SLO2.1/NALCN Functional Complex Activity in Mouse Myometrial Smooth Muscle Cells During Pregnancy.

At the end of pregnancy, the uterus transitions from a quiescent to a highly contractile state. This is partly due to depolarization of the resting membrane potential in uterine (myometrial) smooth muscle cells (MSMCs). Experiments with human MSMCs showed that the membrane potential is regulated by a functional complex between the sodium (Na + )-activated potassium (K + ) channel SLO2.1 and the Na + Leak Channel Non-Selective (NALCN). In human MSMCs, Na + entering through NALCN activates SLO2.1, leading to K + efflux, membrane hyperpolarization (cells become more negative inside), and reduced contractility. Decreased SLO2.1/NALCN activity results in reduced K + efflux, leading to membrane depolarization, Ca 2+ influx via voltage-dependent calcium channels, and increased MSMC contractility. However, all of these experiments were performed with MSMCs isolated from women at term, so the role of the SLO2.1/NALCN complex early in pregnancy was speculative. To address this question here, we examined the role of the SLO2.1/NALCN complex in regulating mouse MSMC membrane potential across pregnancy. We report that Slo2 . 1 and Nalcn expression change along pregnancy, being more highly expressed in MSMCs from non-pregnant and early pregnant mice than in those from late-pregnant mice. Functional studies revealed that SLO2.1 channels mediate a significant portion of the K + current in mouse MSMCs, particularly in cells from non-pregnant and early pregnant mice. Activation of SLO2.1 by Na + influx through NALCN led to membrane hyperpolarization in MSMCs from early pregnancy but not in MSMCs from later pregnancy. Moreover, we found that the NALCN/SLO2.1 complex regulates intracellular Ca 2+ responses more in MSMCs from non-pregnant and early pregnancy mice than in MSMCs from late pregnancy. Together, these findings reveal that the SLO2.1/NALCN functional complex is conserved between mouse and humans and functions throughout pregnancy. This work could open avenues for targeted pharmacological interventions in pregnancy-related complications.

[Prevalence of hypophosphatemia in children with diabetic ketoacidosis and treatment with subcutaneous regular insulin. Observational study]. / Prevalencia de Hipofosfatemia en niños con cetoacidosis diabética y tratamiento con insulina regular subcutánea. Estudio observacional.

Diabetic ketoacidosis (DKA) is one of the most serious complications of type 1 diabetes mellitus. Its treatment requires fluid and electrolyte replacement and insulin. Hypophosphatemia as a complication of treatment has been scarcely evaluated. OBJECTIVES: To estimate the incidence of hypophosphatemia in children with DKA, treated with subcutaneous regular insulin (IRS), and to explore factors associated with this complication. PATIENTS AND METHOD: Prospective, observational study. Patients diagnosed with DKA hospitalized in the general care ward were included. Data on phosphatemia, glycemia, acid-base status, and IRS amount (U/kg) received were recorded at baseline and after 24 h of treatment. Hypophosphatemia was defined as values below 2.5 mg/dl. The correlation between initial phosphate and at 24 h of treatment was evaluated; the incidence of hypophosphatemia at 24 h was expressed as a percentage of the total number of patients. RESULTS: 30 patients were included, 15 were female, mean age 11.4 ± 3.2 years. At 24 h of treatment with IRS, 36.7% (95%CI 22-55%) presented hypophosphatemia, mean value 1.9 ± 1.5 mg/dl. Initial bicarbonate < 10 mmol/L acted as a predictor of hypophosphatemia (OR 7.5; 95%CI 1.4-39.8%; p = 0.01). No patient required intravenous phosphate correction, and no associated clinical complications were observed. CONCLUSION: In the group studied, the incidence of hypophosphatemia reached 36.7% at 24 hours of treatment. Initial bicarbonate lower than 10 mmol/L was significantly associated with hypophosphatemia. No complications associated with hypophosphatemia were observed.

Identification of consistent QTL and candidate genes associated with seed traits in common bean by combining GWAS and RNA-Seq.

KEY MESSAGE: Association analysis, colocation study with previously reported QTL, and differential expression analyses allowed the identification of the consistent QTLs and main candidate genes controlling seed traits. Common beans show wide seed variations in shape, size, water uptake, and coat proportion. This study aimed to identify consistent genomic regions and candidate genes involved in the genetic control of seed traits by combining association and differential expression analyses. In total, 298 lines from the Spanish Diversity Panel were genotyped with 4,658 SNP and phenotyped for seven seed traits in three seasons. Thirty-eight significant SNP-trait associations were detected, which were grouped into 23 QTL genomic regions with 1,605 predicted genes. The positions of the five QTL regions associated with seed weight were consistent with previously reported QTL. HCPC analysis using the SNP that tagged these five QTL regions revealed three main clusters with significantly different seed weights. This analysis also separated groups that corresponded well with the two gene pools described: Andean and Mesoamerican. Expression analysis was performed on the seeds of the cultivar 'Xana' in three seed development stages, and 1,992 differentially expressed genes (DEGs) were detected, mainly when comparing the early and late seed development stages (1,934 DEGs). Overall, 91 DEGs related to cell growth, signaling pathways, and transcriptomic factors underlying these 23 QTL were identified. Twenty-two DEGs were located in the five QTL regions associated with seed weight, suggesting that they are the main set of candidate genes controlling this character. The results confirmed that seed weight is the sum of the effects of a complex network of loci, and contributed to the understanding of seed phenotype control.

