A dual program for translation regulation in cellular proliferation and differentiation.

A dichotomous choice for metazoan cells is between proliferation and differentiation. Measuring tRNA pools in various cell types, we found two distinct subsets, one that is induced in proliferating cells, and repressed otherwise, and another with the opposite signature. Correspondingly, we found that genes serving cell-autonomous functions and genes involved in multicellularity obey distinct codon usage. Proliferation-induced and differentiation-induced tRNAs often carry anticodons that correspond to the codons enriched among the cell-autonomous and the multicellularity genes, respectively. Because mRNAs of cell-autonomous genes are induced in proliferation and cancer in particular, the concomitant induction of their codon-enriched tRNAs suggests coordination between transcription and translation. Histone modifications indeed change similarly in the vicinity of cell-autonomous genes and their corresponding tRNAs, and in multicellularity genes and their tRNAs, suggesting the existence of transcriptional programs coordinating tRNA supply and demand. Hence, we describe the existence of two distinct translation programs that operate during proliferation and differentiation.

Tracing the origin of adult intestinal stem cells.

Adult intestinal stem cells are located at the bottom of crypts of Lieberkühn, where they express markers such as LGR51,2 and fuel the constant replenishment of the intestinal epithelium1. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells3,4, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we show, using unbiased quantitative lineage-tracing approaches, biophysical modelling and intestinal transplantation, that all cells of the mouse intestinal epithelium-irrespective of their location and pattern of LGR5 expression in the fetal gut tube-contribute actively to the adult intestinal stem cell pool. Using 3D imaging, we find that during fetal development the villus undergoes gross remodelling and fission. This brings epithelial cells from the non-proliferative villus into the proliferative intervillus region, which enables them to contribute to the adult stem-cell niche. Our results demonstrate that large-scale remodelling of the intestinal wall and cell-fate specification are closely linked. Moreover, these findings provide a direct link between the observed plasticity and cellular reprogramming of differentiating cells in adult tissues following damage5-9, revealing that stem-cell identity is an induced rather than a hardwired property.

Genome-wide cell-free DNA fragmentation in patients with cancer.

Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular features of cell-free DNA are poorly understood. Here we developed an approach to evaluate fragmentation patterns of cell-free DNA across the genome, and found that profiles of healthy individuals reflected nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. We used this method to analyse the fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric or bile duct cancer and 245 healthy individuals. A machine learning model that incorporated genome-wide fragmentation features had sensitivities of detection ranging from 57% to more than 99% among the seven cancer types at 98% specificity, with an overall area under the curve value of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cell-free DNA analyses detected 91% of patients with cancer. The results of these analyses highlight important properties of cell-free DNA and provide a proof-of-principle approach for the screening, early detection and monitoring of human cancer.

Incidence of late recurrence and second primary cancers 5-10 years after non-metastatic colorectal cancer.

The fraction of patients who are cancer-free survivors 5 years after curative-intended surgery for colorectal cancer (CRC) is increasing, suggesting that extending surveillance beyond 5 years may be indicated. Here we estimate the incidence of late recurrence, metachronous CRC, and second primary cancers 5-10 years postoperative. All patients resected for UICC stage I-III CRC in Denmark through 2004-2013 were identified. Through individual-level linkage of nationwide health registry data, recurrence status was determined using a validated algorithm. Cancer-free survivors 5 years after surgery, were included. Cumulative incidence functions (CIF) of late recurrence, metachronous CRC, and second primary cancer 5-10 years postoperative were constructed. Subdistribution hazards ratios (sHR) were computed using Fine-Gray regression. Among 8883 patients, 370 developed late recurrence (5-10-year CIF = 4.1%, 95%CI: 3.7%-4.6%), 270 metachronous CRC (5-10-year CIF = 3.0%, 95%CI: 2.7%-3.4%), and 635 a second primary cancer (5-10-year CIF = 7.2%, 95%CI: 6.7%-7.7%). The risk of late recurrence was reduced for patients operated in 2009-2013 compared to 2004-2008 (2.9% vs. 5.6%, sHR = 0.52, 95% CI: 0.42-0.65). The risk of metachronous CRC was likewise reduced from 4.1% to 2.1% (sHR = 0.50, 95%CI: 0.39-0.65). While the risk of second primary cancer did not change between 2009-2013 and 2004-2008 (7.1% vs. 7.1%, sHR = 0.98, 95% CI: 0.84-1.15). Using nation-wide 10-year follow-up data, we document that the incidences of late recurrence and metachronous CRC are low and decreasing from 2004 to 2013. Thus, despite increasing numbers of long-term cancer survivors, the data do not advocate for extending CRC-specific surveillance beyond 5 years.

Beyond basics: Key mutation selection features for successful tumor-informed ctDNA detection.

