The fabrication of a zinc hydroxide nitrate-sodium dodecylsulfate bispyribac modified with multi-walled carbon nanotube (ZHN-SDS-BP/MWCNT) paste electrode for uric acid and bisphenol A detection was presented in this study. Electrochemical impedance spectroscopy, chronocoulometry, square-wave voltammetry, and cyclic voltammetry were all used to examine the electrocatalytic activities of modified paste electrodes. The modified electrode’s sensitivity and selectivity have been considered in terms of the composition of the modifier in percentages, the types of supporting electrolytes used, the pH of the electrolyte, and square-wave voltammetry parameters like frequency, pulse size, and step increment. Square-wave voltammetry is performed by applying a small amplitude square-wave voltage to a scanning potential from −0.3 V to +1.0 V, demonstrating a quick response time and high sensitivity. The ZHN-SDS-BP/MWCNT sensor demonstrated a linear range for uric acid and bisphenol A from 5.0 µM to 0.7 mM, with a limit of detection of 0.4 µM and 0.8 µM, respectively, with good reproducibility, repeatability, and stability as well. The modified paste electrode was successfully used in the determination of uric acid and bisphenol A in samples of human urine and lake water. Uric acid (UA) is an end product of purine metabolism and is considered a species of great importance in human diagnosis. UA is important for the detection of abnormal levels of UA in the human body. Extremely high UA levels can build up in the body, and excessive amounts in bodily fluids can cause solid-state urate, which can cause kidney or gout stones, as well as hyperuricemia, which is a separate risk factor for the onset of cardiovascular and renal disease as well as Lesh–Nyhan syndrome. Meanwhile, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, and optic neuritis have all been associated with reduced UA concentration. Gout develops when sodium urate crystals accumulate in the tendons, bursae, soft tissues, and joints ,2. UrateBisphenol A (BPA) is most commonly used in the production of polycarbonate and epoxy resin in the chemical industry worldwide. BPA, with the chemical name 2,2- propane, is an organic compound consisting of two phenolic rings connected by a single carbon carrying two methyl groups. Exposure to BPA triggers an adverse effect in the natural environment and brings major concern, as it induces negative effects on human health. BPA has been found to leach out into the environment from bottles, packaging, plastic plants, and landfill leachates when exposed to certain conditions, such as high temperatures (heating) or when cleansed with rough or acidic detergents. Consequently, BPA migrates into food and drinking water from a wide variety of food contact materials, and when humans routinely ingest trace amounts of BPA, it will accumulate in the body system  containing 0.1 M KCl at frequencies ranging from 10 MHz to 1 Hz. The spectrum consists of a semicircle portion and a linear part. The diameter of the semicircle in the Nyquist diagram indicates the electron transfer resistance (Rct), which works as a kinetic control parameter for electron transfer at the electrode interface, and the linear component depicts the diffusion process 6 solution.The semicircle diameter, which stands for electron transfer resistance 6] solution.For bare MWCNT, the kThe SWV responses of 0.1 mM UA and BPA in the presence of 0.1 M PBS at pH 6.0 were registered on the ZHN-SDS-BP/MWCNT paste electrode and the unmodified MWCNT paste electrodes, as shown in w/w), which were prepared and examined for their voltammetric signals under identical conditions.It is well known that the electrochemical characteristics of the modified electrode depend greatly on the paste composition. The effect of the composition ratios of ZHN-SDS-BP, used as a modifier in carbon paste composition, on the electrochemical response of 0.1 mM of UA and BPA solutions was investigated using SWV. The results show that when the amount of modifier was increased from 0 to 15%, the peak current intensity was increased until it reached the maximum value obtained at 10% of the ZHN-SDS-BP paste electrode. In higher compositions of the modifier, the current significantly decreased, and the electrode became less conductive and unstable. These conditions can be presumably due to the reduction in the conductive area, which is a carbon particle at the surface electrode .Furthermore, the peak current started to decrease after 15% of the ZHN-SDS-BP, probably due to the repelling effect of the hydrophobicity of the pasting liquid (paraffin oil) that blocks the electron penetration of analytes towards the electrode surface. Yet, the amount of paraffin oil used has a substantial impact on the conductivity. Thus, the 10% composition of the ZHN-SDS-BP modifier was selected as the optimum composition with the highest peak current and was used for further research and study ,21.3COONa), sodium sulfate (Na2SO4), and PBS. The rationale for using supporting electrolytes is to inhibit the migration of electro-active species via electrostatic attractions to the electrodes and thereby obtain diffusion-controlled currents . Thus, tThe effect of the pH values on the current responses in modified ZHN-SDS-BP/MWCNT paste electrodes was investigated for 0.1 mM UA and BPA in 0.1 M PBS over a range from 5.8 to 7.8 . The resThe selectivity of the paste electrode ZHN-SDS-BP/MWCNT was based on the configuration and design of the modifier used. Meanwhile, BPA is an electro-active material due to containing phenolic hydroxyl groups. The potential reaction mechanism of the ZHN-SDS-BP/MWCNT paste electrode and BPA solution is shown in Since they control the signal’s intensity and the method’s sensitivity, the SWV parameters are highly significant in SWV. To determine the optimal resolution and sensitivity of analytes towards electrodes, the SWV parameters have been evaluated. To achieve the greatest sensitivity at a rapid scan rate, the effects of factors, including frequency, pulse size, and step size, were examined in a solution containing 1.0 mM UA and BPA in the presence of 0.1 M PBS at pH 6.2. The influence of frequency was analyzed by raising the frequency from 40 Hz to 240 Hz, as shown in 3[Fe(CN)6] (7.6 × 10−6 cm2 s−1 at 25 °C), and t is the time (s). Qdl is the double-layer charge, which could be eliminated by background subtraction, and Qads is the Faradaic charge. According to the plots of Q vs. t1/2  of unmodified MWCNT and ZHN-SDS-BP-modified MWCNT paste electrodes was investigated via chronocoulometry. The measurement of charge (coulombs) as a function of time (second), noted as Q (t), was calculated according to Equation (2), given by Anson .Q (t) =vs. t1/2 A, the Q 3[Fe(CN)6] in the presence of 0.1M KCl as a model complex. From the slopes of Q vs. t1/2 6]. The results showed that the modification of the MWCNT paste electrode with ZHN-SDS-BP improved the effective surface area of the electrode.The effective surface area (A) of unmodified MWCNT and ZHN-SDS-BP paste electrodes was determined via double-potential-step chronocoulumetry using 4.0 mM Kvs. t1/2 B, the ef1/2)  against the square root of time (t1/2) A was obt−1 cm2 s−1 and 1.48 × 10−1 cm2 s−1, respectively, were determined as the diffusion coefficients, D, for UA and BPA. The adsorption capacities, Γs for UA and BPA, were calculated using the Cottrell equation, Qads = nFAΓs, as 3.47 × 10−9 mol cm2 and 3.92 × 10−9 mol cm2, respectively  and I = 0.3966 [BPA] + 7.8428 (R2 = 0.9966), where I, [UA], and [BPA] represent the response current (µA), uric acid, and bisphenol A concentrations (µM), respectively. The limits of detection, calculated