Membrane potential hyperpolarization: a critical factor in acrosomal exocytosis and fertilization in sperm within the female reproductive tract.

Hyperpolarization of the membrane potential (Em), a phenomenon regulated by SLO3 channels, stands as a central feature in sperm capacitation-a crucial process conferring upon sperm the ability to fertilize the oocyte. In vitro studies demonstrated that Em hyperpolarization plays a pivotal role in facilitating the mechanisms necessary for the development of hyperactivated motility (HA) and acrosomal exocytosis (AE) occurrence. Nevertheless, the physiological significance of sperm Em within the female reproductive tract remains unexplored. As an approach to this question, we studied sperm migration and AE incidence within the oviduct in the absence of Em hyperpolarization using a novel mouse model established by crossbreeding of SLO3 knock-out (KO) mice with EGFP/DsRed2 mice. Sperm from this model displays impaired HA and AE in vitro. Interestingly, examination of the female reproductive tract shows that SLO3 KO sperm can reach the ampulla, mirroring the quantity of sperm observed in wild-type (WT) counterparts, supporting that the HA needed to reach the fertilization site is not affected. However, a noteworthy distinction emerges-unlike WT sperm, the majority of SLO3 KO sperm arrive at the ampulla with their acrosomes still intact. Of the few SLO3 KO sperm that do manage to reach the oocytes within this location, fertilization does not occur, as indicated by the absence of sperm pronuclei in the MII-oocytes recovered post-mating. In vitro, SLO3 KO sperm fail to penetrate the ZP and fuse with the oocytes. Collectively, these results underscore the vital role of Em hyperpolarization in AE and fertilization within their physiological context, while also revealing that Em is not a prerequisite for the development of the HA motility, essential for sperm migration through the female tract to the ampulla.

Two-year efficacy and safety of relugolix combination therapy in women with endometriosis-associated pain: SPIRIT open-label extension study.

STUDY QUESTION: What is the efficacy and safety of long-term treatment (up to 2 years) with relugolix combination therapy (CT) in women with moderate to severe endometriosis-associated pain? SUMMARY ANSWER: For up to 2 years, treatment with relugolix CT improved menstrual and non-menstrual pain, dyspareunia, and function in women with endometriosis; after an initial decline of <1%, the mean bone mineral density (BMD) remained stable with continued treatment. WHAT IS KNOWN ALREADY: Endometriosis is a chronic condition characterized by symptoms of dysmenorrhea, non-menstrual pelvic pain (NMPP), and dyspareunia, which have a substantial impact on the lives of affected women, their partners, and families. SPIRIT 1 and 2 were phase 3, randomized, double-blind, placebo-controlled studies of once-daily relugolix CT (relugolix 40 mg, oestradiol 1 mg, norethisterone acetate 0.5 mg) in premenopausal women (age 18-50 years) with endometriosis and moderate-to-severe dysmenorrhea and NMPP. These trials demonstrated a significant improvement of dysmenorrhea, NMPP, and dyspareunia in women treated with relugolix CT, with minimal decline (<1%) in BMD versus placebo at 24 weeks. STUDY DESIGN, SIZE, DURATION: Patients participating in this open-label, single-arm, long-term extension (LTE) study of the 24-week SPIRIT pivotal studies (SPIRIT 1 and 2) received up to an additional 80 weeks of once-daily oral relugolix CT treatment between May 2018 and January 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: Premenopausal women with confirmed endometriosis and moderate to severe dysmenorrhea and NMPP who completed the 24-week pivotal studies (SPIRIT 1 and 2 trials; Giudice et al., 2022) and who met all entry criteria were eligible to enrol. Two-year results were analysed by treatment group based on original randomization in pivotal studies: relugolix CT, delayed relugolix CT (relugolix 40 mg monotherapy for 12 weeks, followed by relugolix CT), or placebo→relugolix CT (placebo for 24 weeks followed by relugolix CT). The primary endpoints of the LTE study were the proportion of dysmenorrhea and NMPP responders at Week 52 and Week 104/end-of-treatment (EOT). A responder was a participant who achieved a predefined, clinically meaningful reduction from baseline in Numerical Rating Scale (NRS) scores (0 = no pain, 10 = worst pain imaginable) for the specific pain type with no increase in analgesic use. The predefined clinically meaningful threshold for dysmenorrhea was 2.8 points and for NMPP was 2.1 points. Secondary efficacy endpoints included change from baseline in Endometriosis Health Profile-30 (EHP-30) pain domain scores, a measure of the effects of endometriosis-associated pain on daily activities (function), NRS scores for dysmenorrhea, NMPP, dyspareunia, and overall pelvic pain, and analgesic/opioid use. Safety endpoints included adverse events and changes in BMD. MAIN RESULTS AND THE ROLE OF CHANCE: Of 1261 randomized patients, 1044 completed the pivotal studies, 802 enrolled in the LTE, 681 completed 52 weeks of treatment, and 501 completed 104 weeks of treatment. Demographics and baseline characteristics of the extension population were consistent with those of the original randomized population. Among patients randomized to relugolix CT at pivotal study baseline who continued in the LTE (N = 277), sustained improvements in endometriosis-associated pain were demonstrated through 104 weeks. The proportion of responders at Week 104/EOT for dysmenorrhea and NMPP was 84.8% and 75.8%, respectively. Decreases in dyspareunia and improvement in function assessed by EHP-30 pain domain were also sustained over 2 years. At Week 104/EOT, 91% of patients were opioid-free and 75% of patients were analgesic-free. Relugolix CT over 104 weeks was well tolerated with a safety profile consistent with that observed over the first 24 weeks. After initial least squares mean BMD loss <1% at Week 24, BMD plateaued at Week 36 and was sustained for the duration of 104 weeks of treatment. Efficacy and safety results were generally consistent in women in the placebo→relugolix CT and delayed relugolix CT groups. LIMITATIONS, REASONS FOR CAUTION: The study was conducted as an open-label study without a control group over the 80 weeks of the extension period. Of the 802 patients who were enrolled in this LTE study, 681 patients (84.9%) and 501 patients (62.5%) of patients completed 52 and 104 weeks of treatment, respectively. In addition, there currently are no comparative data to other hormonal medications. Finally, a third (37.4%) of the study population terminated participation early. WIDER IMPLICATIONS OF THE FINDINGS: In conclusion, relugolix CT offers an additional option to help address an important unmet clinical need for effective, safe, and well-tolerated medical treatments for endometriosis that can be used longer-term, reducing the need for opioids and improving quality of life. The findings from this study may help support the care of women with endometriosis seeking longer-term effective medical management of their symptoms. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by Myovant Sciences GmbH (now Sumitomo Pharma Switzerland GmbH). C.M.B. reports fees from Myovant, grants from Bayer Healthcare, fees from ObsEva, and Chair of ESHRE Endometriosis Guideline Group (all funds went to the University of Oxford); N.P.J. reports personal fees from Myovant Sciences, during the conduct of the study, personal fees from Guerbet, personal fees from Organon, personal fees from Roche Diagnostics; S.A.-S. reports personal fees from Myovant Sciences, personal fees from Bayer, personal fees from Abbvie, personal fees from UpToDate; J.S.P., and R.B.W. are employees and shareholders of Myovant Sciences; J.C.A.F. and S.J.I. are shareholders of Myovant Sciences (but at time of publicaion are no longer employess of Myovant Sciences); M.S.A. and K.W. have no conflicts to declare; V.M. is a consultant to Myovant; L.C.G. reports personal fees from Myovant Sciences, Inc and Bayer. The authors did not receive compensation for manuscript writing, review, and revision. TRIAL REGISTRATION NUMBER: NCT03654274.

