Female Serum Research Articles

Age and sex effects of a validated LC-MS/MS method for the simultaneous quantification of testosterone, allopregnanolone, and its isomers in human serum

Despite the great relevance of the neurosteroid allopregnanolone and related isomers to various health conditions, quantification typically involves immunoassay, which suffers from serious issues with cross-reactivity of closely related molecules. This article describes the development and partial validation of a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantification of allopregnanolone, pregnanolone, isopregnanolone, epi-allopregnanolone, and testosterone in the human serum of healthy males and females aged 5–85 years. 1-amino-4-methylpiperazine (AMP) was used as a derivatisation reagent to enhance the ionisation signal. Linearity was calculated at 0.99 with a lower limit of quantification of 10.08 pg/mL for allopregnanolone, along with a linearity of 0.98 and a lower limit of quantification of 42.32 pg/mL for testosterone. Application of the method showed sex and age effects across the lifespan for both allopregnanolone and testosterone, whereas a comparative immunoassay for allopregnanolone was not able to detect differences in the same samples. Our partial validation of this method should provide a new tool for researchers to discover the role of allopregnanolone and its isomers in human health, and how it compares to testosterone and sex hormones.

Discriminating fingerprints of chronic neuropathic pain following spinal cord injury using artificial neural networks and mass spectrometry analysis of female mice serum

Spinal cord injury (SCI) often leads to central neuropathic pain, a condition associated with significant morbidity and is challenging in terms of the clinical management. Despite extensive efforts, identifying effective biomarkers for neuropathic pain remains elusive. Here we propose a novel approach combining matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with artificial neural networks (ANNs) to discriminate between mass spectral profiles associated with chronic neuropathic pain induced by SCI in female mice. Functional evaluations revealed persistent chronic neuropathic pain following mild SCI as well as minor locomotor disruptions, confirming the value of collecting serum samples. Mass spectra analysis revealed distinct profiles between chronic SCI and sham controls. On applying ANNs, 100% success was achieved in distinguishing between the two groups through the intensities of m/z peaks. Additionally, the ANNs also successfully discriminated between chronic and acute SCI phases. When reflexive pain response data was integrated with mass spectra, there was no improvement in the classification. These findings offer insights into neuropathic pain pathophysiology and underscore the potential of MALDI-TOF MS coupled with ANNs as a diagnostic tool for chronic neuropathic pain, potentially guiding attempts to discover biomarkers and develop treatments.

6914 Footing the Bill: Analysis of DFU-related amputation cost expenditure in a sample of HealthFirst Insured Patients

Abstract Disclosure: M. Hashmi: None. C. Schechter: None. A. Herrera Chancay: None. H. Tabassum: None. N. Shiraliyeva: None. J. Daily: None. A.K. Myers: None. Objective Diabetes complications, especially diabetic foot ulcers (DFU), are a leading cause of health care expenditures. This study examined the total cost of amputations and subsequent procedures in Health First insured (HF) patients initially treated at Montefiore Medical Center (MMC) for DFU. Using the database of HF, a large-scale Medicaid managed insurance organization, we captured healthcare utilization beyond the scope of one hospital system, obtaining a more comprehensive understanding of expenditures. We also explored the associations of demographic and lab values with expenditures. Methods The HF data extraction covered calendar years 2021 and 2022 plus YTD 2023. Amputations were identified by CPT codes 27880, 27590, 28810, and 28820. One hundred twenty patients were included in the sample as they had HF insurance and their initial admission for DFU was at one of three hospitals at MMC. Demographic and laboratory data were extracted from the EHR. Demographics, lab values within 3 months of the index admission, and total costs were summarized, and paid amounts per capita per year were calculated. Expenditures included amputation, labs, wound care, surgery, hyperbaric therapy, interpreter services, emergency department visits, home care visits, and outpatient follow-up (in-person and telehealth). We explored demographic, comorbidity, and lab values as predictors of expenditure levels using a bivariate analysis. Results The mean age was 60.6 ± 12 years with majority being male (n=79, 65.3%) and having Type 2 Diabetes (n=103, 96.3%). A significant portion identified their race as other (n=58, 53.7%) or ethnicity as Hispanic-Latino (n=60, 56.1%). Common comorbidities in this group were current or former tobacco use (n=64, 59.2%), chronic kidney disease (n=62, 57.9%), and hypertension (n=94, 86.2%). Glycemic control was suboptimal with an average Hemoglobin A1c of 9.1% (SD 3.1%) and average admission glucose of 232.2 mg/dL. C-reactive protein 17.1 (SD 34.1) and erythrocyte sedimentation rate 88.8 (SD 36.3) were both elevated for most patients. The DFU-related amputation cost was higher for Hispanic-Latino individuals, RR 1.97 (CI 1.04, 3.71) and for those with chronic kidney disease, RR 1.36 (1, 1.86). Female sex, serum glucose and BMI were moderately associated with increased relative cost. The study was limited by not having access to subsequent visits outside our health system and the retrospective nature of the study. Conclusions Our study highlights disparities among patients who underwent a DFU related amputation, with higher costs for Hispanic-Latino individuals, those with chronic kidney disease , and women, emphasizing the need for tailored interventions for these populations to ameliorate the high cost of DFU-related care. Presentation: 6/2/2024