based on 3σ/S , were 0.4 µM and 0.8 µM for UA and BPA, respectively. The high sensitivity and limit of detection of the ZHN-SDS BP/MWCNT paste electrode to UA and BPA are comparable to those obtained for several other electrodes by different electroanalytical methods, and this is presented in A good linear relationship between the anodic peak current and the concentration of BPA and UA can be seen in −4 M UA and BPA were investigated by using SWV under optimal experimental conditions. The reproducibility of the sensor was examined using three electrodes that were prepared the same way as the previous electrode to detect UA and BPA in the 0.1M PBS solution. The reproducibility of the method was demonstrated by the relative standard deviation (RSD), where the RSD is estimated by dividing the standard deviation by the average and expressed in the form of a percentage. The RSDs for the UA and BPA obtained from the sensors were 0.87% and 2.59%, respectively, which indicates that all the sensors possess similar current responses and are stable.The reproducibility, repeatability, and storage stability of the sensor for determination of 1.0 × 10n = 3) with the same electrode. The electrode was prepared with the same procedure, and the RSDs obtained for the five replicate measurements of UA and BPA were 3.01% and 3.34%, respectively, indicating that they can be used for repeated measurements [The repeatability was evaluated using five replicate measurements (urements .The storage stability of the electrodes was investigated by measuring the current responses of UA and BPA. The electrode was stored in the laboratory at room temperature (25 °C) for 4 weeks. The current response changes were measured during the first week. −4 M UA and BPA in 1 × 10−1 M PBS at pH 6.2. The result indicates that the presence of selected interferences at a 20-fold excess did not interfere, with the exception of glucose  measurements for the diluted samples that were spiked with specified concentrations of UA and BPA. The results of the percent recoveries were found effective for real sample analysis  and (0.17) for the urine and lake water samples are greater than the significance level p > 0.05, the null hypothesis failed to be rejected, indicating there is no significant difference between the data obtained from the SWV and HPLC techniques, as shown in For comparison purposes, measurements of the urine and lake water samples were also obtained using HPLC . In ordeIn this experiment, the sensing material was simple, highly sensitive, and reasonably priced and was proposed for the testing of UA and BPA with a low LOD. These useful sensing electrode characteristics were developed through the combination of the unique properties of ZHN-SDS BP and MWCNT. Based on electrochemical studies, the effective electrochemical surface area, and the high adsorption capacity of UA and BPA, the ZHN-SDS-BP/MWCNT electrode paste exhibits significant catalytic activity for UA and BPA with higher peak currents.With good selectivity, stability, and repeatability results, the produced electrodes demonstrated considerable electrocatalytic activity against UA and BPA oxidation, demonstrating that the ZHN-SDS BP/MWCNT paste electrode is a promising candidate for real-world uses. In lakes and urine samples, the ZHN-SDS-BP/MWCNT paste electrode demonstrated excellent recovery for UA and BPA.


Human T-cell lymphotropic virus type 1 (HTLV-1)–associated myelopathy (HAM) is a well-recognized neurologic complication of HTLV-1. Beyond HAM, several other neurologic manifestations are increasingly recognized, including acute myelopathy, encephalopathy, and myositis. The clinical and imaging features of these presentations are less well understood and potentially underdiagnosed. In this study, we summarize the imaging features of HTLV-1–related neurologic disease, providing both a pictorial review and pooled series of the less well-recognized presentations.35 cases of acute/subacute HAM and 12 cases of HTLV-1–related encephalopathy were found. In subacute HAM, cervical and upper thoracic longitudinally extensive tranverse myelitis was noted, while in HTLV-1–related encephalopathy, confluent lesions in the frontoparietal white matter and along the corticospinal tracts were the most prevalent finding.There are varied clinical and imaging presentations of HTLV-1–related neurologic disease. Recognition of these features aids early diagnosis where therapy may have the greatest benefit. So far, only HTLV-1 is securely linked to human disease.2 It is estimated that 10 million people are infected worldwide with HTLV-1. It is endemic in diverse geographical regions, including southwestern Japan, equatorial Africa, and South America.3 The virus is transmitted through breastfeeding, sexual intercourse, direct blood contact, and organ transplantation.5 Infection is lifelong, but most persons living with it are asymptomatic. The most feared complications are HTLV-1–associated myelopathy (HAM), also known as tropical spastic paraparesis (TSP) and adult T-cell leukemia/lymphoma (ATL), the lifetime risk of each being 3–5%. Recently, there has been renewed attention to the broader spectrum of potential HTLV-1–related CNS complications and an increased focus on the prevention and improved treatment of HTLV-1–related disease.6 This paradigm shift highlights the need for better awareness of the potential imaging presentations of HLTV-1 and for more robust systematic approaches to imaging and follow-up. This review focuses on the documented non-ATL neurologic associations, including HAM, acute myelopathy, and HTLV-1–related cerebral disease.7 To inform this review, we have pooled our own and published cases for the less-characterized acute myelopathy and encephalopathy presentations.In the early 1980s, the first human retrovirus was described—human T-cell lymphotropic virus type-1 (HTLV-1).After local institutional board review, a retrospective case note review was performed on all patients with HTLV-1 presenting to the National Center for Human Retrovirology, Imperial College Healthcare NHS Trust, London. Routinely acquired retrospective data were obtained and anonymized before analysis, and informed patient consent was deemed not required.Patients who presented with acute to subacute myelopathy or encephalopathy were pooled with previously published cases, which were identified through searching MEDLINE and EMBASE using Ovid online. The search date range was from inception to week 1 of 2022, and the search enquiry used the following keywords “HTLV” OR “HTLV-1” OR “HTLV-I″ AND “neurologic” OR “neuro*” OR “brain” OR “cerebral” OR “spine” OR “myelopathy” OR “HAM” OR “TSP” OR “paraparesis” OR “encephalopathy” OR “encephalitis” OR “enceph*.” Cases where there was another potential cause of myelopathy or encephalopathy were excluded. Please see supplementary material for a detailed summary of the pooled cases.