Endocrine profile of the kisspeptin receptor agonist MVT-602 in healthy premenopausal women with and without ovarian stimulation: results from 2 randomized, placebo-controlled clinical tricals.

BACKGROUND: Kisspeptin is an essential regulator of hypothalamic gonadotropin-releasing hormone release and is required for physiological ovulation. Native kisspeptin-54 can induce oocyte maturation during in vitro fertilization treatment, including in women who are at high risk of ovarian hyperstimulation syndrome. MVT-602 is a potent kisspeptin receptor agonist with prospective utility to treat anovulatory disorders by triggering oocyte maturation and ovulation during medically assisted reproduction (MAR). Currently, the endocrine profile of MVT-602 during ovarian stimulation is unreported. OBJECTIVE: To determine the endocrine profile of MVT-602 in the follicular phase of healthy premenopausal women (phase-1 trial), and after minimal ovarian stimulation to more closely reflect the endocrine milieu encountered during MAR (phase-2a trial). DESIGN: Two randomized, placebo-controlled, parallel-group, dose-finding trials. SETTING: Clinical trials unit. PATIENTS: Healthy women aged 18-35 years, either without (phase-1; n = 24), or with ovarian stimulation (phase-2a; n = 75). INTERVENTIONS: Phase-1: single subcutaneous dose of MVT-602 (0.3, 1.0, or 3.0 µg) or placebo, (n = 6 per dose). Phase-2a: single subcutaneous dose of MVT-602 (0.1, 0.3, 1.0, or 3.0 µg; n = 16-17 per dose), triptorelin 0.2 mg (n = 5; active comparator), or placebo (n = 5). MAIN OUTCOME MEASURES: Phase-1: safety/tolerability; pharmacokinetics; and pharmacodynamics (luteinizing hormone [LH] and other reproductive hormones). Phase-2a: safety/tolerability; pharmacokinetics; pharmacodynamics (LH and other reproductive hormones); and time to ovulation assessed by transvaginal ultrasound. RESULTS: In both the trials, MVT-602 was safe and well tolerated across the entire dose range. It was rapidly absorbed and eliminated, with a mean elimination half-life of 1.3-2.2 hours. In the phase-2a trial, LH concentrations increased dose dependently; mean maximum change from baseline of 82.4 IU/L at 24.8 hours was observed after administration of 3 µg MVT-602 and remained >15 IU/L for 33 hours. Time to ovulation after drug administration was 3.3-3.9 days (MVT-602), 3.4 days (triptorelin), and 5.5 days (placebo). Ovulation occurred within 5 days of administration in 100% (3 µg), 88% (1 µg), 82% (0.3 µg), and 75% (0.1 µg), of women after MVT-602, 100% after triptorelin and 60% after placebo. CONCLUSIONS: MVT-602 induces LH concentrations of similar amplitude and duration as the physiological midcycle LH surge with potential utility for induction of oocyte maturation and ovulation during MAR. CLINICAL TRIAL REGISTRATION NUMBER: EUDRA-CT: 2017-003812-38, 2018-001379-20.