Tumor-informed mutation-based approaches are frequently used for detection of circulating tumor DNA (ctDNA). Not all mutations make equally effective ctDNA markers. The objective was to explore if prioritizing mutations using mutational features-such as cancer cell fraction (CCF), multiplicity, and error rate-would improve the success rate of tumor-informed ctDNA analysis. Additionally, we aimed to develop a practical and easily implementable analysis pipeline for identifying and prioritizing candidate mutations from whole-exome sequencing (WES) data. We analyzed WES and ctDNA data from three tumor-informed ctDNA studies, one on bladder cancer (Cohort A) and two on colorectal cancer (Cohorts I and N). The studies included 390 patients. For each patient, a unique set of mutations (median mutations/patient: 6, interquartile 13, range: 1-46, total n = 4023) were used as markers of ctDNA. The tool PureCN was used to assess the CCF and multiplicity of each mutation. High-CCF mutations were detected more frequently than low-CCF mutations (Cohort A: odds ratio [OR] 20.6, 95% confidence interval [CI] 5.72-173, p = 1.73e-12; Cohort I: OR 2.24, 95% CI 1.44-3.52, p = 1.66e-04; and Cohort N: OR 1.78, 95% CI 1.14-2.79, p = 7.86e-03). The detection-likelihood was additionally improved by selecting mutations with multiplicity of two or above (Cohort A: OR 1.55, 95% CI 1. 14-2.11, p = 3.85e-03; Cohort I: OR 1.78, 95% CI 1.23-2.56, p = 1.34e-03; and Cohort N: OR 1.94, 95% CI 1.63-2.31, p = 2.83e-14). Furthermore, selecting the mutations for which the ctDNA detection method had the lowest error rates, additionally improved the detection-likelihood, particularly evident when plasma cell-free DNA tumor fractions were below 0.1% (p = 2.1e-07). Selecting mutational markers with high CCF, high multiplicity, and low error rate significantly improve ctDNA detection likelihood. We provide free access to the analysis pipeline enabling others to perform qualified prioritization of mutations for tumor-informed ctDNA analysis.

Results from the first autologous grafting of adult human testis tissue: a case report.

Fertility restoration using autologous testicular tissue transplantation is relevant for infertile men surviving from childhood cancer and, possibly, in men with absent or incomplete spermatogenesis resulting in the lack of spermatozoa in the ejaculate (non-obstructive azoospermia, NOA). Currently, testicular tissue from pre-pubertal boys extracted before treatment with gonadotoxic cancer therapy can be cryopreserved with good survival of spermatogonial stem cells. However, strategies for fertility restoration, after successful cancer treatment, are still experimental and no clinical methods have yet been developed. Similarly, no clinically available treatments can help men with NOA to become biological fathers after failed attempts of testicular surgical sperm retrieval. We present a case of a 31-year-old man with NOA who had three pieces of testis tissue (each ∼2 × 4 × 2 mm3) extracted and cryopreserved in relation to performing microdissection testicular sperm extraction (mTESE). Approximately 2 years after mTESE, the thawed tissue pieces were engrafted in surgically created pockets bilaterally under the scrotal skin. Follow-up was performed after 2, 4, and 6 months with assessment of reproductive hormones and ultrasound of the scrotum. After 6 months, all engrafted tissue was extracted and microscopically analyzed for the presence of spermatozoa. Furthermore, parts of the extracted tissue were analyzed histologically and by immunohistochemical analysis. Active blood flow in the engrafted tissue was demonstrated by doppler ultrasound after 6 months. No spermatozoa were found in the extracted tissue. Histological and immunohistochemical analysis demonstrated graft survival with intact clear tubules and normal cell organization. Sertoli cells and spermatocytes with normal morphology were located near the basement membrane. MAGE-A and VASA positive spermatogonia/spermatocytes were detected together with SOX9 positive Sertoli cells. Spermatocytes and/or Sertoli cells positive for γH2AX was also detected. In summary, following autologous grafting of frozen-thawed testis tissue under the scrotal skin in a man with NOA, we demonstrated graft survival after 6 months. No mature spermatozoa were detected; however, this is likely due to the pre-existing spermatogenic failure.

In vitro fertilization outcome based on the detailed early luteal phase trajectory of hormones: a prospective cohort study.

BACKGROUND: Ovarian stimulation and the use of human chorionic gonadotropin (hCG) for triggering oocyte maturation in women undergoing in vitro fertilisation (IVF) introduces several differences in luteal phase hormone levels compared with natural cycles that may negatively impact on endometrial receptivity and pregnancy rates after fresh embryo transfer. Exogenous luteal phase support is given to overcome these issues. The suitability of a pragmatic approach to luteal phase support is not known due to a lack of data on early phase luteal hormone levels and their association with fertility outcomes during IVF with fresh embryo transfer. This study determined early luteal phase profiles of serum progesterone, 17-hydroxyprogesterone and hCG, and associations between hormone levels/hormone level profile after hCG trigger and the live birth rate in women undergoing IVF with fresh embryo transfer. METHODS: This prospective single center, cohort study was conducted in Vietnam from January 2021 to December 2022. Women aged 18-38 years with normal ovarian reserve and undergoing controlled ovarian stimulation using a gonadotropin-releasing hormone antagonist protocol were included. Serum hormone levels were determined before trigger, at 12, 24 and 36 h after hCG, and daily from 1 to 6 days after oocyte pick-up. Serum hormone level profiles were classified as lower or upper. The primary outcome was live birth rate based on early luteal phase hormone level profile. RESULTS: Ninety-five women were enrolled. Live birth occurred in 19/69 women (27.5%) with a lower progesterone profile and 13/22 (59.1%) with an upper progesterone profile (risk ratio [RR] 2.15; 95% confidence interval [CI] 1.28-3.60), and in 6/31 (19.4%) versus 26/60 (43.3%) with a lower versus upper serum 17-hydroxyprogesterone profile (RR 2.24; 95% CI 1.03-4.86). Nearly 20% of women had peak progesterone concentration on or before day 3 after oocyte pick-up, and this was associated with significantly lower chances of having a life birth. CONCLUSIONS: These data show the importance of proper corpus luteum function with sufficient progesterone/17-hydroxyprogesterone production for achievement of pregnancy and to maximize the chance of live birth during IVF. TRIAL REGISTRATION: NCT04693624 ( www. CLINICALTRIALS: gov ).