+ T cells, which cross the blood-brain barrier and activate cytotoxic CD8+ T cells causing neuroglial death and degeneration.9 A high proviral load (PVL) and a PVL greater in the CSF than in the blood are risk factors of developing HAM.10 The PVL and the host's immune response are affected by factors such as the host's human leukocyte antigen genotype and single-nucleotide polymorphisms.11 The CSF concentrations of the cytokines neopterin and CXCL10 (IP-10) correlate with the rate of disease progression in HAM. CXCL10 also drives chronic inflammation by stimulating the migration of infected T cells into the CNS.12 The transmission route also seems to be a determinant factor for different potential complications. ATL has a stronger association with infection during infancy, predominantly through mother-to-child transmission, while HAM is associated with later infection, predominantly through sexual transmission or infected blood transfusions.13HTLV-1 chronically infects T lymphocytes, where it is predominantly found in a proviral form. Most patients remain asymptomatic, with the host's T-cell response primarily dictating the risk of secondary disease. HTLV-1 indirectly damages the CNS through infecting CD414Screening tests detect the presence of antibodies to HTLV-1 or HTLV-2 in sera. Reactive samples undergo confirmatory testing using a Western blot or line immunoassay to type the infection. Molecular testing by PCR can also confirm and type HTLV infection but additionally can provide quantitative evidence of HTLV-1 or HTLV-2 and is mainly reported as a percentage of peripheral blood mononuclear cells infected. PVL less than 1% is considered low and typically unassociated with disease from HTLV compared with that more than 1%. Further risk stratification of asymptomatic carriers into those at an increased risk of myelopathy is aided by measuring markers of T-cell activation, i.e., cell surface markers on CD4 and CD8 cells.15 Proximal weakness of the lower limbs, which eventually spreads distally, is common.16 The onset of HAM is variable, although the chronic, slowly progressive presentation is most characterized. Acute and subacute presentations, with progression to severe paraplegia in only a few months, can occur.17 Poorer prognostic indicators in patients with HAM include a high HTLV-1 PVL, female sex, age at onset of 50 years or older, and an early rapidity of progression.18HAM is a neuroinflammatory disease of the spinal cord. Patients with HAM typically experience chronic lower back pain with early neuropathic urinary bladder symptoms followed by the development of lower limb spasticity.links.lww.com/CPJ/A405).Chronic HAM is diagnosed based on characteristic clinical features and confirming the presence of HTLV-1 infection. The WHO proposed diagnostic criteria for HAM in 1989 have since been modified by De Castro-Costa et al. to separate the degree of certainty of diagnosis as definite, probable, or possible  of our own (n = 6) and published cases (n = 29) found 35 cases of HTLV-1–related acute/subacute myelopathy. In most cases (91%), there was no history of blood transfusion or organ transplantation, suggesting sexual contact may be the predominant transmission route. Of the cases where gender was reported, 76% were female, which mirrors the observation of a worse prognosis in female patients with chronic HAM. Of the cases where CSF PMBC HTLV-1 proviral load were performed, in most patients, it was higher in the CSF than in the peripheral blood. Where a trial of steroid therapy was given, 88% reported a noticeable improvement in clinical symptoms and radiologic appearances.Although HAM has historically been viewed as a chronic unremitting disease, it can have a more acute or subacute onset, which can wax and wane in severity. Awareness of this potentially under-recognized presentation is important because preliminary evidence suggests benefit from early immunosuppressive treatment.Where MR imaging was performed (n = 16), the most frequently observed pattern was acute longitudinally extensive transverse myelitis in the cervical and upper thoracic spinal cord (n = 13) with central high T2w spinal cord signal and swelling . In all 26 This enhancement pattern correlates with the histologic observation of lateral and central dorsal column predominant involvement.27 The pathologic observation of spinal white matter tract vacuolation may partly account for the observed radiologic finding of spinal cord swelling.27The presence and pattern of contrast enhancement in acute HAM are variable, with both dorsolateral and anterior horn involvement noted. In 1 local case, the pattern of dorsolateral and central spinal cord enhancement mirrored the “trident sign” previously described in spinal neurosarcoidosis .26 This 29 While ocular manifestations of HTLV-1 such as uveitis and keratoconjunctivitis are well recognized, there are only minimal reports of HTLV-1–related optic neuritis, suggesting this is more a feature of NMO.30 Current evidence indicates that aquaporin-4 antibodies are not directly related to HTLV-1–associated myelopathy, neither in the pathogenesis of HAM nor in the development of AQP4 antibodies post–HTLV-1 infection.31 In endemic areas, testing for both HTLV-1 and AQP4 antibodies is therefore recommended. Additional clinical features suggestive of HAM over NMO include older median age at onset and lower EDSS scores with a generally slower progressive course.Differentiating acute HAM from neuromyelitis optica (NMO) can be challenging, especially in countries with a high prevalence of both, such as in Brazil and Japan.11C-PBR28, a PET tracer that binds to translocator protein (TSPO) on microglia and is used as a marker of neuroinflammation, showed increased cerebral, thalamic, and brainstem TPSO binding in patients with HAM with severe disease compared with those with milder disease.32Awareness of the clinical and radiologic manifestations of cerebral HTLV-1 is increasing, with chronic white matter lesions and acute encephalopathic presentations now recognized. Patients with HAM have more significant neurocognitive deficits than asymptomatic carriers and age-matched controls. Advanced imaging studies support the presence of brain inflammation in HTLV-1.33 The lesions are believed to be secondary to gliosis from chronic perivascular inflammation. There is a positive correlation between the extent of cerebral white matter lesions and the degree of spinal cord involvement, suggesting synchronous cerebral and spinal disease.34 The clinical significance of WM lesions is uncertain, although a correlation between lesion burden and verbal memory deficits has been noted.35 The WM lesions vary from small to extensive and confluent with typically ill-defined boundaries  with widespread involvement of the median precentral white matter .38Patients with HAM have a more significant number of cerebral white matter (WM) lesions than age-matched controls on MRI and postmortem studies.undaries . They arundaries .35 In oue matter . Involvee cohort . These m39 Brain stem and cervical spine lesions are also purported to be more characteristic of MS.40 In a Japanese cohort, the Fazekas criteria demonstrated reasonable accuracy for differentiating MS from cerebral HTLV-1 .41 Other clinical and laboratory factors aid differentiation of MS from HAM. Primary progressive MS can mimic the caudal rostral pattern of HAM but optic nerve involvement common in MS is rare in HAM. Intrathecal production of oligoclonal IgG bands is found in both conditions, but as described earlier, HTLV-1 proviral measurements in the blood and CSF help identify those with high HTLV-1–related inflammation and CNS disease, respectively.Discriminating the white matter lesions of HTLV-1 from multiple sclerosis (MS) is potentially problematic. In a small series comparing 12 patients with HAM with 13 patients with MS, the combination of a white matter lesion >6 mm and a periventricular lesion >3 mm was found to achieve the highest specificity and sensitivity for MS.42 In this series, the annual incidence of encephalitis in the UK national HTLV-1 cohort (n = 142) was approximately 50 times higher  than the reported annual incidence in the UK general population.42 We have since identified an additional Brazilian case and 2 further UK cases of otherwise unexplained encephalopathy in patients with HTLV-1 .43 Of these 12 cases, most of them had preexisting HAM (83.3%), and all but 1 were female (92%).There is