Genetic control of pod morphological traits and pod edibility in a common bean RIL population.

KEY MESSAGE: QTL mapping, association analysis, and colocation study with previously reported QTL revealed three main regions controlling pod morphological traits and two loci for edible pod characteristics on the common bean chromosomes Pv01 and Pv06. Bean pod phenotype is a complex characteristic defined by the combination of different traits that determine the potential use of a genotype as a snap bean. In this study, the TUM RIL population derived from a cross between 'TU' (dry) and 'Musica' (snap) was used to investigate the genetic control of pod phenotype. The character was dissected into pod morphological traits (PMTs) and edible pod characteristics (EPC). The results revealed 35 QTL for PMTs located on seven chromosomes, suggesting a strong QTL colocation on chromosomes Pv01 and Pv06. Some QTL were colocated with previously reported QTL, leading to the mapping of 15 consensus regions associated with bean PMTs. Analysis of EPC of cooked beans revealed that two major loci with epistatic effect, located on chromosomes Pv01 and Pv06, are involved in the genetic control of this trait. An association study using a subset of the Spanish Diversity Panel (snap vs. non-snap) detected 23 genomic regions, with three regions being mapped at a position similar to those of two loci identified in the TUM population. The results demonstrated the relevant roles of Pv01 and Pv06 in the modulation of bean pod phenotype. Gene ontology enrichment analysis revealed a significant overrepresentation of genes regulating the phenylpropanoid metabolic process and auxin response in regions associated with PMTs and EPC, respectively. Both biological functions converged in the lignin biosynthetic pathway, suggesting the key role of the pathway in the genetic control of bean pod phenotype.

A Plain Language Summary to learn about relugolix combination therapy for the treatment of pain associated with endometriosis.

WHAT IS THIS SUMMARY ABOUT?: This is a summary of research studies (known as clinical trials) called SPIRIT 1 and SPIRIT 2. The SPIRIT 1 and SPIRIT 2 studies compared how well a medicine called relugolix combination therapy worked in relieving pain in women with moderate to severe endometriosis compared to a placebo, a pill with no active medication. Endometriosis occurs when tissue similar to what normally lines the uterus grows in other places, such as the ovaries, fallopian tubes, and bowels. WHAT WERE THE RESULTS?: Researchers looked at 1261 adult women with moderate to severe endometriosis. Randomly, 420 (33%) of these women were assigned to relugolix combination therapy, 420 (33%) were assigned to delayed relugolix combination therapy (relugolix alone first and then relugolix combination therapy for the remainder of the study), and 421 (33%) were assigned to placebo. The SPIRIT 1 and SPIRIT 2 studies showed that more women taking relugolix combination therapy (75% from SPIRIT 1 and 75% from SPIRIT 2) for 24 weeks had both less pelvic or groin pain during menstrual periods from endometriosis and no need for more pain medicines than women who took placebo (27% from SPIRIT 1 and 30% from SPIRIT 2). The SPIRIT 1 and SPIRIT 2 studies also showed that more women taking relugolix combination therapy (59% from SPIRIT 1 and 66% from SPIRIT 2) for 24 weeks had both less pelvic or groin pain between menstrual periods from endometriosis and no need for more pain medicines than women who took placebo (40% from SPIRIT 1 and 43% from SPIRIT 2). Women taking relugolix combination therapy had less pelvic or groin pain during and between menstrual periods within 4 weeks of starting the medicine. The most common side effects were headaches, the common cold, and hot flushes or feeling hot among women taking relugolix combination therapy, delayed relugolix combination therapy, and placebo. Relugolix combination therapy was considered safe for those with no major medical problems. Women taking relugolix combination therapy had little to no loss of bone mineral density (a way of knowing how strong bones are) after 24 weeks of treatment. WHAT DO THE RESULTS OF THESE STUDIES TELL US?: Women with moderate to severe endometriosis taking relugolix combination therapy had much less pain from endometriosis than women taking placebo. Clinical Trial Registration: NCT03204318 (SPIRIT-1); NCT03204331 (SPIRIT-2) (ClinicalTrials.gov).

LIBERTY randomized withdrawal study: relugolix combination therapy for heavy menstrual bleeding associated with uterine fibroids.

