/warmed donor oocytes? SUMMARY ANSWER: We found no significant differences in reproductive outcomes of ICSI cycles within a wide range of times between OPU and ICSI. WHAT IS KNOWN ALREADY: In assisted reproduction, the oocyte is subject to denudation, vitrification/warming and ICSI. As shorter interaction with cumulus cells, oocyte ageing in vitro and insufficient recovery after warming may all impact the resulting embryo developmental competence, strictly controlled times between procedures are often implemented. However, most protocols have not been tested with the aim to improve reproductive results, and little information is available on the ideal times to be followed during these steps in order to optimize fertilization rates and embryo quality, and to achieve the highest pregnancy rate. STUDY DESIGN, SIZE, DURATION: Data from 3986 ICSI cycles performed between December 2012 and May 2014 were included (3178 with fresh and 808 with vitrified/warmed donor oocytes). PARTICIPANTS/MATERIALS, SETTING, METHODS: ICSI was performed using donor oocytes and either partner or donor sperm. Exact times between OPU, denudation, vitrification, warming and ICSI were recorded automatically by a radiofrequency-based system. OPU was performed strictly 36 h after GnRH agonist trigger. Biochemical pregnancy was defined as a positive serum ßHCG 15 days after transfer, clinical pregnancy was defined as a visible embryo with heartbeat 5 weeks after transfer, and ongoing pregnancy was defined as a normally developing pregnancy at 12 weeks after transfer. MAIN RESULTS AND THE ROLE OF CHANCE: Times between OPU and ICSI (OPU-ICSI) ranged from 1 h 25 min to 17 h 13 min (averagefresh ± SD = 4 h 58 m ± 1 h; averagevitrified= 9 h 18 m ± 2 h). We found no effect of OPU-ICSI time on fertilization rate (pfresh=0.39; pvitrified=0.86) or embryo quality at Days 2 and 3 (pfresh=0.08; pvitrified=0.22). There was no difference in average OPU-ICSI times between positive and negative pregnancies (biochemical, clinical, ongoing and live birth rates) in either fresh (P = 0.71, 0.43, 0.79, 0.96) or vitrified (P = 0.59, 0.33, 0.73, 0.87) oocytes, respectively. Data were adjusted for oocyte donor age, semen status, number of motile spermatozoa and sperm concentration, and no effect of OPU-ICSI time on pregnancy and live birth rates for either fresh (P = 0.57, 0.16, 0.11, 0.46) or vitrified (P = 0.80, 0.73, 0.91, 0.95) oocytes was found. Further analysis for linear trend using OPU-ICSI time categorized in deciles showed that pregnancy rates and live birth rates do not increase or decrease across deciles. We found no effect of time taken for denudation to vitrification, warming to ICSI and denudation to ICSI on pregnancy rates. LIMITATIONS, REASONS FOR CAUTION: This is a study with automatically collected times from a high number of ICSI cases; however, its retrospective nature cannot exclude the influence of unaccounted for variables on the results. All oocytes came from oocyte donors (≤35 years old), so results cannot be extended to older or infertile women. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that the effective window of time for insemination by ICSI might be wider than previously thought. It therefore appears that, within appropriate time frames, the management of ICSI cycles involving oocytes from young women in embryology laboratories could be adjusted to accommodate caseloads and workflow with no loss of oocyte viability or cycle efficiency.

Phenotypic identification of subclones in multiple myeloma with different chemoresistant, cytogenetic and clonogenic potential.