increasing evidence that HTLV-1 can be associated with encephalopathy. A prior case series published by our group reported 3 local cases and pooled them with 6 previously published cases.44 The observation of leptomeningeal involvement is further supported by a case-control study that found HTLV-1 had the highest prevalence of leptomeningeal enhancement across a range of chronic neuroinflammatory conditions in endemic tropical areas.45 Abnormal meningeal enhancement in the context of HTLV-1 should also raise the differential of leukemic infiltration.In cases with MR imaging characterization (n = 9), 7 patients had abnormal intracranial imaging . The com47 Both chronic progressive cerebellar atrophy and an isolated case of acute cerebellitis with transient cerebellar swelling have been reported in the context of HTLV-1.49 Broken smooth pursuit eye movements and nystagmus have also been noted in patients with HAM, which may act as a marker for posterior fossa involvement.51Cerebellar syndromes are a rarer presentation of HTLV-1.52 In case-control studies of blood donors, polyneuropathy was described in 9% of seropositive individuals vs 3% in seronegative people.53 Electrodiagnostic studies have also shown that as many as 30% of patients with HAM may have concurrent polyneuropathy.54 Isolated HTLV-1 polyneuropathy without any spinal involvement tends to be predominantly sensory with distal paresthesia and stocking-type sensory loss.7 Inflammatory myositis has also been reported rarely in relation to HTLV-1, and HTLV-1 is myotoxic in vitro.55 The imaging features of HTLV-1–related myositis is poorly characterized, although in our cohort, we have seen a 70-year-old patient with HTLV-1 and an inclusion body myositis-like pattern where there was edema and fatty infiltration in the anterior compartments of both thighs, which relatively spared the rectus femoris  in patients with HAM, but the efficacy is limited, and the treatment is rarely used due to potential risks.HTLV-1 is an underappreciated infection. Even in high-prevalence countries, there is limited awareness of its mode of transmission, disease course, and imaging features. The neuroradiologic manifestations of HTLV-1 go beyond spinal cord atrophy to include acute myelopathy, encephalopathy, and a wide range of associated imaging findings. Although data are too limited to reach definite conclusions, there are a few possible emerging imaging patterns that warrant additional investigation. The observation that immunotherapy may halt and, in some cases, reverse acute HTLV-1–related CNS disease further emphasizes the potential role of imaging in reaching an early diagnosis where therapy may be of greatest benefit. A recent WHO technical report described the pressing need for international collaborative studies to provide further insight into HTLV-1 pathogenesis and disease manifestation. Neuroimaging will likely be a key modality to achieving these aims.→ HTLV-related neurologic diseases are varied and extend beyond chronic HAM to include longitudinally extensive transverse myelitis, cerebral white matter disease, encephalopathy, and acute myositis.→ HTLV-1 should be suspected and tested for in high-risk patient groups who demonstrate supportive imaging features .→ Acute HTLV-1–related neurologic diseases are potentially reversible, and early diagnosis may help prevent irreversible damage.


For women living with HIV (WLH) in serodiscordant partnerships, decisions about childbearing can challenge condom use and antiretroviral adherence. In a prospective cohort of 148 WLH in serodiscordant partnerships, 58 (39%) wanted more children in the future but were not currently trying to conceive (fertility desire), and 32 (22%) were currently trying to become pregnant (fertility intent). Detection of prostate specific antigen (PSA) in vaginal secretions, a marker for recent condomless sex, was lowest in women with fertility desire and highest in women with fertility intent. Detectable viral load followed a similar pattern. Risk of HIV transmission, when condomless sex and PSA detection occurred concurrently, was three to fourfold higher at visits with fertility intent compared to visits with fertility desire. Qualitative interviews underscored the importance women place on childbearing and suggested that they had limited information about the role of antiretroviral therapy in reducing sexual HIV transmission. Sub-Saharan Africa (SSA) has the highest prevalence and incidence of human immunodeficiency virus (HIV) infection worldwide . Nearly While there are available methods for safer conception including use of antiretroviral therapy (ART) for the partner living with HIV and pre-exposure prophylaxis (PrEP) for the partner not living with HIV , discordThe objective of this analysis was to examine the associations between fertility desire and fertility intent, condomless sex, detectable HIV viral load, and when these events occur simultaneously creating the potential for HIV transmission in a cohort of Kenyan WLH in discordant relationships. Three main hypotheses were tested. First, we hypothesized that compared to visits when women expressed no fertility desire, there would be increases in condomless sex at visits when women reported fertility desire and fertility intent, motivated by the goal of becoming pregnant. Second, we hypothesized that compared to visits at which women reported no fertility desire, there would be increases in ART adherence and viral suppression at visits with fertility desire and intent, motivated by the desire to minimize transmission risk to sex partners and infants. Third, we hypothesized that when comparing visits when women reported no fertility desire to those with fertility desire and fertility intent, there would be no difference in transmission potential, since increases in condomless sex would be offset by greater ART adherence and viral suppression.Lifecourse Study, a group of prospective cohort studies examining the relationship between women’s reproductive life course events, condomless sex, and unsuppressed viral load. This analysis utilized data from a cohort of WLH in discordant couples in Nairobi, Kenya. Women living with HIV were eligible to enroll if they were 18 years or older or emancipated minors. Women were enrolled in the discordant couples cohort if their primary partner was HIV-seronegative, and registered at the Kenyatta National Hospital Discordant Couples Centre with their partner at the time of enrollment.This mixed methods analysis was nested within the On enrollment, women completed a face-to-face interview in English or Kiswahili to collect demographic, sexual, behavioral, and partner characteristics. Women were asked if they desired to have more/any children (yes/no) and if they wanted more children, were they currently trying to have children (yes/no). A medical history including HIV history and contraceptive use was collected. Depressive symptoms were assessed using the standardized patient health questionnaire-9 (PHQ-9) , 17, andParticipants returned monthly for ART refills and assessment of adherence. Fertility desire and fertility intent were assessed quarterly. Pelvic examinations, genital sample collection, and urine pregnancy tests were also performed quarterly. Screening for depressive symptoms was completed every 6 months, while screening for alcohol use, IPV, and partner controlling behavior were conducted annually.Case report forms (CRFs) were reviewed daily to ensure that all fields were appropriately completed. Data were entered into an SPSS database. At quarterly intervals, database entries were printed and compared line-by-line to hard copy CRFs to identify and correct any data entry