BACKGROUND: In the pivotal LIBERTY 1 and 2 trials and long-term extension study, once-daily relugolix combination therapy (40 mg relugolix, 1 mg estradiol, 0.5 mg norethindrone acetate) reduced menstrual blood loss volume and pain among women with uterine fibroids. Relugolix combination therapy was well tolerated with preservation of bone mineral density through 52 weeks. OBJECTIVE: This study aimed to report the 2-year relugolix combination therapy efficacy and safety results of the phase 3 LIBERTY randomized withdrawal study. STUDY DESIGN: Women with uterine fibroid-associated heavy menstrual bleeding who completed the 24-week LIBERTY 1 or 2 trials, followed by the 28-week long-term extension study (up to 52 weeks total treatment), and who met the responder criteria (menstrual blood loss volume <80 mL and ≥50% reduction from pivotal study baseline at week 48 [week 24 of long-term extension]) were randomized in a 1:1 ratio to either blinded treatment with relugolix combination therapy or placebo for 52 weeks (total treatment period, 104 weeks). For women who had a relapse of heavy menstrual bleeding during the study (menstrual blood loss volume ≥80 mL), open-label relugolix combination therapy was offered. The primary endpoint was the proportion of women who maintained menstrual blood loss volume <80 mL through week 76 (week 24 of randomized withdrawal study). Secondary endpoints included time to menstrual blood loss volume ≥80 mL, proportion of women who maintained a menstrual blood loss volume of <80 mL through week 104 (over the 52-week randomized treatment period), the proportion of women who achieved or maintained amenorrhea at week 76 at the end of treatment, and the change in Uterine Fibroid Symptom-Quality of Life Bleeding and Pelvic Discomfort Scale and symptom severity scores. Analyses were performed for the modified intent-to-treat population, including all randomized women who received ≥1 dose of the study drug. RESULTS: Of the 229 randomized women (relugolix combination therapy, n=115; placebo, n=114), 228 received the study drug and 175 (76.7%) completed the randomized withdrawal study. Through week 76, 78.4% of women on relugolix combination therapy maintained menstrual blood loss volume <80 mL vs 15.1% in the placebo group (difference, 63.4%; 95% confidence interval, 52.9%-73.9%; P<.0001). At week 104, 69.8% of women on relugolix combination therapy maintained menstrual blood loss volume <80 mL vs 11.8% in the placebo group (difference, 58.0%; 95% confidence interval, 47.0%-69.1%; P<.0001). Through week 104, 88.3% of women on placebo relapsed with heavy menstrual bleeding (median time to relapse, 5.9 weeks). Among the 89 women in the placebo group who relapsed and received open-label rescue treatment, 87 women responded to relugolix combination therapy with a menstrual blood loss volume <80 mL. The proportion of women who achieved or maintained amenorrhea were 57.4% vs 13.3% at week 76 (difference, 44.1%; 95% confidence interval, 33.10%-55.1%; P<.0001) and 58.3% vs 10.6% at week 104 (difference, 47.6%; 95% confidence interval, 37.0%-58.3%; nominal P<.0001) for relugolix combination therapy and the placebo group, respectively. Relugolix combination therapy was generally well tolerated; no new safety signals were identified, and the adverse event profile over the second year was consistent with that reported through the first year of treatment. Bone mineral density remained stable in women who received relugolix combination therapy from week 52 to week 104. In women continuously treated with relugolix combination therapy up to 2 years, bone mineral density was generally preserved. CONCLUSION: After 2 years of treatment with relugolix combination therapy, there was evidence of durability of the effect in maintaining low menstrual blood loss volume in women with symptomatic uterine fibroids. Most women had return of heavy menstrual bleeding and associated symptoms after treatment cessation, which improved upon retreatment with relugolix combination therapy. Relugolix combination therapy was well tolerated, the adverse event profile remained consistent, and the mean bone mineral density was generally preserved through 2 years of treatment.

Genetic erosion within the Fabada dry bean market class revealed by high-throughput genotyping.

The Fabada market class within the dry beans has a well-differentiated seed phenotype with very large white seeds. This work investigated the genetic diversity maintained in the seed collections within this market class and possible genetic erosion over the last 30 years. A panel with 100 accessions was maintained in seed collections for 30 years, 57 accessions collected from farmers in 2021, six cultivars developed in SERIDA, and 16 reference cultivars were gathered and genotyped with 108,585 SNPs using the genotyping-by-sequencing method. Filtering based on genotypic and phenotypic data was carried out in a staggered way to investigate the genetic diversity among populations. The dendrogram generated from genotyping revealed 90 lines forming 16 groups with identical SNP profiles (redundant lines) from 159 lines classified as market-class Fabada according to their passport data. Seed phenotyping indicated that 19 lines were mistakenly classified as Fabada (homonymies), which was confirmed in the dendrogram built without redundant lines. Moreover, this study provides evidence of genetic erosion between the population preserved for 30 years and the currently cultivated population. The conserved population contains 54.6% segregation sites and 41 different SNP profiles, whereas the cultivated population has 19.6% segregation sites and 26 SNP profiles. The loss of genetic variability cannot be attributed to the diffusion of modern cultivars, which increase genetic diversity (six new SNP profiles). The results allow for the more efficient preservation of plant genetic resources in genebanks, minimizing redundant accessions and incorporating new variations based on genotypic and phenotypic data.

SLO3: A Conserved Regulator of Sperm Membrane Potential.

Sperm cells must undergo a complex maturation process after ejaculation to be able to fertilize an egg. One component of this maturation is hyperpolarization of the membrane potential to a more negative value. The ion channel responsible for this hyperpolarization, SLO3, was first cloned in 1998, and since then much progress has been made to determine how the channel is regulated and how its function intertwines with various signaling pathways involved in sperm maturation. Although Slo3 was originally thought to be present only in the sperm of mammals, recent evidence suggests that a primordial form of the gene is more widely expressed in some fish species. Slo3, like many reproductive genes, is rapidly evolving with low conservation between closely related species and different regulatory and pharmacological profiles. Despite these differences, SLO3 appears to have a conserved role in regulating sperm membrane potential and driving large changes in response to stimuli. The effect of this hyperpolarization of the membrane potential may vary among mammalian species just as the regulation of the channel does. Recent discoveries have elucidated the role of SLO3 in these processes in human sperm and provided tools to target the channel to affect human fertility.

A Randomized Open-Label Study of Relugolix Alone or Relugolix Combination Therapy in Premenopausal Women.

