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STUDY QUESTION Is it possible to identify by mass spectrometry a wider range of proteins and key proteins involved in folliculogenesis and oocyte growth and development by studying follicular fluid (FF) from human small antral follicles (hSAF)? SUMMARY ANSWER The largest number of proteins currently reported in human FF was identified in this study analysing hSAF where several proteins showed a strong relationship with follicular developmental processes. WHAT IS KNOWN ALREADY Protein composition of human ovarian FF constitutes the microenvironment for oocyte development. Previous proteomics studies have analysed fluids from pre-ovulatory follicles, where large numbers of plasma constituents are transferred through the follicular basal membrane. This attenuates the detection of low abundant proteins, however, the basal membrane of small antral follicles is less permeable, making it possible to detect a large number of proteins, and thereby offering further insights in folliculogenesis. STUDY DESIGN, SIZE, DURATION Proteins in FF from unstimulated hSAF (size 6.1 ± 0.4 mm) were characterised by mass spectrometry, supported by high-throughput and targeted proteomics and bioinformatics. The FF protein profiles from hSAF containing oocytes, capable or not of maturing to metaphase II of the second meiotic division during an IVM (n = 13, from 6 women), were also analysed. PARTICIPANTS/MATERIALS, SETTING, METHODS We collected FF from hSAF of ovaries that had been surgically removed from 31 women (∼28.5 years old) undergoing unilateral ovariectomy for fertility preservation. MAIN RESULTS AND THE ROLE OF CHANCE In total, 2461 proteins were identified, of which 1108 identified for the first time in FF. Of the identified proteins, 24 were related to follicular regulatory processes. A total of 35 and 65 proteins were down- and up-regulated, respectively, in fluid from hSAF surrounding oocytes capable of maturing (to MII). We found that changes at the protein level occur already in FF from small antral follicles related to subsequent oocyte maturation. LIMITATIONS, REASONS FOR CAUTION A possible limitation of our study is the uncertainty of the proportion of the sampled follicles that are undergoing atresia. Although the FF samples were carefully aspirated and processed to remove possible contaminants, we cannot ensure the absence of some proteins derived from cellular lysis provoked by technical reasons. WIDER IMPLICATIONS OF THE FINDINGS This study is, to our knowledge, the first proteomics characterisation of FF from hSAF obtained from women in their natural menstrual cycle. We demonstrated that the analysis by mass spectrometry of FF from hSAF allows the identification of a greater number of proteins compared to the results obtained from previous analyses of larger follicles. Significant differences found at the protein level in hSAF fluid could predict the ability of the enclosed oocyte to sustain meiotic resumption. If this can be confirmed in further studies, it demonstrates that the viability of the oocyte is determined early on in follicular development and this may open up new pathways for augmenting or attenuating subsequent oocyte viability in the pre-ovulatory follicle ready to undergo ovulation. STUDY FUNDING/COMPETING INTEREST(S) The authors thank the financial support from ReproUnion, which is funded by the Interreg V EU programme. No conflict of interest was reported by the authors. TRIAL REGISTRATION NUMBER N/A

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STUDY QUESTION How does the human granulosa cell (GC) transcriptome change during ovulation? SUMMARY ANSWER Two transcriptional peaks were observed at 12 h and at 36 h after induction of ovulation, both dominated by genes and pathways known from the inflammatory system. WHAT IS KNOWN ALREADY The crosstalk between GCs and the oocyte, which is essential for ovulation and oocyte maturation, can be assessed through transcriptomic profiling of GCs. Detailed transcriptional changes during ovulation have not previously been assessed in humans. STUDY DESIGN, SIZE, DURATION This prospective cohort study comprised 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital-affiliated fertility clinic in 2016–2018. PARTICIPANTS/MATERIALS, SETTING, METHODS From each woman, one sample of GCs was collected by transvaginal ultrasound-guided follicle aspiration either before or 12 h, 17 h or 32 h after ovulation induction (OI). A second sample was collected at oocyte retrieval, 36 h after OI. Total RNA was isolated from GCs and analyzed by microarray. Gene expression differences between the five time points were assessed by ANOVA with a random factor accounting for the pairing of samples, and seven clusters of protein-coding genes representing distinct expression profiles were identified. These were