A-031 Development of novel ELISAs for total GDF-15 and H-specific GDF-15

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total human GDF-15 and H-specific (H202D mutant) GDF-15 in human serum, and other biological fluids. Growth/differentiation Factor 15 (GDF-15) is a divergent member of the TGF-β superfamily of growth factors. It is encoded in humans by a gene in chromosome 19. The human GDF-15 gene encodes for a protein of 308 amino acid residues which consists of a signal sequence (residues 1-29), pro-domain (30-194), and mature growth factor domain (195-308). The molecular weight of a mature GDF-15 dimer is 25 kDa. Approximately 25% of humans have a missense polymorphism in the GDF-15 gene resulting in mutation of histidine 202 to aspartate (histidine 6 in the mature domain), close to the N-terminus of the mature growth factor. This variant is associated with phenotypes in prostate cancer, hyperemesis gravidarum, (severe morning sickness in pregnancy), and rheumatoid arthritis. Methods Highly specific and reproducible total GDF-15 (AL-1014-r) and H-specific GDF-15 (AL-1018-r) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and mutant (DD) concentration in serum in the respective assays. The ELISAs use 10 uL sample volume and are calibrated to recombinant human GDF-15 from R & D Systems (Biotechne, USA). These ELISAs were validated for their specificity using HH, DD, HD recombinant preparations and their circulating levels in male, female, 1st, and 2nd-trimester serum samples. Results The Total GDF-15 ELISA assay detects total GDF-15 including histidine 202 to aspartate mutation (HH, HD & DD variant) in the mature domain in equimolar proportions. The H-specific GDF-15 assay is specific to histidine at 202aa in the GDF-15 sequence and does not detect histidine 202 to aspartate mutation (DD variant). Median Levels of total GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 847.9 pg/mL, 1182.4 pg/mL, 12759.7 pg/mL, and 12951.2 pg/mL. Median Levels of H-specific GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 723.2 pg/mL, 641.3 pg/mL, 8381.4 pg/mL and 4652.1 pg/mL. Method comparison between total GDF-15 and commercial GDF-15 assay using serum samples (HH, wild type) in the range of 400-2500 pg/mL yielded a slope of 0.98 (r=0.97). The HD and DD mutant samples in the total assay recovered at twice the concentrations of the commercial assay. The total and intact assays are highly reproducible with the total coefficient of variation less than 7%. Conclusions Use of total GDF-15 assay in combination with the GDF-15 H-specific assay (DD nondetectable) ELISA can accurately estimate the mutant (DD) concentration in serum. This will enhance the knowledge of the GDF-15 measurements in the literature as the commercial assays were compromised for its immunoreactivity to the HD DD genotypes which is highly prevalent.

B-347 Analytical Performance Evaluation of the High Sensitivity PSA Assay and the Free PSAII Assay on the Atellica IM Analyzer