errors.Women were reimbursed 250 Kenyan shillings (~ $2.50) at each visit to compensate for travel expenses. All women provided written informed consent to take part in this study. Ethical approval was obtained from the Kenyatta National Hospital-University of Nairobi Ethics and Research Committee and the University of Washington Human Subjects Division.Neisseria gonorrhoeae, Chlamydia trachomatis, and Trichomonas vaginalis were diagnosed using nucleic acid amplification testing on vaginal swabs . Prostate specific antigen was detected in genital secretions using a rapid test for the p30 antigen . HIV ribonucleic acid quantification was performed using the Hologic/Gen-Probe 2nd generation assay, with a lower limit of quantification of 30 copies/mL. Some 100 mL blood samples were diluted six-fold to 600 mLs prior to testing, so viral suppression for this study was defined as ≤ 180 c/mL  11–28). The percent of expected visits attended per participant ranged from 42 to 115%, with a median of 94% (IQR 87–100%). Four participants (3%) had < 50% of expected visits; 11 (7%) had between 50 and 74% of expected visits; and 133 (90%) had between 75 and 115% of expected visits. The median age of women enrolled in the cohort was 32 years (IQR range 29–37) (Table 2 1.29), and slightly higher at visits with fertility intent , though this association did not reach statistical significance. Visits with fertility intent had a significantly higher risk of PSA detection than visits with fertility desire in both unadjusted  and adjusted analyses .Prostate specific antigen was detected in vaginal secretions at 11.5% (74/644) of visits when women reported no fertility desire. Detection of PSA was slightly lower at visits with fertility desire .To explore whether differences in modern non-barrier contraceptive use might explain the observation that condomless sex was higher at visits without fertility desire compared to visits with fertility desire, modern non-barrier contraceptive use was compared between visits when women reported no fertility desire and visits when fertility desire was reported. Women reported modern non-barrier contraceptive use at 44% (285/652) of visits when they reported no fertility desire compared to 23% (155/665) of visits when they reported fertility desire (χ2 5.51). This association was stronger in the adjusted analysis . There was not a meaningful difference in reported condomless sex between visits with no fertility desire and visits with fertility intent .Overall, self-reported condomless sex was slightly less common than PSA detection. Women reported condomless sex in the past week at 8.3% (125/1514) of visits when there was no fertility desire compared to 3.4% (36/1060) of visits when there was fertility desire, and at 6.4% (28/440) of visits when there was fertility intent. In inferential analyses, compared to visits without fertility desire, at visits with fertility desire women reported substantially less condomless sex . Similarly, when fertility intent was expressed, abstinence was less frequent when compared to visits without fertility desire .In comparison to the U-shaped relationships seen in PSA detection and condomless sex where the lowest prevalences were in women with fertility desire, there were stepwise declines in abstinence at visits when women reported no fertility desire , compared to visits with fertility desire , and fertility intent . The GEE results highlight these findings. Compared to visits without fertility desire, abstinence in the past week was less common at visits when women reported fertility desire . In contrast, higher sexual frequency did not differ greatly between visits without fertility desire and visits with fertility intent .Reporting a sex frequency of three or more episodes per week (the median for the cohort) varied when comparing visits without fertility desire , to visits with fertility desire , and with fertility intent . Compared to visits without fertility desire, higher sexual frequency was reported more often at visits with fertility desire . Similarly, compared to visits without fertility desire, STIs were diagnosed less frequently at visits with fertility intent , though confidence intervals were wide and this association was not statistically significant.Sexually transmitted infections were relatively uncommon in this population but varied in relation to women’s reported fertility desire and fertility intent. The prevalence of STIs was 3.0% (19/642) at visits without fertility desire compared to 1.3% (6/471) of visits with fertility desire, and 1.1% (2/187) of visits with fertility intent. In inferential analyses, compared to visits without fertility desire, the risk of having an STI was significantly lower at visits with fertility desire . However, after adjustment for age and education, there was a statistical trend suggesting a lower risk of late refills when fertility desire was expressed compared to visits without fertility desire . Similarly, late refills occurred less frequently at visits when women reported fertility intent compared to visits without fertility desire .Late refills occurred at 5.9% (83/1400) of visits when fertility desire was not expressed, at 6.0% (56/935) of visits with fertility desire, and at 3.7% (14/376) of visits with fertility intent. Compared to visits without fertility desire, the risk of late refills was similar at visits with fertility desire in unadjusted analyses . This association was stronger in adjusted analyses , though the difference was not statistically significant. There was a similar risk of detectable viral loads when comparing visits without fertility desire to visits with fertility intent , although this association also was not statistically significant.Detectable viral load patterns were somewhat different than late refills. Detectable viral loads occurred at 15.0% (51/341) of visits when women reported no fertility desire, 12.5% (32/257) of visits with fertility desire, and at 16.7% (17/102) of visits with fertility intent. In GEE analyses, compared to visits without fertility desire, the risk of detectable viral loads was lower at visits with fertility desire  and higher at visits with fertility intent , though confidence intervals were wide and results were nonsignificant for both comparisons.Transmission potential was uncommon and followed a U-shaped pattern that was similar to PSA detection. Specifically, a detectable plasma HIV viral load was present at the same time as detection of PSA in vaginal secretions at 2.1% (7/338) of visits without fertility desire, compared to 1.2% (3/256) of visits with fertility desire, and 3.9% (4/102) of visits with fertility intent. In inferential analyses, compared to visits without fertility desire, the risk of transmission potential was lower at visits with fertility desire “A woman cannot be complete without children.” Themes around community pressure to have children, cultural norms, and a woman’s identity being tied to her children were present in all interviews.These data underscore the fact that fertility and children may be highly important drivers of behavior in this population.No […] We are not planning to have another one just because with the agony I passed through with the pregnancy of this boy, the uncertainty of the status […] That is the main reason: the uncertainty of the status of the boy and even my status too. Carrying the pregnancy, taking care of the pregnancy with the status of HIV, I cannot do it. It’s really stressful. The effect of a woman’s HIV status on fertility desire was mixed. Some women described hesitancy to have more children because of their HIV status and concerns about potential transmission to their baby.It cannot prevent me. The only thing is that I take care of myself and ensure that I follow everything that they have told me, my CD4 is okay, viral load is low so that I can get a baby who is okay at least, so that he can have a good life. However, in other interviews, women articulated that HIV did not change their views about having children.Ultimately, community pressure and individual women’s desire to have children were strong. However, HIV did impact some women’s decision about whether to have additional children.