Investigation of luteal HCG supplementation in GnRH-agonist-triggered fresh embryo transfer cycles: a randomized controlled trial.

RESEARCH QUESTION: Does splitting the human chorionic gonadotrophin (HCG) support in IVF cycles triggered by a gonadotrophin-releasing hormone agonist result in a better progesterone profile? DESIGN: Randomized controlled three-arm study, performed at the Fertility Clinic, Odense University Hospital, Denmark. Patients with 12-25 follicles ≥12 mm were randomized into three groups: Group 1 - ovulation triggered with 6500 IU HCG; Group 2 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1500 IU HCG on the day of oocyte retrieval (OCR); and Group 3 - ovulation triggered with 0.5 mg GnRH agonist, followed by 1000 IU HCG on the day of OCR and 500 IU HCG on OCR + 5. All groups received 180 mg vaginal progesterone. Progesterone concentrations were analysed in eight blood samples from each patient. RESULTS: Sixty-nine patients completed the study. Baseline and laboratory data were comparable. Progesterone concentration peaked on OCR + 4 in Groups 1 and 2, and peaked on OCR + 6 in Group 3. On OCR + 6, the progesterone concentration in Group 2 was significantly lower compared with Groups 1 and 3 (P = 0.003 and P < 0.001, respectively). On OCR + 8, the progesterone concentration in Group 3 was significantly higher compared with the other groups (both P<0.001). Progesterone concentrations were significantly higher in Group 3 from OCR + 6 until OCR + 14 compared with the other groups (all P ≤ 0.003). Four patients developed ovarian hyperstimulation syndrome in Group 3. CONCLUSION: Sequential HCG support after a GnRH agonist trigger provides a better progesterone concentration in the luteal phase.

Epidural analgesia versus oral morphine for postoperative pain management following video-assisted thoracic surgery: A randomised, controlled, double-blind trial.

BACKGROUND: The use of thoracic epidural analgesia for postoperative pain management in video-assisted thoracic surgery (VATS) is controversial. Still, the evidence on omitting it in favour of systemic opioids is inconclusive, and studies are small and non-blinded. OBJECTIVE: We aimed to compare pain after VATS using epidural analgesia or enteral opioids for postoperative pain management. DESIGN/SETTING/PATIENTS/INTERVENTION: A randomised, double-blind, controlled trial at a Danish tertiary hospital. Adult patients scheduled for VATS were assigned to multimodal non-opioid baseline analgesia supplemented with either thoracic epidural analgesia (TE Group) or oral morphine (OM Group) for postoperative pain management. We recorded pain five times a day, both at rest and during activity, using the Numeric Rating Scale (NRS) and categorised it into "acceptable pain" or "unacceptable pain". Unacceptable pain was defined as NRS (at rest) ≥3 or NRS (with activity) ≥5 when supplementary analgesics were given. MAIN OUTCOME MEASURES: The primary outcomes were the proportions of patients experiencing "unacceptable pain" during the postoperative period and the use of intravenous "rescue" opioids. RESULTS: Of the 161 included patients, 146 received the allocated treatment and their data were analysed. At rest, 34% of patients in the TE Group and 64% of patients in the OM Group experienced unacceptable pain during the study period, a significant between-group difference of 30% ( P  < 0.0005). During activity these percentages were 32% of patients in the TE Group and 59% in the OM group, a difference of 27% ( P  < 0.005). The median intravenous rescue morphine consumption during the study period was 4.5 [interquartile range (IQR), 0-10.0] mg in the TE Group and 7.5 [0-19.0] mg in the OM Group ( P  < 0.005). CONCLUSION: Epidural analgesia provided better pain relief after VATS than oral morphine. The between-group difference in rescue intravenous morphine consumption was statistically significant but clinically irrelevant. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02359175).

Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

Impact of letrozole co-treatment during ovarian stimulation on oocyte yield, embryo development, and live birth rate in women with normal ovarian reserve: secondary outcomes from the RIOT trial.