Abstract Background Measurement of serum prostate specific antigen (PSA) levels may be used in prostate cancer (PC) screening, but elevated PSA levels also are observed in patients with other etiologies such as benign prostatic hypertrophy. Studies suggest that measuring PSA with or before digital rectal examination (DRE) provides a better method of detecting PC than DRE alone. The free prostate specific antigen (fPSA) assay is an aid for distinguishing PC from benign prostate conditions in men 50 years or older with PSA values 4.0-10.0 ng/mL (µg/L) and DRE findings nonsuspicious for PC. The goal of this study was to evaluate the analytical performance of the high sensitivity PSA (hsPSA)* and free PSA (fPSAII)* assays on the Atellica® IM Analyzer. Methods Detection capability studies were performed per CLSI EP17-A2. Precision testing was performed per CLSI EP05-A3 with native human serum and QC samples, 3 reagent lots, 1 analyzer, 2 runs/day, 2 replicates/run for 20 days. For Atellica® hsPSA and fPSAII assays a multi-site reproducibility study was conducted per CLSI EP-05 A3 using native and contrived serum samples, 3 reagent lots, and 3 different sites and analyzers; samples were assayed in triplicate for five days, two runs/day ∼2 hours apart. Linearity testing was performed per CLSI EP06-A on a dilution series made from a high PSA sample (native human male serum pool) and a low sample (native human female serum), 4 replicates, 3 reagent lots, and 1 analyzer. Specimen equivalency was determined by Weighted Deming regression model per CLSI EP09c-ed3. Results Results are presented in the table. Conclusions The Atellica IM hsPSA assay and fPSAII assays detection capability, precision, linearity, and specimen equivalency were acceptable. *CAUTION: Investigational Device. Not available for sale. Future availability cannot be guaranteed. The results are preliminary and were achieved in unique setting with no expected timeline for completion.

Correlation of Recombinant FSH Doses with Follicular and Serum Level Resolvin E1 in Polycystic Ovarian Syndrome Women Undergoing ICSI

Background: Reproduction is a intricately structured process achieved by interaction of the higher centers in the brain, the gonads, and different body parts; Whereas infertility is a multifactorial pathological condition affecting approximately 10–15% of couples, especially in industrialized region.RESOLVIN E1 is a member of specialized pro resolving mediator family of bio active lipids. It has anti-inflammatory effects in various inflammatory diseases. A promising researches in their way to prove its positive role in reproduction through direct effect on folliculogenesis. It has a fundamental role in regulating follicular growth and maturation at central and peripheral levels highlighting its possible role in diagnosing and treating infertility problems. High level of RESOLVIN E1 which might play critical role in poly cystic ovarian syndrome due to its anti-inflammatory effects. Objectives: To investigate the correlation of serum and follicular RESOLVIN E1 to gonadotropin (recombinant FSH doses) in PCOS patient undergoing ICSI. Subject, Patients, Materials, and Method: A prospective cross-sectional study conducted in the high institute for infertility diagnosis and assisted reproductive technology /Al Nahrain University /Baghdad /Iraq, from November 2023 till March 2024. Twenty-nine infertile PCOS women who were undergoing intracytoplasmic sperm injection were enrolled in the study. Serum and follicular RESOLVIN E1 at day of oocyte pickup were measured using ELISA technique. Results: The current study shows significantly lower gonadotropins stimulation dose needed for PCOS females (p <0.001). It also revealed no significant correlations between total gonadotropins dose with both serum and follicular fluids resolvin E1, while there were insignificant negative correlations between serum and follicular fluids resolvin E1 with patients’ ages and body mass indices among PCOS females. Conclusions: There is no correlation between the serum and follicular RvE1 levels in PCOS infertile females.

Evaluation of Serum Interleukin 35 and Interleukin 38 and Electrolyte Level in the Sera of Rheumatoid Arthritis and Osteoarthritis Iraqi Females

Background: Rheumatoid arthritis (RA), is a disease that happened because autoimmune and inflammatory effects, it is mean that the human immune system offence healthy cells in the body mistakenly, resulting in inflammation (swelling) in the impacted portions of the body. Osteoarthritis (OA) is treated as the most famous form of joint arthritis. Objective: It is a slowly developed, disabling joint problem that decrease quality of life (QOL) and affects 14% of adults ages 25 and older and nearly 34% of those ages 65 and older. Interleukin elevation levels have been linked to autoimmune and degenerative diseases, thus in the current study. Methods: We tried to evaluate the level of interleukins (IL-35 and IL-38) in the two diseases and found the relationship between immune system response and disease severity. Three groups (RA, OA and control) have been used in the study. Results: The comparison between the three groups has been done and the results show a high significant increase in the level of IL-35 and IL-38 in both patients groups (RA and OA) as compared with control (p<0.001). Furthermore, a highly significant (p<0.001) elevated Na level with a significant (p<0.001) decrease in K and Ca levels was found in the RA and OA patients compared with the control. Conclusions: The study shows that the rise in the levels of IL-35 and IL-38 declares the instinctive role of interleukins and the immune system in the regulation and severity of RA and OA diseases, the role of this parameter may be clear in the controlling and monitoring the progression of diseases.