Knowledge about clonal diversity and selection is critical to understand multiple myeloma (MM) pathogenesis, chemoresistance and progression. If targeted therapy becomes reality, identification and monitoring of intraclonal plasma cell (PC) heterogeneity would become increasingly demanded. Here we investigated the kinetics of intraclonal heterogeneity among 116 MM patients using 23-marker multidimensional flow cytometry (MFC) and principal component analysis, at diagnosis and during minimal residual disease (MRD) monitoring. Distinct phenotypic subclones were observed in 35/116 (30%) newly diagnosed MM patients. In 10/35 patients, persistent MRD was detected after 9 induction cycles, and longitudinal comparison of patient-paired diagnostic vs MRD samples unraveled phenotypic clonal tiding after therapy in half (5/10) of the patients. After demonstrating selection of distinct phenotypic subsets by therapeutic pressure, we investigated whether distinct fluorescence-activated cell-sorted PC subclones had different clonogenic and cytogenetic profiles. In half (5/10) of the patients analyzed, distinct phenotypic subclones showed different clonogenic potential when co-cultured with stromal cells, and in 6/11 cases distinct phenotypic subclones displayed unique cytogenetic profiles by interphase fluorescence in situ hybridization, including selective del(17p13). Collectively, we unravel potential therapeutic selection of preexisting diagnostic phenotypic subclones during MRD monitoring; because phenotypically distinct PCs may show different clonogenic and cytogenetic profiles, identification and follow-up of unique phenotypic-genetic myeloma PC subclones may become relevant for tailored therapy.

The immunophenotype of different immature, myeloid and B-cell lineage-committed CD34+ hematopoietic cells allows discrimination between normal/reactive and myelodysplastic syndrome precursors.

Occurrence of phenotypic abnormalities in CD34(+) hematopoietic progenitor and precursor cells (HPC) and their major B-cell and nonlymphoid compartments has been frequently reported in myelodysplastic syndromes (MDS). Here, we analyze for the first time the numerical and phenotypic abnormalities of different maturation-associated subsets of bone marrow (BM) CD34(+) HPC from 50 newly diagnosed MDS patients in comparison to normal/reactive BM (n=29). Our results confirm the existence of heterogeneously altered phenotypes among CD34(+) HPC from MDS and indicate that such variability depends both on the relative distribution of the different subsets of CD34(+) HPC committed into the different myeloid and B-lymphoid compartments, and their immunophenotype (for example, higher reactivity for CD117 and CD13 and lower expression of CyMPO, CD64 and CD65 on CD34(+) immature and neutrophil precursors), a clear association existing between the accumulation of CD34(+) HPC and that of immature CD34(+) HPC. Interestingly, expansion of erythroid- and neutrophil-lineage CD34(+) cells is detected in low-grade MDS at the expense of CD34(+) plasmacytoid dendritic cell and B-cell precursors, while expansion of immature CD34(+) precursors occurs in high-grade MDS. On the basis of the number and severity of the phenotypic abnormalities detected, a scoring system is proposed that efficiently discriminates between normal/reactive and MDS CD34(+) HPC, the mean score significantly increasing from low- to high-grade MDS.

TCRgammadelta+ large granular lymphocyte leukemias reflect the spectrum of normal antigen-selected TCRgammadelta+ T-cells.

T-cell large granular lymphocytes (LGL) proliferations range from reactive expansions of activated T cells to T-cell leukemias and show variable clinical presentation and disease course. The vast majority of T-LGL proliferations express TCRalphabeta. Much less is known about the characteristics and pathogenesis of TCRgammadelta+ cases. We evaluated 44 patients with clonal TCRgammadelta+ T-LGL proliferations with respect to clinical data, immunophenotype and TCR gene rearrangement pattern. TCRgammadelta+ T-LGL leukemia patients had similar clinical presentations as TCRalphabeta+ T-LGL leukemia patients. Their course was indolent and 61% of patients were symptomatic. The most common clinical manifestations were chronic cytopenias - neutropenia (48%), anemia (23%), thrombocytopenia (9%), pancytopenia (2%) - and to a lesser extent splenomegaly (18%). Also multiple associated autoimmune (34%) and hematological (14%) disorders were found. Leukemic LGLs were predominantly positive for CD2, CD5, CD7, CD8, and CD57, whereas variable expression was seen for CD16, CD56, CD11b, and CD11c. The Vgamma9/Vdelta2 immunophenotype was found in 48% of cases and 43% of cases was positive for Vdelta1, reflecting the TCR-spectrum of normal TCRgammadelta+ T-cells in adult PB. Identification of the well-defined post-thymic Vdelta2-Jdelta1 selection determinant in all evaluable Vgamma9+/Vdelta2+ patients, is suggestive of common (super)antigen involvement in the pathogenesis of these TCRgammadelta+ T-LGL leukemia patients.