STUDY QUESTION: Does letrozole (LZ) co-treatment during ovarian stimulation with gonadotropins for in IVF impact follicle recruitment, oocyte number and quality, embryo quality, or live birth rate (LBR)? SUMMARY ANSWER: No impact of LZ was found in follicle recruitment, number of oocytes, quality of embryos, or LBR. WHAT IS KNOWN ALREADY: Multi-follicle stimulation for IVF produces supra-physiological oestradiol levels. LZ is an aromatase inhibitor that lowers serum oestradiol thus reducing negative feedback and increasing the endogenous gonadotropins in both the follicular and the luteal phases, effectively normalizing the endocrine milieu during IVF treatment. STUDY DESIGN, SIZE, DURATION: Secondary outcomes from a randomized, double-blind placebo-controlled trial (RCT) investigating once-daily 5 mg LZ or placebo during stimulation for IVF with FSH. The RCT was conducted at four fertility clinics at University Hospitals in Denmark from August 2016 to November 2018 and pregnancy outcomes of frozen-thawed embryo transfers (FET) registered until May 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: One hundred fifty-nine women with expected normal ovarian reserve (anti-Müllerian hormone 8-32 nmol/l) were randomized to either co-treatment with LZ (n = 80) or placebo (n = 79). In total 1268 oocytes were aspirated developing into 386 embryos, and morphology and morphokinetics were assessed. One hundred twenty-nine embryos were transferred in the fresh cycle and 158 embryos in a subsequent FET cycle. The effect of LZ on cumulative clinical pregnancy rate (CPR), LBR, endometrial thickness in the fresh cycle, and total FSH consumption was reported. MAIN RESULTS AND THE ROLE OF CHANCE: The proportion of usable embryos of retrieved oocytes was similar in the LZ group and the placebo group with 0.31 vs 0.36 (mean difference (MD) -0.05, 95% CI (-0.12; 0.03), P = 0.65). The size and number of aspirated follicles at oocyte retrieval were similar with 11.8 vs 10.3 follicles per patient (MD 1.5, 95% CI (-0.5; 3.1), P = 0.50), as well as the number of retrieved oocytes with 8.0 vs 7.9 oocytes (MD 0.1, 95% CI (-1.4; 1.6), P = 0.39) in the LZ and placebo groups, respectively. The chance of retrieving an oocyte from the 13 to 16 mm follicles at trigger day was 66% higher (95% CI (24%; 108%), P = 0.002) in the placebo group than in the LZ group, whilst the chance of retrieving an oocyte from the ≥17 mm follicles at trigger day was 50% higher (95% CI (2%; 98%), P = 0.04) in the LZ group than in the placebo group. The proportion of fertilized oocytes with two-pronuclei per retrieved oocytes or per metaphase II oocytes (MII) (the 2PN rates) were similar regardless of fertilization with IVF or ICSI with 0.48 vs 0.57 (MD -0.09, 95% CI (-0.24; 0.04), P = 0.51), and 0.62 vs 0.64 (MD -0.02, 95% CI (-0.13; 0.07), P = 0.78) in the LZ and placebo groups, respectively. However, the MII rate in the ICSI group was significantly lower with 0.75 vs 0.88 in the LZ vs the placebo group (MD -0.14, 95% CI (-0.22; -0.06), P = 0.03). Blastocysts on Day 5 per patient were similar with 1.5 vs 2.0, P = 0.52, as well as vitrified blastocysts per patient Day 5 with 0.8 vs 1.2 in (MD -0.4, 95% CI (-1.0; 0.2), P = 0.52) and vitrified blastocysts per patient Day 6 with 0.6 vs 0.6 (MD 0, 95% CI (-0.3; 0.3), P = 1.00) in the LZ vs placebo group, respectively. Morphologic evaluation of all usable embryos showed a similar distribution in 'Good', 'Fair', and 'Poor', in the LZ vs placebo group, with an odds ratio (OR) of 0.8 95% CI (0.5; 1.3), P = 0.68 of developing a better class embryo. Two hundred and ninety-five of the 386 embryos were cultured in an embryoscope. Morphokinetic annotations showed that the odds of having a high KIDscore™ D3 Day 3 were 1.2 times higher (CI (0.8; 1.9), P = 0.68) in the LZ group vs the placebo group. The CPR per transfer was comparable with 31% vs 39% (risk-difference of 8%, 95% CI (-25%; 11%), P = 0.65) in the LZ and placebo group, respectively, as well as CPR per transfer adjusted for day of transfer, oestradiol and progesterone levels at trigger, progesterone levels mid-luteal, and number of oocytes retrieved (adjusted OR) of 0.8 (95% CI (0.4; 1.6), P = 0.72). Comparable LBR were found per transfer 28% vs 37% (MD -9%, 95% CI (-26%; 9%), P = 0.60) and per randomized women 24% vs 30% (MD of -6%, CI (-22%; 8%), P = 0.60) in the LZ group and placebo group, respectively. Furthermore, 4.8 years since the last oocyte aspiration, a total of 287 of 386 embryos have been transferred in the fresh or a subsequently FET cycle, disclosing the cumulative CPR, which is similar with 38% vs 34% (MD 95% CI (8%; 16%), P = 0.70) in the LZ vs placebo group. LIMITATIONS, REASONS FOR CAUTION: Both cleavage stage and blastocyst transfer and vitrification were permitted in the protocol, making it necessary to categorize their quality and pool the results. The study was powered to detect hormonal variation but not embryo or pregnancy outcomes. WIDER IMPLICATIONS OF THE FINDINGS: The similar utilization rate and quality of the embryos support the use of LZ co-treatment for IVF with specific indication as fertility preservation, patients with previous cancer, or poor responders. The effect of LZ on mature oocytes from different follicle sizes and LBRs should be evaluated in a meta-analysis or a larger RCT. STUDY FUNDING/COMPETING INTEREST(S): Funding was received from EU Interreg for ReproUnion, Sjaelland University Hospital, Denmark, Ferring Pharmaceuticals, and Gedeon Ricther. Roche Diagnostics contributed with assays. A.P. has received grants from Ferring, Merck Serono, and Gedeon Richter, consulting fees from Preglem, Novo Nordisk, Ferring, Gedeon Richter, Cryos, & Merck A/S, speakers fees from Gedeon Richter, Ferring, Merck A/S, Theramex, & Organon, and travel support from Gedeon Richter. The remaining authors declare that they have no competing interests in the research or publication. TRIAL REGISTRATION NUMBERS: NCT02939898 and NCT02946684.