Abstract P141: Salt tips the balance of tryptophan metabolism between host and gut microbiota in a sex specific manner

Introduction: Tryptophan (Trp) diet attenuates hypertension in male Dahl salt sensitive (S) rats with unknown mechanisms. Kynurenine (a vasodilator) and serotonin (mixed effects on blood pressure) are host-derived Trp metabolites while indole is produced exclusively by the gut microbiota. We have previously shown that the gut microbiota of male, but not female, S rats is susceptible to salt, causing a higher indole level that elevated blood pressure. In this study, we reported the sex differences in all Trp metabolites with a focus on the impact of salt and the mechanisms by which female gut microbiota is more resilient. Methodology: S rats of both sexes (10–13-week-old, n = 4-7/group) were maintained on either a low-salt (0.3 % NaCl, LS) or high-salt (2 % NaCl, HS) diet for 3 weeks. Trp metabolites were analyzed in serum and cecum by LC-MS/MS. To investigate if estrogen shape gut microbiota, gonadectomy was performed on 7-week-old Spontaneously Hypertensive Rats (SHR) of both sexes (n=5/group). β-estradiol (2.8 mg/kg) was administered subcutaneously for 12 weeks before fecal 16S rRNA gene sequencing. Results: Females had elevated levels of kynurenine (Kyn) compared to male S rats on both LS (5.5 ± 1.4 vs. 2.1 ± 0.5 µM; P <0.05) and HS (7 ± 1.3 vs. 4.2 ± 1 µM; P <0.05). Kyn metabolites including kynurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid were elevated in the serum of females compared with males, and HS amplified this difference (all P <0.05). Further, HS significantly decreased the serum serotonin levels in both sexes ( P <0.01 in females, P <0.0001 in males). Intriguingly, HS increased cecal indole levels in male ( P <0.05), but not in female rats. Microbiota analysis of gonadectomized (GDX) SHR rats revealed (1) Significant differences in β-diversity between intact vs. GDX rats ( P <0.01), and between GDX male vs. female rats ( P =0.01); (2) Estrogen supplementation eliminates the gut microbiota differences between GDX females and males ( P =0.18). Conclusion: We demonstrated sex differences in Trp metabolism in response to high salt. Further, estrogen masked the microbial differences between GDX male and female SHR rats. In summary, salt led to an elevated Trp-Kyn metabolism in female host, and an enhanced Trp-indole metabolism in male gut microbiota, which could be due to the estrogen’s effect on shaping gut microbiota. Thus, the Trp metabolism in salt-sensitive hypertension is regulated in a sex-specific manner.

Isolation of donor gamma globulin obtained from multiparous women and its effects upon expression of HLA-G and HLA-DR molecules on lymphocytes from mothers of children with septal congenital heart defects

A common pathogenetic mechanism of reproductive losses and congenital heart disease (CHD) is associated with immune inflammation in the “mother-embryo” system which affects differentiation and proliferation of cardiovascular progenitor cells. It is hypothesized that this link may be blocked by regulatory auto- and alloimmune antibodies to HLA-G and HLA-DR molecules. Moreover, these antibodies may be present at sufficient amounts in donor immunoglobulins, especially those obtained from the blood of multiparous women. Based on this suggestion, the aim of our study was to obtain enriched gamma globulin fraction from the blood of multiparous women and evaluate its functional effects towards HLA-DR and HLA-G molecules. Isolation of the gammaglobulin fraction (GGF) from the blood plasma of multiparous women was performed using affinity chromatography in several sessions. Purity grade of the resulting protein was analyzed by immunoelectrophoresis, electrophoretic separation of the protein fraction of blood serum and electrophoresis in 4.12% polyacrylamide gel with the addition of SDS (PAGE electrophoresis). PAAG electrophoresis showed that this GGF did not differ from commercial therapeutic intravenous immunoglobulin (IVIG). Assessment of the functional activity of GGF upon HLA-DR and HLA-G molecules was performed in the main group of women and their children with congenital heart disease (n = 38), and control group of women who gave birth to conditionally healthy children (n = 21). To determine the specificity of GGF with respect to HLA-G, HLA-DR molecules, as well as to compare its effect with autologous and allogeneic sera and IVIG, we developed an immunological testing protocol using flow cytometry. The protocol was arranged on the basis of the methodology of “cross-match” approach and Russian patent “Method for determining antibodies to HLA-G”. It was found that the blocking activity of female serum towards autologous (intrinsic) and allogeneic (embryo/fetus/child) HLA-G and HLA-DR molecules may determine the protective effect on development of congenital heart defects in the next generation. Donor human immunoglobulin showed a similar blocking effects to these molecules, possibly due to the presence of alloimmune antibodies to HLA classes I and II. The gammaglobulin fraction obtained from the donor blood of multiparous women has a more pronounced blocking effect on the HLA-G and HLA-DR expression. Hence, this immunobiological preparation can be considered a prototype of therapeutic and prophylactic agent blocking the genesis of congenital heart defects.