“We use a condom because I don’t want to infect him […] About family planning, I get injections...” Interviewer: What is the main reason you two use a condom?Interviewee: “So that I don’t infect him” .Another common theme was that condoms were often used to prevent transmission to women’s HIV-seronegative partners, rather than explicitly for pregnancy prevention.“Because he is very keen when it comes to using condoms. He couldn’t accept even if I told him otherwise. So getting pregnant is not that easy.” However, using condoms to prevent transmission of HIV to sexual partners was challenging if women were intending to get pregnant.“Condoms also help prevent unwanted pregnancies and I don’t want to use ‘drugs’ [contraceptive pills].” Less commonly, women reported using condoms specifically for pregnancy prevention.“Looking at my children I thought if I was not to live positively, I would die leaving them young, and my husband is not very supportive towards our family. So, I felt that if I was to die, my children may suffer a lot. So, I decided if I was to raise my children then I needed to adopt that positive attitude, avoid stress, eat right so that I may live like a normal person.” “That’s when I was told I will have to take the medication so that I may live and raise my child. That’s when I accepted my status.” A common theme of ART use was women’s desire to improve their own health so they could continue caring for their existing children.“[…] I asked here and there, and I got to learn that there’s a risk [of HIV being transmitted to your baby], albeit small, because nowadays there are drugs which can be taken to minimize transmission. Also, I learnt that when you are going to give birth, it’s better to be free with your doctors and tell them about your condition so that they can do something to prevent your infected blood from mixing with your baby’s during childbirth.” Antiretroviral therapy was also recognized as a method for decreasing mother-to-child transmission of HIV during pregnancy.Interviewee: “ […]if I took the medicine I was told my child will not get infected. [So] I started taking the medication when I was two months pregnant.” Interviewer: What steps were taken to ensure that your child will not be infected with HIV?“My CD4 count used to be 253 now it’s risen to almost 1000, I’m okay. The drugs are okay and they’re helping me so it’s harder to infect him.” Women also discussed the role of ART and decreasing transmission to seronegative partners. However, this benefit of ART was less commonly discussed than the other reasons for ART use.However, women did not always verbalize the link between ART adherence, undetectable viral load, and prevention of sexual transmission of HIV. Even in the setting of viral suppression, there were concerns about HIV transmission if condoms were not being used. This is contrary to the Undetectable = Untransmissable (U = U) messaging that stresses to people living with HIV that an undetectable viral load means that HIV is not transmissible to others.Interviewee: “No, not really, because the virus is still present […] they hide, they are still present.” Interviewer: […] If you use ARTs, can you have unprotected sex, if you have reduced your viral load?“He actually reminds me. I usually take the ARV at nine o’ clock. He calls me at eight fifty-nine. Even if he doesn’t have credit, he will flash me. When he flashes and I check my phone, it is nine o’ clock.” “My husband knows, he used to wake me up before midnight he used to put the alarm for me and wake me up and tell me wake up and take your drugs…” Partners were supportive of women taking ART and were facilitators of adherence.In this population of WLH in serodiscordant partnerships, children were often viewed as a central element of womanhood. At baseline, two out of five women reported that they would like to have more children in the future, and another one in five was actively trying to become pregnant. Detection of PSA, a biomarker for recent condomless sex, was lowest in women with fertility desire and highest in women with fertility intent, with a significant increase when comparing visits with fertility desire to those with fertility intent. Women expressed a strong reliance on condom use for prevention of sexual HIV transmission but seemed to know less about how ART can also reduce sexual transmission risk.Other studies have documented similarly complex relationships between fertility desire, fertility intent, and HIV transmission risk in African HIV-serodiscordant couples , 36. ForWhile current guidance for discordant couples highlights the effectiveness of ART to prevent HIV transmission –40, womeIt is important to stress how these findings fit into the larger discussion about safer conception in discordant partnerships and preventing-mother-to-child transmission (PMTCT) programs. These results highlight the fear and stress that women feel about the risks of transmitting HIV to their baby when thinking of future pregnancies. The U = U messaging was not widely disseminated at the time of this study, and U = U is based on preventing sexual transmission of HIV . Safer c. The observed differences in condomless sex could be explained by differences in modern non-barrier contraceptive use, since visits where women reported no fertility desire were associated with more modern non-barrier contraceptive use than at visits when women reported fertility desire. Given these differences in modern non-barrier contraceptive use, it is possible that condoms are used more commonly for pregnancy prevention when women have fertility desire but are not currently trying to become pregnant. Future research could help to determine whether a similar phenomenon is observed in other populations of serodiscordant couples. Regardless, these results make it clear that for WLH, the transition from desiring more children in the future to actively trying to become pregnant may represent an important reproductive life course event during which additional measures could be helpful in minimizing the risk of sexual HIV transmission.While the observed differences were modest, we noted that visits with fertility desire were associated with the lowest prevalences of both condomless sex and detectable viral loadThis study had several strengths. The longitudinal cohort allowed for repeated assessment of exposures and outcomes over time, providing insight into the temporal relationships between variables. The use of biologic measures of condomless sex and viral load suppression avoided the potential for recall and social desirability biases. In addition, combining these biological measures with self-reported sexual behaviors and medication refill data provided a more comprehensive picture of behaviors. Finally, despite the relatively small number of in-depth interviews, these qualitative data provided insight into women’s reasons for condom use and adhering to ART, offering context and illustrating potential mechanisms that help to explain the quantitative findings.There are also some limitations to note. First, the reproductive plans of WLH may be a sensitive topic due to negative societal perceptions