Human platelet lysate improves the growth and survival of cultured human pre-antral follicles.

RESEARCH QUESTION: How do platelet-rich plasma products like human platelet lysate (HPL) and umbilical cord plasma (UCP) affect the growth and survival of isolated human pre-antral follicles in vitro? DESIGN: Human pre-antral follicles (n = 724; mean diameter: 75 µm; range: 46-237 µm) were isolated from ovarian medulla donated by 14 patients undergoing unilateral oophorectomy for ovarian tissue cryopreservation. Follicles were encapsulated in 0.5% alginate and cultured for 8 days in media supplemented with 5% fetal bovine serum (FBS) (n = 171), 2.5% human serum albumin (HSA) (n = 159), 5% HPL (n = 223) or 5% UCP (n = 171). RESULTS: The survival probability was significantly higher in the group supplemented with HPL (80%) compared with the other three groups: FBS (54%, P < 0.001); HSA (63%, P = 0.004) and UCP (29%, P < 0.001). Surviving follicles in the UCP group had less defined follicular membranes and decompacted granulosa cell layers. The median growth of surviving follicles was significantly (P < 0.001) larger in the HPL group (73 µm) compared with any of the other three groups: HSA (43 µm); FBS (40 µm) UCP (54 µm). A descriptive analysis of follicular secretion of anti-Müllerian hormone and oestradiol did not reveal any difference between the groups. The detectability of follicular genes was high for AR (100%), AMHR2 (100%) and FSHR (76%), whereas few follicles expressed LHR (20%). CONCLUSION: Human platelet lysate significantly improved survival and growth of cultured human pre-antral follicles compared with FBS, HSA and UCP. The use of HPL is a valuable improvement to culture human pre-antral follicles but further studies will have to prove whether the superiority of HPL translates into better quality oocytes.

Acute normal tissue responses in a murine model following fractionated irradiation of the head and neck with protons or X-rays.

BACKGROUND: The purpose of this study was to investigate acute normal tissue responses in the head and neck region following proton- or X-irradiation of a murine model. MATERIALS AND METHODS: Female C57BL/6J mice were irradiated with protons (25 or 60 MeV) or X-rays (100 kV). The radiation field covered the oral cavity and the major salivary glands. For protons, two different treatment plans were used, either with the Bragg Peak in the middle of the mouse (BP) or outside the mouse (transmission mode; TM). Delivered physical doses were 41, 45, and 65 Gy given in 6, 7, and 10 fractions for BP, TM, and X-rays, respectively. Alanine dosimetry was used to assess delivered doses. Oral mucositis and dermatitis were scored using CTC v.2.0-based tables. Saliva was collected at baseline, right after end of irradiation, and at day 35. RESULTS: The measured dose distribution for protons (TM) and X-rays was very similar. Oral mucositis appeared earlier, had a higher score and was found in a higher percentage of mice after proton irradiation compared to X-irradiation. Dermatitis, on the other hand, had a similar appearance after protons and X-rays. Compared to controls, saliva production was lower right after termination of proton- and X-irradiation. The BP group demonstrated saliva recovery compared to the TM and X-ray group at day 35. CONCLUSION: With lower delivered doses, proton irradiation resulted in similar skin reactions and increased oral mucositis compared to X-irradiation. This indicates that the relative biological effectiveness of protons for acute tissue responses in the mouse head and neck is greater than the clinical standard of 1.1. Thus, there is a need for further investigations of the biological effect of protons in normal tissues.

Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100-130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E2), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E2 and FSH to the culture medium.

Circulating tumor DNA for prognosis assessment and postoperative management after curative-intent resection of colorectal liver metastases.