Effect of prematurely elevated late follicular progesterone on pregnancy outcomes following ovarian stimulation-intrauterine insemination for unexplained infertility: secondary analysis of the AMIGOS trial.

What is the relationship between late follicular phase progesterone levels and clinic pregnancy and live birth rates in couples with unexplained infertility undergoing ovarian stimulation with IUI (OS-IUI)? Late follicular progesterone levels between 1.0 and <1.5 ng/ml were associated with higher live birth and clinical pregnancy rates while the outcomes in groups with higher progesterone levels did not differ appreciably from the <1.0 ng/ml reference group. Elevated late follicular progesterone levels have been associated with lower live birth rates after fresh embryo transfer following controlled ovarian stimulation and egg retrieval, but less is known about whether an association exists with outcomes in OS-IUI cycles. Existing studies are few and have been limited to ovarian stimulation with gonadotrophins, but the use of oral agents, such as clomiphene citrate and letrozole, is common with these treatments and has not been well studied. The study was a prospective cohort analysis of the Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation (AMIGOS) randomized controlled trial. Frozen serum was available for evaluation from 2121 cycles in 828 AMIGOS participants. The primary pregnancy outcome was live birth per cycle, and the secondary pregnancy outcome was clinical pregnancy rate per cycle. Couples with unexplained infertility in the AMIGOS trial, for whom female serum from day of trigger with hCG was available in at least one cycle of treatment, were included. Stored frozen serum samples from day of hCG trigger during treatment with OS-IUI were evaluated for serum progesterone level. Progesterone level <1.0 ng/ml was the reference group for comparison with progesterone categorized in increments of 0.5 ng/ml up to ≥3.0 ng/ml. Unadjusted and adjusted risk ratios (RR) and 95% CI were estimated using cluster-weighted generalized estimating equations to estimate modified Poisson regression models with robust standard errors. Compared to the reference group with 110/1363 live births (8.07%), live birth rates were significantly increased in cycles with progesterone 1.0 to <1.5 ng/ml (49/401 live births, 12.22%) in both the unadjusted (RR 1.56, 95% CI 1.14, 2.13) and treatment-adjusted models (RR 1.51, 95% CI 1.10, 2.06). Clinical pregnancy rates were also higher in this group (55/401 clinical pregnancies, 13.72%) compared to reference group with 130/1363 (9.54%) (unadjusted RR 1.46, 95% CI 1.10, 1.94 and adjusted RR 1.42, 95% CI 1.07, 1.89). In cycles with progesterone 1.5 ng/ml and above, there was no evidence of a difference in clinical pregnancy or live birth rates relative to the reference group. This pattern remained when stratified by ovarian stimulation treatment group but was only statistically significant in letrozole cycles. The AMIGOS trial was not designed to answer this clinical question, and with small numbers in some progesterone categories our analyses were underpowered to detect differences between some groups. Inclusion of cycles with progesterone values above 3.0 ng/ml may have included those wherein ovulation had already occurred at the time the IUI was performed. These cycles would be expected to experience a lower success rate but pregnancy may have occurred with intercourse in the same cycle. Compared to previous literature focusing primarily on OS-IUI cycles using gonadotrophins, these data include patients using oral agents and therefore may be generalizable to the wider population of infertility patients undergoing IUI treatments. Because live births were significantly higher when progesterone ranged from 1.0 to <1.5 ng/ml, further study is needed to clarify whether this progesterone range may truly represent a prognostic indicator in OS-IUI cycles. Oklahoma Shared Clinical and Translational Resources (U54GM104938) National Institute of General Medical Sciences (NIGMS). AMIGOS was funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development: U10 HD077680, U10 HD39005, U10 HD38992, U10 HD27049, U10 HD38998, U10 HD055942, HD055944, U10 HD055936, and U10HD055925. Research made possible by the funding by American Recovery and Reinvestment Act. Dr Burks has disclosed that she is a member of the Board of Directors of the Pacific Coast Reproductive Society. Dr Hansen has disclosed that he is the recipient of NIH grants unrelated to the present work, and contracts with Ferring International Pharmascience Center US and with May Health unrelated to the present work, as well as consulting fees with May Health also unrelated to the present work. Dr Diamond has disclosed that he is a stockholder and a member of the Board of Directors of Advanced Reproductive Care, Inc., and that he has a patent pending for the administration of progesterone to trigger ovulation. Dr Anderson, Dr Gavrizi, and Dr Peck do not have conflicts of interest to disclose. N/A.