used as input for subsequent bioinformatic analyses to identify enriched pathways and suggest upstream regulators. Subsets of genes were assessed to explore specific ovulatory functions. MAIN RESULTS AND THE ROLE OF CHANCE We identified 13 345 differentially expressed transcripts across the five time points (false discovery rate, <0.01) of which 58% were protein-coding genes. Two clusters of mainly downregulated genes represented cell cycle pathways and DNA repair. Upregulated genes showed one peak at 12 h that resembled the initiation of an inflammatory response, and one peak at 36 h that resembled the effector functions of inflammation such as vasodilation, angiogenesis, coagulation, chemotaxis and tissue remodelling. Genes involved in cell–matrix interactions as a part of cytoskeletal rearrangement and cell motility were also upregulated at 36 h. Predicted activated upstream regulators of ovulation included FSH, LH, transforming growth factor B1, tumour necrosis factor, nuclear factor kappa-light-chain-enhancer of activated B cells, coagulation factor 2, fibroblast growth factor 2, interleukin 1 and cortisol, among others. The results confirmed early regulation of several previously described factors in a cascade inducing meiotic resumption and suggested new factors involved in cumulus expansion and follicle rupture through co-regulation with previously described factors. LARGE SCALE DATA The microarray data were deposited to the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/gds/, accession number: GSE133868). LIMITATIONS, REASONS FOR CAUTION The study included women undergoing ovarian stimulation and the findings may therefore differ from a natural cycle. However, the results confirm significant regulation of many well-established ovulatory genes from a series of previous studies such as amphiregulin, epiregulin, tumour necrosis factor alfa induced protein 6, tissue inhibitor of metallopeptidases 1 and plasminogen activator inhibitor 1, which support the relevance of the results. WIDER IMPLICATIONS OF THE FINDINGS The study increases our understanding of human ovarian function during ovulation, and the publicly available dataset is a valuable resource for future investigations. Suggested upstream regulators and highly differentially expressed genes may be potential pharmaceutical targets in fertility treatment and gynaecology. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by EU Interreg ÔKS V through ReproUnion (www.reprounion.eu) and by a grant from the Region Zealand Research Foundation. None of the authors have any conflicts of interest to declare.

Introduction Young women with a cancer diagnosis often have very little time to decide whether or not to commence fertility‐preserving strategies before initiating potentially sterilizing cancer treatment. Minimizing the interval from opting for fertility preservation to completion of the procedure will reduce the potential risk of delaying cancer treatment. In the current study, we have evaluated the period of time from referral to ovarian tissue cryopreservation (OTC) to actual freezing of the tissue in a cohort of Danish women. Material and methods The study population comprised 277 consecutive patients with both malignant and nonmalignant diseases referred for OTC from four centers in the Danish network. Statistical analysis was conducted to analyze the impact of age, diagnosis, and referring center on the time from OTC‐referral to OTC. A literature search for “random start” protocols for controlled ovarian stimulation (COS) for fertility preservation in cancer patients was performed. Results The time from OTC‐referral to OTC was significantly influenced by diagnosis, age, and referring center. Women with malignant diseases other than breast cancer, such as sarcomas, pelvic cancers, and hematological cancers, experienced a significantly shorter interval to OTC (5 days) than women with breast cancer (7 days) and nonmalignant diseases including systemic, ovarian, and hereditary conditions (13‐17.5 days). Women over the age of 30 years experienced a significantly longer time to OTC (P < 0.03), and the diagnosis determined the length of the interval (P < 0.001). According to the literature, fertility preservation by oocyte vitrification requires 13‐14 days, as the average time for 1 round of COS was 11 days and oocyte collection can be performed 2 days later. Conclusions It is in the interest of both cancer patients and clinicians to perform fertility preservation as quickly and safely as possible. In a Danish setting, OTC provides a short interval of around 6 days from the patient choosing this option to completion of the procedure. This is considerably less time than what is needed to perform COS and oocyte vitrification, and therefore OTC might be considered the preferred choice of fertility preservation when urgency is needed.