The recurrence rate of colorectal liver metastases (CRLM) patients treated with curative intent is above 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging as well as carcinoembryonic antigen (CEA) measurements. Nonetheless, relapse detection often happens too late to resume curative treatment. This longitudinal cohort study enrolled 115 patients with plasma samples (N = 439) prospectively collected before surgery, postoperatively at day 30 and every third month for up to 3 years. Droplet digital PCR (ddPCR) was used to monitor serial plasma samples for somatic mutations. Assessment of ctDNA status either immediately after surgery, or serially during surveillance, stratified the patients into groups of high and low recurrence risk (hazard ratio [HR], 7.6; 95% CI, 3.0-19.7; P < .0001; and HR, 4.3; 95% CI, 2.3-8.1; P < .0001, respectively). The positive predictive value (PPV) of ctDNA was 100% in all postoperative analyses. In multivariable analyses, postoperative ctDNA status was the only consistently significant risk marker associated with relapse (P < .0001). Indeterminate CT findings were observed for 30.8% (21/68) of patients. All patients (9/21) that were ctDNA positive at the time of the indeterminate CT scan later relapsed, contrasting 42.6% (5/12) of those ctDNA negative (P = .0046). Recurrence diagnoses in patients with indeterminate CT findings were delayed (median 2.8 months, P < .0001). ctDNA status is strongly associated with detection of minimal residual disease and early detection of relapse. Furthermore, ctDNA status can potentially contribute to clinical decision-making in case of indeterminate CT findings, reducing time-to-intervention.

Error Characterization and Statistical Modeling Improves Circulating Tumor DNA Detection by Droplet Digital PCR.

BACKGROUND: Droplet digital PCR (ddPCR) is a widely used and sensitive application for circulating tumor DNA (ctDNA) detection. As ctDNA is often found in low abundance, methods to separate low-signal readouts from noise are necessary. We aimed to characterize the ddPCR-generated noise and, informed by this, create a sensitive and specific ctDNA caller. METHODS: We built 2 novel complimentary ctDNA calling methods: dynamic limit of blank and concentration and assay-specific tumor load estimator (CASTLE). Both methods are informed by empirically established assay-specific noise profiles. Here, we characterized noise for 70 mutation-detecting ddPCR assays by applying each assay to 95 nonmutated samples. Using these profiles, the performance of the 2 new methods was assessed in a total of 9447 negative/positive reference samples and in 1311 real-life plasma samples from colorectal cancer patients. Lastly, performances were compared to 7 literature-established calling methods. RESULTS: For many assays, noise increased proportionally with the DNA input amount. Assays targeting transition base changes were more error-prone than transversion-targeting assays. Both our calling methods successfully accounted for the additional noise in transition assays and showed consistently high performance regardless of DNA input amount. Calling methods that were not noise-informed performed less well than noise-informed methods. CASTLE was the only calling method providing a statistical estimate of the noise-corrected mutation level and call certainty. CONCLUSIONS: Accurate error modeling is necessary for sensitive and specific ctDNA detection by ddPCR. Accounting for DNA input amounts ensures specific detection regardless of the sample-specific DNA concentration. Our results demonstrate CASTLE as a powerful tool for ctDNA calling using ddPCR.

ctDNA for Risk of Recurrence Assessment in Patients Treated with Neoadjuvant Treatment: A Systematic Review and Meta-analysis.

BACKGROUND: We wanted to investigate the association between circulating tumor DNA (ctDNA) detection at baseline, during and after neoadjuvant treatment, after surgery, and recurrence, in patients with nonmetastatic cancer. PATIENTS AND METHODS: In this systematic review and meta-analysis, we included studies that investigated patients undergoing neoadjuvant treatment for nonmetastatic cancer and provided recurrence indices stratified for ctDNA status at the following timepoints: baseline, during treatment, posttreatment, and postsurgery. Study quality was reported with the Newcastle-Ottawa scale, REMARK checklist, and GRADE approach. PubMed, Embase, Cochrane Library, and Web of Science were our data sources (inception to 3 June 2021). The main outcome was risk of recurrence. RESULTS: We identified ten studies including 727 patients with rectal, breast, gastric, and bladder cancer. All studies reported posttreatment ctDNA analysis, while seven, four, and six reported baseline, during treatment, and postsurgery ctDNA analysis, respectively. ctDNA detection was associated to recurrence across all timepoints [baseline: risk ratio (RR) 2.86, 95% confidence interval (CI) 1.33-6.14, during treatment: RR 3.81, 95% CI 2.09-6.92, posttreatment: RR 4.29, 95% CI 2.79-6.60, postsurgery: RR 8.03, 95% CI 3.16-20.43]. Heterogeneity was low to moderate. CONCLUSIONS: This meta-analysis of observational studies found that ctDNA detection in patients undergoing neoadjuvant treatment for nonmetastatic cancer was associated with recurrence. A stronger association was evident in posttreatment and postsurgery timepoints. However, some studies reported low negative predictive value (NPV) of pathological complete response, showing that ctDNA-detection-guided escalation and de-escalation studies following neoadjuvant treatment regimens are needed before its role as a treatment guidance can be affirmed.

Effects of letrozole cotreatment on endocrinology and follicle development in women undergoing ovarian stimulation in an antagonist protocol.

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 < 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 < 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 >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.

A randomized, controlled, first-in-patient trial of choriogonadotropin beta added to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol.