High-precision K isotopic analysis of cerebrospinal fluid and blood serum microsamples via multicollector inductively coupled plasma-mass spectrometry equipped with 1013 Ω faraday cup amplifier resistors

BackgroundPotassium isotopic analysis is increasingly performed in both geological and biological contexts as a result of the introduction of MC-ICP-MS instrumentation either equipped with a collision/reaction cell or having the capability of working at “extra-high” mass resolution in order to deal with spectral interference caused by argon hydride (ArH+) ions. Potassium plays an important role in the central nervous system, and its isotopic analysis could provide an enhanced insight into the corresponding processes, but K isotopic analysis of cerebrospinal fluid is challenging due to the small volume, a few microliter only, typically available. This work aimed at developing a method for determining the K isotopic signature of serum and cerebrospinal fluid at a final K concentration of 25 ng mL−1 using Faraday cup amplifiers equipped with a 1013 Ω resistor. ResultsPotassium isotope ratios obtained for reference materials measured at a final K concentration of 25 ng mL−1 were in excellent agreement with the corresponding reference values and the internal and external precision for the δ41K value was 0.11 ‰ (2SE, N = 50) and 0.10 ‰ (2SD, N = 6), respectively. The robustness against the presence of matrix elements and the concentration mismatch between sample and standard observed at higher K concentrations is preserved at low K concentration. Finally, K isotopic analysis of serum and cerebrospinal fluid (3–12 μL of sample) of healthy mice of both sexes was performed, revealing a trend towards an isotopically lighter signature for serum and cerebrospinal fluid from female individuals, however being significant for serum only. SignificanceThis work provides a robust method for high-precision K isotopic analysis at a concentration of 25 ng mL−1. By monitoring both K isotopes, 39K and 41K, with Faraday cups connected to amplifiers with 1013 Ω resistors, accurate K isotope ratio results are obtained with a two-fold improvement in internal and external precision compared to those obtained with the set-up with traditional 1011 Ω resistors. The difference in the K isotope ratio in CSF and serum between the sexes, is possibly indicating an influence of the sex or hormones on the fractionation effects accompanying cellular uptake/release.

Integrated liver and serum proteomics uncover sexual dimorphism and alteration of several immune response proteins in an aging Werner syndrome mouse model.

Werner syndrome (WS) is a progeroid disorder caused by mutations in a protein containing both a DNA exonuclease and DNA helicase domains. Previous studies indicated that males lacking the helicase domain of the Wrn protein orthologue exhibited hepatic transcriptomic and metabolic alterations. In this study, we used a label-free liquid chromatography-tandem mass spectrometry approach to uncover proteins abundance associated with specific biological processes that differed depending on the age (four or ten months) and/or the genotype (wild type or Wrn mutant) in the serum and liver of mice. Principal component analysis of the proteomic data from both serum and hepatic tissue revealed a sexual dimorphism regardless of the age and the genotype of the mice. Moreover, although all Wrn mutant mice exhibited fatty liver by the age of ten months, a significant age and genotype dependent enrichment of proteins involved in lipid and fatty acid metabolic processes were uncovered only in males. Also, a genotype dependent increase in serum oxidant detoxification processes was observed in the serum of Wrn mutant males. Despite these sexual differences, several aspects of the immune system were affected in both females and males. Finally, an increase of specific immunoglobulin molecules was common in the liver and serum of both older Wrn mutant females and males. Such results suggest that specific immunoglobulin variants maybe associated with fatty liver progression in WS.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry.

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

The Role of One-Carbon Metabolism and Methyl Donors in Medically Assisted Reproduction: A Narrative Review of the Literature.

One-carbon (1-C) metabolic deficiency impairs homeostasis, driving disease development, including infertility. It is of importance to summarize the current evidence regarding the clinical utility of 1-C metabolism-related biomolecules and methyl donors, namely, folate, betaine, choline, vitamin B12, homocysteine (Hcy), and zinc, as potential biomarkers, dietary supplements, and culture media supplements in the context of medically assisted reproduction (MAR). A narrative review of the literature was conducted in the PubMed/Medline database. Diet, ageing, and the endocrine milieu of individuals affect both 1-C metabolism and fertility status. In vitro fertilization (IVF) techniques, and culture conditions in particular, have a direct impact on 1-C metabolic activity in gametes and embryos. Critical analysis indicated that zinc supplementation in cryopreservation media may be a promising approach to reducing oxidative damage, while female serum homocysteine levels may be employed as a possible biomarker for predicting IVF outcomes. Nonetheless, the level of evidence is low, and future studies are needed to verify these data. One-carbon metabolism-related processes, including redox defense and epigenetic regulation, may be compromised in IVF-derived embryos. The study of 1-C metabolism may lead the way towards improving MAR efficiency and safety and ensuring the lifelong health of MAR infants.