BACKGROUND AND OBJECTIVE: Relugolix is a gonadotropin-releasing hormone receptor antagonist. Relugolix 40-mg monotherapy is associated with vasomotor symptoms and long-term bone mineral density loss due to hypoestrogenism. This study assessed whether the addition of estradiol (E2) 1 mg and norethindrone acetate (NETA) 0.5 mg to relugolix 40 mg (relugolix combination therapy) provides systemic E2 concentrations in the 20-50 pg/mL range to minimize these undesirable effects. METHODS: This was a randomized, open-label, parallel-group study to assess the pharmacokinetics, pharmacodynamics, safety, and tolerability of relugolix 40 mg alone or in combination with E2 1 mg and NETA 0.5 mg in healthy premenopausal women. Eligible women were randomized 1:1 to receive relugolix alone or relugolix plus E2/NETA for 6 weeks. Study assessments included pharmacokinetic parameters of E2, estrone, and relugolix in both treatment groups, and norethindrone in the relugolix plus E2/NETA treatment group at weeks 3 and 6. RESULTS: Median E2 24 h average concentrations with the relugolix plus E2/NETA group (N = 23) were 31.5 pg/mL, 26 pg/mL higher compared with the relugolix-alone group (6.2 pg/mL) (N = 25). There were 86.4% of participants in the relugolix plus E2/NETA group who had E2 average concentrations exceeding 20 pg/mL, the threshold expected to minimize bone mineral density loss, compared with 21.1% in the relugolix-alone group. Both treatments were generally safe and well tolerated. CONCLUSIONS: Relugolix 40 mg in combination with E2 1 mg and NETA 0.5 mg provided systemic E2 concentrations within a range expected to minimize the risk of undesirable effects of hypoestrogenism associated with the administration of relugolix alone. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov identifier no. NCT04978688. Trial registration date: 27 July, 2021; retrospectively registered.

Ética en investigación: desafíos durante la pandemia / Research ethics: challenges during the pandemic

Durante la pandemia de Covid-19 los hospitales pediatricos se vieron menos afectados, debido a la menor infección en niños, y sus recursos fueron reasignados en distintas tareas.. El Comité de Ética en Investigación del Hospital General de Niños Pedro de Elizalde presenta los distintos procedimientos implementados en esta emergencia, para sostener diferentes investigaciones, y que les permitió una rápida respuesta a esta situación.

Development of relugolix combination therapy as a medical treatment option for women with uterine fibroids or endometriosis.

Treatment of uterine fibroids (UF) and endometriosis (EM) has relied on the surgical skills of gynecologists to improve symptoms and potentially alter the course of these debilitating diseases. Medical management of symptoms for both diseases leverages combined hormonal contraceptives used off label as a first-line treatment, with nonsteroid anti-inflammatory drugs and opioids to manage pain as needed. Gonadotropin-releasing hormone (GnRH) receptor agonists (peptide analogs) have been used as short-term therapy to manage severe symptoms of UF or EM, treat anemia, and reduce fibroid size before surgery. The introduction of oral GnRH receptor antagonists opened the door for the development of new treatment options for UF, EM, and other estrogen-driven diseases. Relugolix is an orally active, nonpeptide, GnRH receptor antagonist that competitively binds to GnRH receptors, preventing the release of follicle-stimulating hormone and luteinizing hormone (LH) into the systemic circulation. In women, the reduction in follicle-stimulating hormone concentrations prevents natural follicular development, suppressing ovarian production of estrogen, and together with reductions in LH concentrations, prevent ovulation, corpus luteum formation, and, thereby, the production of progesterone (P). By reducing circulating concentrations of estradiol (E2) and P, relugolix improves heavy menstrual bleeding and other symptoms associated with UF and moderate to severe pain associated with EM, including dysmenorrhea, nonmenstrual pelvic pain (NMPP) and dyspareunia. However, as monotherapy, the use of relugolix is associated with signs and symptoms of a hypoestrogenic state, including bone mineral density loss and vasomotor symptoms. The clinical development of relugolix incorporated the addition of a 1 mg dose of E2 and a 0.5-mg dose of norethindrone acetate (NETA) to achieve systemic E2 concentrations that remain in a therapeutic range while mitigating the risk for bone mineral density loss and vasomotor symptoms, enabling the longer-term treatment and reducing the impact of symptoms on quality of life, and potentially delaying or preventing the need for surgery. Relugolix 40 mg in combination with estradiol (E2) 1 mg and NETA 0.5 mg as a single fixed-dose combination tablet (relugolix combination therapy [relugolix-CT]) approved in the United States as MYFEMBREE is the first and only once daily oral GnRH antagonist combination therapy indicated for the management of heavy menstrual bleeding associated with UF and moderate to severe pain associated with EM. In the European Union (EU) and the United Kingdom (UK), relugolix-CT is approved as RYEQO for the management of symptoms associated with UF. In Japan, relugolix 40 mg, as monotherapy, was the first GnRH receptor antagonist approved to improve symptoms associated with UF or pain associated with EM under the brand name RELUMINA. In men, relugolix suppresses testosterone production. Relugolix 120 mg (ORGOVYX) was developed by Myovant Sciences and is approved in the United States, EU, and UK as the first and only oral androgen-deprivation therapy for the treatment of advanced prostate cancer. This review is focused on the development of relugolix and relugolix-CT in women's health indications.

