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STUDY QUESTION Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo ? SUMMARY ANSWER The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-β family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016–2018. PARTICIPANTS/MATERIALS, SETTING, METHODS We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12–17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12–17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus–oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared. [ABSTRACT FROM AUTHOR]

Protein microarrays are powerful tools to quantify and characterize proteins in multiplex assays. They have great potential within clinical diagnostics and prognostics, as they minimize consumption of both analyte and biological sample. Assays that do not require labeling of the biological specimen, henceforth called label-free, are vital for ease of clinical sample processing. Here, we evaluate two label-free techniques, reverse-phase and sandwich antibody assays, using microarrays on high-performance porous silicon surfaces and fluorescence detection. In view of increasing interest in reverse microarrays, this paper focuses on analytical sensitivity of the reverse assays compared to the more complex but highly sensitive sandwich assay. Sensitivity, linear range, and reproducibility of the two assays were compared using prostatespecific antigen (PSA) in buffer. The sandwich assay displayed 5 orders of magnitude lower detection limit (0.7 ng/mL) compared to the reverse assay (70 [micro]g/mL). PSA at 50 nM (1.5 [micro]g/mL) in cell lysates was detected by the sandwich assay but not by the reverse assay, demonstrating again a far lower detection limit for sandwich microarrays. In independent assay runs of PSA spiked in female serum, the sandwich assay had good linearity ([R.sup.2] > 0.99) and reproducibility (coefficient of variation [less than or equal to] 15%), and the detection limit could be improved to 0.14 ng/mL. Without further signal amplification, the sandwich assay would be our choice for PSA analysis of clinical samples using a microarray technology platform.

Sulfidopeptide leukotrienes, which are important biomarkers for several diseases, are commonly measured by microtiter plate immunoassays. These immunoassays, however, cannot distinguish between several structurally similar leukotrienes and their cross-reactive metabolites and, therefore, need extensive sample handling and fractionation by means of liquid chromatography (LC). This paper describes the development and automation of a continuous-flow immunochemical detection (ICD) system and its subsequent on-line coupling to LC. The on-line LC-ICD system based on fluorescence-labeled leukotriene [E.sub.4] ([LTE.sub.4]) was used to determine sulfidopeptide leukotrienes and their cross-reactive metabolites in a single run. Furthermore, biological matrices, e.g., urine and human cell extracts, were analyzed, the only sample pretreatment being on-line solid-phase extraction (SPE) on a novel RP-[C.sub.4] restricted-access support. The determination limit of [LTE.sub.4] in urine was 0.2 ng/mL (800 fmol; injection volume, 2000 [[micro]liter]; signal-to-noise ratio, 10). The system was linear from 0.2 to 1.0 ng/mL [LTE.sub.4]. Using nonlinear curve-fitting, the range could be expanded to 2.5 ng/mL. It is shown that, besides quantitation of known analytes, on-line LC-ICD is useful in the discovery of cross-reactive [LTE.sub.4] metabolites.