of pregnancy in this population. This social desirability bias might be expected to result in under-reporting of fertility intent, causing bias in the risk ratios for our outcomes that are not easy to predict. This limitation was minimized by having experienced research staff administer the questionnaires in a non-judgmental way that was supportive of all reproductive choices, and in the setting of a discordant couples care center where participants are encouraged to discuss their reproductive plans. Second, PSA detection in vaginal secretions only detects condomless sex during the past 24–48 h. This does not bias comparisons of PSA detection between groups but means that the rate of PSA detection reflects only a fraction of all episodes of condomless sex. Third, this study did not include HIV resistance testing, so it is not possible to differentiate viremia due to current poor adherence from viremia caused by resistance to ART. If fertility desire and fertility intent impact viral load through effects on ART adherence, the relation between these reproductive choices and viral load could be attenuated by the effects of ART resistance. Fourth, the cohort study was conducted between 2013 and 2017 and the interviews were conducted in 2014, prior to widespread dissemination of the U = U messaging. While this does not impact the internal validity of the results, it will be important to consider the changing context when comparing these results to contemporary populations. Finally, because the analysis of qualitative data was conducted after the cohort study was completed, a member check of the qualitative findings with participants was not possible.In conclusion, these results suggest that in WLH women in serodiscordant partnerships, the transition from wanting more children in the future to actively trying to conceive is a potential point for intervention to reduce the risk of sexual HIV transmission. Fortunately, overall transmission risk was relatively low. However, supporting WLH by improving their understanding of the role of ART in preventing sexual transmission, performing viral load monitoring with resistance testing if indicated, providing adherence support with evidence-based interventions, and timing condomless sex to the period of peak fertility could help to support safer conception.


While cancer treatment advancements have increased the number of reproductive-aged women survivors, they can harm reproductive function. Despite national guidelines, oncofertility service uptake remains low. This review explores interventions for fertility preservation alignment with American Society of Clinical Oncology (ASCO) guidelines and consideration of a multilevel framework.We systematically reviewed literature from 2006 to 2022 across four databases. Identified interventions were assessed and scored for quality based on CONSORT and TREND statement checklists. Results were synthesized to assess for intervention alignment with ASCO guidelines and four multilevel intervention framework characteristics: targeted levels of influence, conceptual clarity, methodologic pragmatism, and sustainability.n=8) interventions included provider-led discussions of treatment-impaired fertility. Fewer noted discussions on fertility preservation approaches (n=5) and specified discussion timing (n=4). Most (n=8) referred patients to reproductive specialists, and few (n=2) included psychosocial service referrals. Most (n=8) were multilevel, with five targeting three levels of influence. Despite targeting multiple levels, all analyses were conducted at the individual level. Intervention strategies included: educational components (n=5), decision aids (n=2), and nurse navigators (n=2). Five interventions considered stakeholders’ views. All interventions were implemented in real-world contexts, and only three discussed sustainability.Of 407 articles identified, this review includes nine unique interventions. The average quality score was 7.7 out of 11. No intervention was guided by theory. Per ASCO guidelines, most (This review identifies key gaps in ASCO guideline-concordant fertility preservation that could be filled by updating and adhering to standardized clinical practice guidelines and considering multilevel implementation frameworks elements.The online version contains supplementary material available at 10.1007/s00520-023-08133-3. Approximately one million women are diagnosed with cancer each year in the USA. As of 2020, 10% of these cancer cases occurred among women under the age of 40, with more than 48,000 new cancer diagnoses in adolescent and young adult (AYA) women ages 15 to 39 [To achieve these impressive outcomes, cancer treatments typically comprise extensive chemotherapy, radiotherapy, hormone therapies, and/or surgical procedures. While life-saving, these therapies can damage reproductive function –7. For wMoreover, loss of endocrine support for hormonally responsive tissues can trigger a cascade of long-term medical problems in addition to infertility . For exaIn 2006, the American Society of Clinical Oncology (ASCO) endorsed evidence-based clinical practice guidelines on fertility preservation for healthcare providers, publishing subsequent updates in 2013 and 2018. Aligning with other national and international recommendations, ASCO guidelines recommend that all patients undergoing potential gonadotoxic treatments should receive information and counseling about the impact of their disease or treatment on future fertility and fertility preservation options as part of their initial comprehensive care plan. Specifically, the guidelines encourage (1) provider discussion of potential impairment to fertility, (2) provider discussion of fertility preservation approaches, (3) all discussions occurring as early as possible prior to beginning treatment, (4) patient referral to reproductive specialists for fertility preservation, (5) documentation of fertility-related discussions, and (6) patient referral to psychosocial services for additional support [To further facilitate care of patients at risk of exposure to gonadotoxic agents, fertility preservation options are outlined as part of these guidelines. Standard fertility preservation options vary based on several factors including patient’s biological sex, age and cancer type . For exaAccordingly, many fertility preservation interventions have been implemented in recent years to increase the uptake of fertility preservation. However, approximately 30–50% of patients do not receive adequate information regarding infertility risks and preservation options prior to beginning cancer treatments . A recenAs envisioned by ASCO, guideline-concordant fertility preservation care is inherently multifaceted and influenced by a complex and interconnected set of barriers and facilitators operating at individual, interpersonal and organizational level. For example, in addition to the patient, various family members  may need to be involved with decision-making. Further, a variety of health care providers  are also involved to address patients’ needs . At the To evaluate the range of evidence-based interventions being used to implement ASCO guideline- concordant fertility preservation care, we conducted a systematic review assessing: (1) strategies and outcomes aligned with ASCO 2018 practice guidelines, (2) consideration of multilevel influences on service provision, and (3) sustainability of the evaluated interventions for healthcare system integration. To this end, we reviewed the literature published from 2006 to 2022 focusing on reproductive age women.In this review, we define interventions to include those testing strategies to increase consideration of and