In situ fabrication of covalent organic frameworks on solid-phase microextraction probes coupled with electrospray ionization mass spectrometry for enrichment and determination of androgens in biosamples.

Solid-phase microextraction (SPME) coupled with electrospray ionization mass spectrometry (ESI-MS) was developed for rapid and sensitive determination of endogenous androgens. The SPME probe is coated with covalent organic frameworks (COFs) synthesized by reacting 1,3,5-tri(4-aminophenyl)benzene (TPB) with 2,5-dioctyloxybenzaldehyde (C8PDA). This COFs-SPME probe offers several advantages, including enhanced extraction efficiency and stability. The analytical method exhibited wide linearity (0.1-100.0µg L-1), low limits of detection (0.03-0.07µg L-1), high enrichment factors (37-154), and satisfactory relative standard deviations (RSDs) for both within one probe (4.0-14.8%) and between different probes (3.4-12.7%). These remarkable performance characteristics highlight the reliability and precision of the COFs-SPME-ESI-MS method. The developed method was successfully applied to detect five kinds of endogenous androgens in female serum samples, indicating that the developed analytical method has great potential for application in preliminary clinical diagnosis.

Dynamically stiffening biomaterials reveal age- and sex-specific differences in pulmonary arterial adventitial fibroblast activation

Respiratory diseases like pulmonary arterial hypertension (PAH) frequently exhibit sexual dimorphism. Female PAH patients are more susceptible to the disease but have increased survival rates. This phenomenon is known as the estrogen paradox, and the underlying mechanisms are not fully understood. During PAH progression in vivo, human pulmonary arterial adventitial fibroblasts (hPAAFs) differentiate into an activated phenotype. These cells produce excess, aberrant extracellular matrix proteins that stiffen the surrounding pulmonary arterial tissues. Here, we employed dynamic poly(ethylene glycol)-alpha methacrylate (PEGαMA)-based biomaterials to study how the age and sex of human serum influenced hPAAF activation in response to microenvironmental stiffening in vitro. Results showed female and male cells responded differently to increases in microenvironmental stiffness and serum composition. Male hPAAFs were less activated than female cells on soft hydrogels and more responsive to increases in microenvironmental stiffness regardless of serum composition. Female hPAAF activation followed this pattern only when cultured in younger (age < 50) female serum or when older (age ≥ 50) female serum was supplemented with estradiol. Otherwise, female hPAAF activation was relatively high on both soft and stiffened hydrogels, with little difference in activation between the two conditions. Collectively, these results suggest that it may be possible to model the estrogen paradox observed in PAH in vitro and that it is critical for researchers to report cell sex and serum source when conducting in vitro experimentation.

Ascertaining sensitive exposure biomarkers of various metal(loid)s to embryo implantation

Close relationships exist between metal(loid)s exposure and embryo implantation failure (EIF) from animal and epidemiological studies. However, there are still inconsistent results and lacking of sensitive metal(loid) exposure biomarkers associated with EIF risk. We aimed to ascertain sensitive metal(loid) biomarkers to EIF and provide potential biological explanations. Candidate metal(loid) biomarkers were measured in the female hair (FH), female serum (FS), and follicular fluid (FF) with various exposure time periods. An analytical framework was established by integrating epidemiological association results, comprehensive literature searching, and knowledge-based adverse outcome pathway (AOP) networks. The sensitive biomarkers of metal(loid)s along with potential biological pathways to EIF were identified in this framework. Among the concerned 272 candidates, 45 metal(loid)s biomarkers across six time periods and three biomatrix were initially identified by single-metal(loid) analyses. Two biomarkers with counterfactual results according to literature summary results were excluded, and a total of five biomarkers were further determined from 43 remained candidates in mixture models. Finally, four sensitive metal(loid) biomarkers were eventually assessed by overlapping AOP networks information, including Se and Co in FH, and Fe and Zn in FS. AOP networks also identified key GO pathways and proteins involved in regulation of oxygen species biosynthetic, cell proliferation, and inflammatory response. Partial dependence results revealed Fe in FS and Co in FH at their low levels might be potential sensitive exposure levels for EIF. Our study provided a typical framework to screen the crucial metal(loid) biomarkers and ascertain that Se and Co in FH, and Fe and Zn in FS played an important role in embryo implantation.