A plain language summary of the safety of relugolix combination therapy and improvement in symptoms in women with uterine fibroids from the LIBERTY 1 and LIBERTY 2 studies.

WHAT IS THIS SUMMARY ABOUT?: This is a summary of research studies (known as clinical trials) called LIBERTY 1 and LIBERTY 2. The LIBERTY 1 and LIBERTY 2 studies looked at how well a medication called relugolix combination therapy worked to reduce heavy bleeding at the time of menstruation compared with placebo. The studies also looked at what side effects were reported in women with uterine fibroids and heavy menstrual bleeding. WHAT WERE THE RESULTS?: Researchers looked at 388 adult women in the LIBERTY 1 study and 382 adult women in the LIBERTY 2 study. All women had heavy menstrual bleeding with uterine fibroids before the start of the LIBERTY 1 and LIBERTY 2 studies. The women were given one of three treatments during the studies: relugolix combination therapy or placebo for 24 weeks, or delayed relugolix combination therapy (relugolix alone for the first 12 weeks, then relugolix combination therapy for the last 12 weeks of the studies). More women taking relugolix combination therapy in the LIBERTY 1 study (73%) and LIBERTY 2 study (71%) had menstrual blood loss of less than one-third of a cup (80 mL) and had reduction of at least 50% less blood loss during their last menstrual period after 24 weeks of taking the medicine compared with placebo (LIBERTY 1: 19% and LIBERTY 2: 15%). The women taking relugolix combination therapy also had less pain than those taking placebo. Side effects were similar across treatment groups. Headaches and hot flushes were the most common side effects. WHAT DO THE RESULTS MEAN?: More women with uterine fibroids taking relugolix combination therapy for 24 weeks were likely to have fewer uterine fibroid symptoms than women receiving placebo. Clinical Trial Registration: NCT03049735 (LIBERTY 1); NCT03103087 (LIBERTY 2).

Selection and adaptive introgression guided the complex evolutionary history of the European common bean.

Domesticated crops have been disseminated by humans over vast geographic areas. Common bean (Phaseolus vulgaris L.) was introduced in Europe after 1492. Here, by combining whole-genome profiling, metabolic fingerprinting and phenotypic characterisation, we show that the first common bean cultigens successfully introduced into Europe were of Andean origin, after Francisco Pizarro's expedition to northern Peru in 1529. We reveal that hybridisation, selection and recombination have shaped the genomic diversity of the European common bean in parallel with political constraints. There is clear evidence of adaptive introgression into the Mesoamerican-derived European genotypes, with 44 Andean introgressed genomic segments shared by more than 90% of European accessions and distributed across all chromosomes except PvChr11. Genomic scans for signatures of selection highlight the role of genes relevant to flowering and environmental adaptation, suggesting that introgression has been crucial for the dissemination of this tropical crop to the temperate regions of Europe.

Genome-wide association study for the extractable phenolic profile and coat color of common bean seeds (Phaseolus vulgaris L.).

BACKGROUND: A large variation in seed coat colors and seed phenolic metabolites is present in common bean (Phaseolus vulgaris L.). The study of the relationships between seed coat color phenotype and the phenolic profile is an important step in the elucidation of the gene network involved in the phenylpropanoid biosynthetic pathway. However, this relationship is still poorly understood in this species. RESULTS: A genome-wide association study (GWAS) was used to investigate the genomic regions associated with the synthesis of 10 flavonoids (5 anthocyanins and 5 flavonols) and with 10 seed coat color traits using a set of 308 common bean lines of the Spanish Diversity Panel (SDP) which have been genotyped with 11,763 SNP markers.. A total of 31 significant SNP-trait associations (QTNs) were identified, grouped in 20 chromosome regions: 6 for phenolic metabolites on chromosomes Pv01, Pv02, Pv04, Pv08, and Pv09, 13 for seed coat color on chromosomes Pv01, Pv02, Pv06, Pv07, and Pv10, and 1 including both types of traits located on chromosome Pv08. In all, 58 candidate genes underlying these regions have been proposed, 31 of them previously described in the phenylpropanoid pathway in common bean, and 27 of them newly proposed in this work based on the association study and their homology with Arabidopsis anthocyanin genes. CONCLUSIONS: Chromosome Pv08 was identified as the main chromosome involved in the phenylpropanoid pathway and in consequence in the common bean seed pigmentation, with three independent chromosome regions identified, Phe/C_Pv08(2.7) (expanding from 2.71 to 4.04 Mbp), C_Pv08(5.8) (5.89-6.59 Mbp), and Phe_Pv08(62.5) (62.58 to 63.28 Mbp). Candidate genes previously proposed by other authors for the color genes V and P were validated in this GWAS. Candidate genes have been tentatively proposed from this study for color genes B and Rk on Pv02, Asp on Pv07, and complex C on Pv08. These results help to clarify the complex network of genes involved in the genetic control of phenolic compounds and seed color in common bean and provide the opportunity for future validation studies.

A selective inhibitor of the sperm-specific potassium channel SLO3 impairs human sperm function.