STUDY QUESTION: Does addition of choriogonadotropin beta (recombinant CG beta) to follitropin delta increase the number of good-quality blastocysts following ovarian stimulation in a long GnRH agonist protocol? SUMMARY ANSWER: At the doses investigated, the addition of CG beta reduced the number of intermediate follicles and related down-stream parameters including the number of oocytes and blastocysts. WHAT IS KNOWN ALREADY: CG beta is a novel recombinant hCG (rhCG) molecule expressed by a human cell line (PER.C6®) and has a different glycosylation profile compared to urinary hCG or rhCG derived from a Chinese Hamster Ovary (CHO) cell line. In the first-in-human trial, the CG beta pharmacokinetics were similar between men and women. In women, the AUC and the peak serum concentration (Cmax) increased approximately dose proportionally following single and multiple daily doses. In men, a single dose of CG beta provided higher exposure with a longer half-life and proportionately higher testosterone production than CHO cell-derived rhCG. STUDY DESIGN, SIZE, DURATION: This placebo-controlled, double-blind, randomized trial (RAINBOW) was conducted in five European countries to explore the efficacy and safety of CG beta as add-on treatment to follitropin delta in women undergoing ovarian stimulation in a long GnRH agonist protocol. Randomization was stratified by centre and age (30-37 and 38-42 years). The primary endpoint was the number of good-quality blastocysts (Grade 3 BB or higher). Subjects were randomized to receive either placebo or 1, 2, 4, 8 or 12 µg CG beta added to the daily individualized follitropin delta dose during ovarian stimulation. PARTICIPANTS/MATERIALS, SETTING, METHODS: In total, 620 women (30-42 years) with anti-Müllerian hormone (AMH) levels between 5 and 35 pmol/l were randomized in equal proportions to the six treatment groups and 619 subjects started treatment. All 619 subjects were treated with an individualized dose of follitropin delta determined based on AMH (Elecsys AMH Plus Immunoassay) and body weight. Triggering with rhCG was performed when 3 follicles were ≥17 mm but no more than 25 follicles ≥12 mm were reached. MAIN RESULTS AND THE ROLE OF CHANCE: The demographic characteristics were comparable between the six treatment groups and the overall mean age, body weight and AMH were 35.6 ± 3.3 years, 65.3 ± 10.7 kg and 15.3 ± 7.0 pmol/l, respectively. The incidence of cycle cancellation (range 0-2.9%), total follitropin delta dose (mean 112 µg) and duration of stimulation (mean 10 days) were similar across the groups. At stimulation Day 6, the number and size of follicles was similar between the treatment groups, whereas at the end-of-stimulation dose-related decrease of the intermediate follicles between 12 and 17 mm was observed in comparison to the placebo group. In contrast, the number of follicles ≥17 mm was similar between the CG beta dose groups and the placebo group. A reduced number of intermediate follicles (12 to 17 mm) and fewer oocytes (mean range 9.7 to 11.2) were observed for all doses of CG beta compared to the follitropin delta only group (mean 12.5). The mean number of good-quality blastocysts was 3.3 in the follitropin delta group and ranged between 2.1 and 3.0 across the CG beta groups. The incidence of transfer cancellation was higher in the 4, 8 and 12 µg group, mostly as no blastocyst was available for transfer. In the group receiving only follitropin delta, the ongoing pregnancy rate (10-11 weeks after transfer) was 43% per started cycle versus 28-39% in CG beta groups and 49% per transfer versus 38-50% in the CG beta groups. There was no apparent effect of CG beta on the incidence of adverse events, which was 48.1% in the placebo group and 39.6-52.3% in the CG beta dose groups. In line with the number of collected oocytes, the overall ovarian hyperstimulation syndrome incidence remained lower following follitropin delta with CG beta (2.0-10.3%) compared with follitropin delta only treatment (11.5%). Regardless of the dose, CG beta was safe and well-tolerated with low risk of immunogenicity. LIMITATIONS, REASONS FOR CAUTION: The effect of the unique glycosylation of CG beta and its associated potency implications in women were not known prior to this trial. Further studies will be needed to evaluate optimal doses of CG beta for this and/or different indications. WIDER IMPLICATIONS OF THE FINDINGS: The high ongoing pregnancy rate in the follitropin delta group supports the use of individualized follitropin delta dosing in a long GnRH agonist protocol. The addition of CG beta reduced the presence of intermediate follicles with the investigated doses and negatively affected all down-stream parameters. Further clinical research will be needed to assess the optimal dose of CG beta in the optimal ratio to follitropin delta to develop this novel combination product containing both FSH and LH activity for ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study was funded by Ferring Pharmaceuticals, Copenhagen, Denmark. B.M. and P.L. are employees of Ferring Pharmaceuticals. M.F.S., H.V., C.Y.A., M.F., C.B., A.P. and Y.K. have received institutional clinical trial fees from Ferring Pharmaceuticals. C.B. has received payments for lectures from Organon, Ferring Pharmaceuticals, Merck A/S and Abbott. M.F.S. has received payment for lectures from Ferring Pharmaceuticals. Y.K. has received payment for lectures from Merck and travel support from Gedeon Richter. H.V. has received consulting fees from Oxo and Obseva and travel support from Gedeon Richter, Ferring Pharmaceuticals and Merck. C.Y.A. has received payment for lectures from IBSA, Switzerland. M.F and C.Y.A. were reimbursed as members of the Data Monitoring Board in this trial. M.F. has an issued patent about unitary combination of FSH and hCG (EP1633389). TRIAL REGISTRATION NUMBER: 2017-003810-13 (EudraCT Number). TRIAL REGISTRATION DATE: 21 May 2018. DATE OF FIRST PATIENT'S ENROLMENT: 13 June 2018.