STUDY QUESTION What are the downstream endocrine and paracrine consequences of letrozole (LZ) cotreatment during ovarian stimulation and is follicle growth and recruitment affected? SUMMARY ANSWER Letrozole cotreatment induces marked changes in both the follicular and luteal phase endocrinology causing potentiation of follicle diameter and an improved corpus luteum function without affecting the secondarily recruited follicle cohort. WHAT IS KNOWN ALREADY Letrozole is a third-generation aromatase inhibitor that is well-established as an effective ovulatory agent, while its possible benefits in standard in vitro fertilization protocols are less thoroughly investigated. STUDY DESIGN, SIZE, DURATION This study included a double-blinded, placebo-controlled, randomized study with LZ or placebo intervention during ovarian stimulation for IVF treatment, an observational preceding baseline natural cycle and a succeeding follow-up visit. Participants were enrolled between August 2016 and November 2018. Data from the randomized, stimulated cycle were part of a larger RCT, which was previously published. PARTICIPANTS/MATERIALS, SETTING, METHODS The study was conducted at a public fertility clinic at Herlev Hospital, Denmark, including 31 healthy, normo-responding women eligible for IVF treatment. They underwent a natural baseline cycle and were subsequently randomized to receive either LZ 5 mg (n = 16) or placebo (n = 15) daily during ovarian stimulation from cycle day (CD) 2–3 until induction of ovulation. Throughout both cycles, monitoring was performed every third day with transvaginal ultrasound for assessment of follicle count and diameter, and blood analyses for the determination of twelve endocrine and paracrine parameters. A follow-up assessment was performed at CD2–3 in the succeeding cycle. In the randomized part of the study, we determined differences in blood parameters, follicle recruitment, and follicle diameter. In the observational part of the study, we assessed follicle recruitment in between cycles and its correlation to endocrine parameters. MAIN RESULTS AND THE ROLE OF CHANCE Letrozole cotreatment significantly suppressed oestradiol (E2) concentrations in the follicular phase (area under the curve (AUC) −58% (95% CI [−70%; −43%], P &lt; 0.001)) and luteal phase (AUC −39% [−63%; −1%], P = 0.046). This had a marked effect on the endocrine and paracrine output with increased follicular phase luteinizing hormone (AUC +37% [3%; 82%], P = 0.033), androstenedione (AUC +36% [6%; 74%], P = 0.016), testosterone (AUC +37% [7%; 73%], P = 0.013) and 17-OH-progesterone (AUC +114% [10%; 318%], P = 0.027). Furthermore, follicle-stimulating hormone (FSH) was increased at stimulation day 5 in the LZ group (P &lt; 0.05). In the luteal phase, increased corpus luteum output was reflected by elevated progesterone (AUC +44% [1%; 104%], P = 0.043), inhibin A (AUC +52% [11%; 108%], P = 0.011), androstenedione (AUC +31% [9%; 58%], P = 0.006) and testosterone (AUC +29% [6%; 57%], P = 0.012) in the LZ group. The altered balance between oestrogens and androgens was reflected in a markedly reduced SHBG concentration in the LZ group throughout the luteal phase (AUC −35% [−52%; −11%], P = 0.009). Endocrine and paracrine parameters were similar between groups at the follow-up visit. Letrozole cotreatment significantly increased the mean number of follicles &gt;16 mm at oocyte retrieval (7.2 vs 5.2, difference: 2.0, 95% CI [0.1; 3.8], P = 0.036), while the mean total number of follicles at oocyte retrieval was the same (23.7 vs 23.5, difference: 0.2 [−5.8; 6.1], P = 0.958), and the mean FSH consumption during the stimulated cycle was similar (1500 vs 1520 IU, difference −20 IU [−175; 136], P = 0.794). Between cycles, the mean antral follicle count at CD2–3 was unchanged (natural cycle 19.0, stimulated cycle 20.9, follow-up cycle 19.7, P = 0.692) and there was no effect of LZ cotreatment on the recruitment of the next follicle cohort (test for interaction, P = 0.821). LIMITATIONS, REASONS FOR CAUTION This study included a relatively small, selected group of healthy women with an expected normal ovarian function and reserve, and the effects of LZ may therefore be different in other patient groups. WIDER IMPLICATIONS OF THE FINDINGS We confirm some previous findings concerning increased follicle growth and increased endogenous FSH and androgen production, which support the rationale for further studies on the use of LZ cotreatment, for example, as a form of endogenous androgen priming sensitizing the follicle to FSH. Letrozole appears to improve the luteal phase with better stimulation of corpus luteum and progesterone secretion. STUDY FUNDING/COMPETING INTEREST(s) The authors declare no conflicts of interest relating to the present work. TRIAL REGISTRATION NUMBER NCT02939898.

PurposeUltrasonography is a noninvasive, cheap, and fast way of assessing abdominal pain in an emergency department. Many physicians working in emergency departments do not have pre-existing ultrasound experience. The purpose of this study was to investigate the ability of first-year internship doctors to perform a reliable ultrasound examination on patients with abdominal pain in an emergency setting.Materials and methodsThis study took place in an emergency department in Denmark. Following a 1-day ultrasound introduction course, three doctors without prior ultrasound experience scanned 45 patients during a 2-month period. The applicability of the examinations was evaluated by subsequent control examination: computed tomography, operation, or ultrasound by a trained radiologist or gynecologist or, in cases where the patient was immediately discharged, by ultrasound image evaluation.ResultsIn 14 out of 21 patients with a control examination, there was diagnostic agreement between the project ultrasound examination and the control. Image evaluation of all patients showed useful images of the gallbladder, kidneys, liver, abdominal aorta, and urinary bladder, but no useful images for either the pancreas or colon.ConclusionWith only little formal training, it is possible for first-year internship doctors to correctly visualize some abdominal organs with ultrasonography. However, a longer study time frame, including more patients, and an ultrasound course specifically designed for the purpose of use in an emergency department, is needed to enhance the results.