Partitioning of PFAS to serum, tissues, eggs, and hatchlings of an Australian freshwater turtle

Turtles are a potential sentinel species of aquatic ecosystem health as they inhabit aquatic ecosystems, are long lived, and potentially have high exposure to anthropogenic chemicals via food and water. This study investigated per- and polyfluoroalkyl substances (PFAS) tissue partitioning in female Emydura macquarii macquarii turtle, and the maternal offloading of (PFAS) into eggs and then hatchlings as well as the accumulation of PFAS in male and female Emydura macquarii macquarii serum. Significantly higher levels of perfluorosulfonic acids (PFSAs) and perfluorocarboxylic acids (PFCAs) were measured in the male serum compared to the female turtle serum, whereas perfluoroalkane sulfonamides (FASAs) were significantly higher in the female turtle serum. Perfluorooctane sulfonate (PFOS) was the predominant PFAS in the turtles whereas PFHxA was the predominant PFAS found in the surrounding water. PFHxA was not reported in any turtle tissue or the serum. The short-chain PFSAs and FASAs appeared to be highly associated with blood; long-chain PFSAs and PFCAs were more likely to be associated with tissue. Half of the PFHxS and all the long-chain PFSAs and PFCAs reported in the yolks were transferred into the hatchlings (by mass), suggesting a potential intergenerational effect.

Evaluation of fish pituitary spheroids to study annual endocrine reproductive control

The pituitary gland is a small endocrine gland located below the hypothalamus. This gland releases several important hormones and controls the function of many other endocrine system glands to release hormones. Fish pituitary hormonal cells are controlled by neuroendocrine and sex steroid feedback. To study the complex pituitary function in vivo, we established an in vitro pituitary spheroid assay and evaluated its suitability for monitoring the annual reproductive physiological conditions in Takifugu rubripes, also known as torafugu, is one of the most economically important species distributed in the northwestern part of the Pacific Ocean, in the western part of the East China Sea, and in more northern areas near Hokkaido, Japan. Fish pituitary spheroids can be easily constructed in liquid or solid plates. The culture medium (L-15) made the aggregation faster than MEM (Hank’s). A Rho-kinase inhibitor (Y-27632, 10 μM) and/or fish serum (2.5 %) also promoted spheroid formation. Laser confocal microscopy analysis of spheroids cultured with annual serum of both sexes revealed that luteinizing hormone (LH) synthesis has the highest peak in the final maturation stage (3 years old, May) in accordance with the highest serum sex steroid levels; in contrast, follicle stimulating hormone (FSH) synthesis has no correlation with the dose of serum or nutrients. Similarly, 3D cell propagation assays using female serum showed that total pituitary cells displayed the highest proliferation at puberty onset (2 years old, October) before half a year of the spawning season. These results indicate that pituitary spheroids are useful in vitro models for monitoring the reproductive physiological status of fish in vivo and may be applicable to the in vitro screening of environmental chemicals and bioactive compounds affecting reproductive efficiency in aquaculture.

Lipopolysaccharide-induced chronic inflammation increases female serum gonadotropins and shifts the pituitary transcriptomic landscape.

Female reproductive function depends on a choreographed sequence of hormonal secretion and action, where specific stresses such as inflammation exert profound disruptions. Specifically, acute LPS-induced inflammation inhibits gonadotropin production and secretion from the pituitary, thereby impacting the downstream production of sex hormones. These outcomes have only been observed in acute inflammatory stress and little is known about the mechanisms by which chronic inflammation affects reproduction. In this study we seek to understand the chronic effects of LPS on pituitary function and consequent luteinizing and follicle stimulating hormone secretion. A chronic inflammatory state was induced in female mice by twice weekly injections with LPS over 6 weeks. Serum gonadotropins were measured and bulk RNAseq was performed on the pituitaries from these mice, along with basic measurements of reproductive biology. Surprisingly, serum luteinizing and follicle stimulating hormone was not inhibited and instead we found it was increased with repeated LPS treatments. Analysis of bulk RNA-sequencing of murine pituitary revealed paracrine activation of TGFβ pathways as a potential mechanism regulating FSH secretion in response to chronic LPS. These results provide a framework with which to begin dissecting the impacts of chronic inflammation on reproductive physiology.