Impact of letrozole co-treatment during ovarian stimulation with gonadotrophins for IVF: a multicentre, randomized, double-blinded placebo-controlled trial.

STUDY QUESTION: Does letrozole co-treatment during ovarian stimulation with gonadotrophins for IVF reduce the proportion of women with premature progesterone levels above 1.5 ng/ml at the time of triggering final oocyte maturation? SUMMARY ANSWER: The proportion of women with premature progesterone above 1.5 ng/ml was not significantly affected by letrozole co-treatment. WHAT IS KNOWN ALREADY: IVF creates multiple follicles with supraphysiological levels of sex steroids interrupting the endocrine milieu and affects the window of implantation. Letrozole is an effective aromatase inhibitor, normalizing serum oestradiol, thereby ameliorating some of the detrimental effects of IVF treatment. STUDY DESIGN, SIZE, DURATION: A randomized, double-blinded placebo-controlled trial investigated letrozole intervention during stimulation for IVF with FSH. The trial was conducted at four fertility clinics at University Hospitals in Denmark from August 2016 to November 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A cohort of 129 women with expected normal ovarian reserve (anti-Müllerian hormone 8-32 nmol/l) completed an IVF cycle with fresh embryo transfer and received co-treatment with either 5 mg/day letrozole (n = 67) or placebo (n = 62), along with the FSH. Progesterone, oestradiol, FSH, LH and androgens were analysed in repeated serum samples collected from the start of the stimulation to the mid-luteal phase. In addition, the effect of letrozole on reproductive outcomes, total FSH consumption and adverse events were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: The proportion of women with premature progesterone >1.5 ng/ml was similar (6% vs 0% (OR 0.0, 95% CI [0.0; 1.6], P = 0.12) in the letrozole versus placebo groups, respectively), whereas the proportion of women with mid-luteal progesterone >30 ng/ml was significantly increased in the letrozole group: (59% vs 31% (OR 3.3, 95% CI [1.4; 7.1], P = 0.005)). Letrozole versus placebo decreased oestradiol levels on the ovulation trigger day by 68% (95% CI [60%; 75%], P < 0.0001). Other hormonal profiles, measured as AUC, showed the following results. The increase in LH in the letrozole group versus placebo group was 38% (95% CI [21%; 58%], P < 0.0001) and 34% (95% CI [11%; 61%], P = 0.006) in the follicular and luteal phases, respectively. In the letrozole group versus placebo group, testosterone increased by 79% (95% CI [55%; 105%], P < 0.0001) and 49% (95% CI [30%; 72%], P < 0.0001) in the follicular and luteal phases, respectively. In the letrozole group versus placebo group, the increase in androstenedione was by 85% (95% CI [59%; 114%], P < 0.0001) and 69% (95% CI [48%; 94%], P < 0.0001) in the follicular and luteal phases, respectively. The ongoing pregnancy rate was similar between the letrozole and placebo groups (31% vs 39% (risk-difference of 8%, 95% CI [-25%; 11%], P = 0.55)). No serious adverse reactions were recorded in either group. The total duration of exogenous FSH stimulation was 1 day shorter in the intervention group, significantly reducing total FSH consumption (mean difference -100 IU, 95% CI [-192; -21], P = 0.03). LIMITATIONS, REASONS FOR CAUTION: Late follicular progesterone samples were collected on the day before and day of ovulation triggering for patient logistic considerations, and the recently emerged knowledge about diurnal variation of progesterone was not taken into account. The study was powered to detect hormonal variations but not differences in pregnancy outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Although the use of letrozole has no effect on the primary outcome, the number of women with a premature increase in progesterone on the day of ovulation triggering, the increased progesterone in the mid-luteal phase due to letrozole may contribute to optimizing the luteal phase endocrinology. The effect of letrozole on increasing androgens and reducing FSH consumption may be used in poor responders. However, the effect of letrozole on implantation and ongoing pregnancy rates should be evaluated in a meta-analysis or larger randomized controlled trial (RCT). STUDY FUNDING/COMPETING INTEREST(S): Funding was received from EU Interreg for ReproUnion and Ferring Pharmaceuticals, and Roche Diagnostics contributed with assays. N.S.M. and A.P. have received grants from Ferring, Merck Serono, Anecova and Gedeon Richter, and/or personal fees from IBSA, Vivoplex, ArtPred and SPD, outside the submitted work. The remaining authors have no competing interests. TRIAL REGISTRATION NUMBERS: NCT02939898 and NCT02946684. TRIAL REGISTRATION DATE: 15 August 2016. DATE OF FIRST PATIENT'S ENROLMENT: 22 August 2016.