To fertilize an oocyte, the membrane potential of both mouse and human sperm must hyperpolarize (become more negative inside). Determining the molecular mechanisms underlying this hyperpolarization is vital for developing new contraceptive methods and detecting causes of idiopathic male infertility. In mouse sperm, hyperpolarization is caused by activation of the sperm-specific potassium (K+) channel SLO3 [C. M. Santi et al., FEBS Lett. 584, 1041-1046 (2010)]. In human sperm, it has long been unclear whether hyperpolarization depends on SLO3 or the ubiquitous K+ channel SLO1 [N. Mannowetz, N. M. Naidoo, S. A. S. Choo, J. F. Smith, P. V. Lishko, Elife 2, e01009 (2013), C. Brenker et al., Elife 3, e01438 (2014), and S. A. Mansell, S. J. Publicover, C. L. R. Barratt, S. M. Wilson, Mol. Hum. Reprod. 20, 392-408 (2014)]. In this work, we identified the first selective inhibitor for human SLO3-VU0546110-and showed that it completely blocked heterologous SLO3 currents and endogenous K+ currents in human sperm. This compound also prevented sperm from hyperpolarizing and undergoing hyperactivated motility and induced acrosome reaction, which are necessary to fertilize an egg. We conclude that SLO3 is the sole K+ channel responsible for hyperpolarization and significantly contributes to the fertilizing ability of human sperm. Moreover, SLO3 is a good candidate for contraceptive development, and mutation of this gene is a possible cause of idiopathic male infertility.

Differentially expressed genes against Colletotrichum lindemuthiamum in a bean genotype carrying the Co-2 gene revealed by RNA-sequencing analysis.

Anthracnose is responsible for large yield losses in common bean crops. RNA-sequencing was used to investigate the differentially expressed genes (DEGs) in response to race 38 of Colletotrichum lindemuthianum in two near-isogenic lines (A25 and A4804) that differ in the presence of a resistance gene located in the cluster Co-2. Their responses were analyzed at different hours after inoculation (0, 24, and 48) and within and between genotypes. In all, 2,850 DEGs were detected, with 2,373 assigned to at least one functional GO term. Enriched GO terms in the resistant genotype were mainly related to functions as a response to stimulus, hormone signaling, cellular component organization, phosphorylation activities, and transcriptional regulation. The region containing the Co-2 cluster was delimited at the end of chromosome Pv11 (46.65-48.65 Mb) through a comparison with the SNP genotypes, obtained using 'Genotyping by Sequencing,' among seven resistant lines harboring the Co-2 gene and the susceptible line A25. The delimited region contained 23 DEGs, including 8 typical R genes, that showed higher expression levels in the resistant genotype and non-changes in the susceptible genotype after inoculation. Six R genes encoding protein kinases and an LRR domain formed a cluster in a core region between 46.98 and 47.04 Mb. The alignment of the raw transcriptome reads in the core region revealed structural changes that were used to design four potential breeder-friendly DNA markers, and it revealed some alignments with the intergenic regions, suggesting the presence of genes in addition to those annotated in the reference genome.

Relugolix Combination Therapy for Uterine Leiomyoma-Associated Pain in the LIBERTY Randomized Trials.

OBJECTIVE: To assess the effect of once-daily relugolix combination therapy (relugolix-CT: relugolix 40 mg, estradiol 1 mg, and norethindrone acetate 0.5 mg) compared with placebo on moderate-to-severe pain in women with uterine leiomyomas and heavy menstrual bleeding. METHODS: Two replicate, multinational, double-blind, 24-week, randomized, phase 3 studies (LIBERTY 1 and 2) were conducted in premenopausal women with uterine leiomyoma-associated heavy menstrual bleeding (80 mL or greater per cycle for two cycles or 160 mL or greater during one cycle). A predefined secondary objective was to determine the effect of relugolix-CT on moderate-to-severe uterine leiomyoma-associated pain in the pain subpopulation (women with maximum pain scores of 4 or higher on the 0-10 numerical rating scale at baseline, with pain score reporting compliance of 80% (ie, 28 days or more over the last 35 days of treatment). This key secondary endpoint was defined as the proportion of women achieving minimal-to-no uterine leiomyoma-associated pain (maximum numerical rating scale score 1 or lower) at week 24; menstrual and nonmenstrual pain were evaluated in prespecified secondary analyses. Treatment comparisons were performed in the pooled LIBERTY 1 and 2 pain subpopulation using the Cochran-Mantel-Haenszel test stratified by baseline menstrual blood loss volume. RESULTS: Across both trials, 509 women were randomized to relugolix-CT or placebo (April 2017-December 2018). Of these, 277 (54.4%) met pain subpopulation requirements. With relugolix-CT, 45.2% (95% CI 36.4-54.3) of women achieved minimal-to-no pain compared with 13.9% (95% CI 8.8-20.5) with placebo (nominal P<.001). The proportions of women with minimal-to-no pain during menstrual days and during nonmenstrual days were significantly higher with relugolix-CT (65.0% [95% CI 55.6-73.5] and 44.6% [95% CI 32.3-57.5], respectively) compared with placebo (19.3% [95% CI 13.2-26.7], nominal P<.001, and 21.6% [95% CI 12.9-32.7], nominal P=.004, respectively). CONCLUSION: Over 24 weeks, relugolix-CT significantly reduced moderate-to-severe uterine leiomyoma-associated pain with a more pronounced effect on menstrual pain. These data support that relugolix-CT had clinically meaningful effects on women's experience of uterine leiomyoma-associated pain. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov: LIBERTY 1, NCT03049735; LIBERTY 2, NCT03103087. FUNDING SOURCE: Myovant Sciences GmbH.