access to fertility preservation. Further, only articles in which interventions were evaluated relative to a comparison group are included.In conducting the review, we followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRSMA) guidelines . We compThe intervention study inclusion criteria were: English language, published between 2006 and 2022, that described an intervention including women of reproductive age (ages 13–49) with a cancer diagnosis and/or the providers that care for them. Recent data show the average age for the onset of menarche is at 12.4 years old. Thus, we selected 13 years old as the lower age limit to identify women of “reproductive age” . AdditioUsing these criteria, we conducted initial title searches to identify articles for initial abstract review. Keywords were clarified to ensure that all relevant articles appeared in the search. Next, one author (SP) reviewed all titles and abstracts to identify articles for full-text review. Finally, we completed full-text reviews to determine the interventions to be included. Two authors  completed data abstraction on all full-text review articles. All discrepancies were resolved through discussion to reach consensus.We applied the CONSORT  and TRENWe used the population, intervention, comparator, outcomes, timeframe, and study design (PICOTS) framework  to selecFor each intervention, we determined the alignment of intervention targets and relevant outcomes with the ASCO guidelines for fertility preservation prior to cancer treatment. A “yes” was assigned for the following guideline elements were targeted as an intervention strategy or assessed as an outcome: (1) provider-led discussion of potential impairment to fertility, (2) provider-led discussion of fertility preservation approaches, (3) timing of discussions occurring prior to beginning treatment, (4) patient was referred to reproductive specialist for fertility preservation, (5) fertility-related discussion was documented, and (6) patient was referred to psychosocial services for additional support .We coded interventions for their alignment with a parsimony-based operational framework based on complexity theory and pragmatic trials . The fraExtracted data were organized by quality assessments for: general intervention characteristics, alignment of intervention strategies and outcomes with ASCO guidelines and consideration of multiple levels of influence. Within each category, we rated intervention quality from 0 to 11.We identified 438 articles from four electronic databases, after removing duplicates, there were 407 unique articles, 32 were eligible for full-text review after title and abstract screening, and 11 articles fit inclusion criteria for final data abstraction  and the lowest was 5 (n=1). All interventions reported inclusion criteria, described group assignment, reported baseline and group comparisons, and reported measured outcomes. Most interventions detailed sampling methods (n=7) [n=7) [n=5) [n=4) [n=2) [Among the nine included interventions, the highest quality score assigned was 9 (ds (n=7) –31, prov7) [n=7) –31, and 7) [n=5) , 32, 33.5) [n=4) , 32, 33 4) [n=2) , 28. No n=7) [n=6) [Descriptions of the interventions are detailed in Table n=7) , 30–34 wn=7) , 26, 29.n=7) –27. Two n=7) , 31. Thrn=7) , 28, 29.n=7) , 26. Twon=7) , 32, 33.We assessed the extent to which the intervention strategies and relevant outcomes aligned with ASCO guidelines’ six expectations of fertility preservation service provision  included strategies to assist providers in initiating discussion of potential treatment effects on fertility and measured the occurrence of these discussions as an outcome [ outcome , 27–34.  outcome . Additio outcome .Guideline Element 2: Providers discuss fertility preservation with patients. Over half (n=5) of the interventions noted discussions regarding fertility preservation approaches [proaches –29, withproaches , 26. Theproaches –27 and pproaches , 28, 29.Guideline Element 3: Provide fertility preservation services as early as possible prior to the patient beginning treatment. Only four interventions specified that discussions occurred prior to treatment, and all four mentioned this as part of an intervention strategy but not as an outcome [ outcome , 29, 31.Guideline Element 4: Discussions of fertility effects and preservation be documented. Four interventions noted that fertility-related discussions were documented [cumented , 29–31, cumented . In one Guideline Element 5: Referral to fertility preservation services. In the majority of interventions, providers referred patients to reproductive specialists either to discuss fertility preservation options or to receive fertility preservation treatment (n=8) [nt (n=8) –31, 34. Guideline Element 6: Refer patients for psychosocial services. This was noted in only two interventions , 31 withAll interventions except one  were muln=4) [When considering inter-level mechanisms for conceptual clarity, approximately half of the interventions included stakeholders’ views on the assumed process chains being targeted (n=4) , 31, 34.When considering methodologic pragmatism, all interventions were implemented in real world contexts as they took place in cancer centers, healthcare practices, and fertility clinics. Across the targeted levels of influence, seven interventions considered the sociodemographic characteristics of the patients as background factors , 31, 34,Only three of the 11 interventions discussed sustainability –28. ExamWe reviewed fertility preservation intervention studies targeting reproductive-aged women to assess their alignment with ASCO practice guidelines; and whether they considered multilevel influences on service provision, effectiveness, and sustainability for healthcare system integration. Previous studies have shown that not adhering to evidence-based guidelines leads to practice variation and subsequently to a suboptimal quality of care and quality of life in survivors .We found very few fertility preservation interventions included comparison groups which limits the rigor of the current evidence base. This review additionally identified gaps in the provision of ASCO guideline-concordant care. Our review suggests that while most interventions targeted some elements of the ASCO guideline, overall guideline adherence was limited and variable. Moreover, some interventions employed guideline relevant strategies but did not assess related outcomes. In these cases, it was unclear whether the interventions were effective in promoting guideline adherence.The variability in alignment with ASCO guidelines may be associated with health care provider specialty and practice setting. Cancer care is provided by an interdisciplinary team of providers who see patients at varying points in the diagnosis and treatment process. Mapping fertility preservation intervention strategies to the standard course of care and the interchange between different care providers should be considered and tested in future interventions.Additionally, greater attention should be given to specifically linking intervention strategies and evaluation outcomes to clinical practice and national guidelines. For example, Anazodo et al (2019) suggested the following: that the role and scope of practice for fertility care be defined for all healthcare providers, communication paths between different healthcare providers be mapped, discussions be initiated in a specified time frame, patients receive high quality communication to convey fertility risk and preservation options in different formats and at appropriate literacy levels; and that all communications and referrals to supportive care be documented . InterveProvision of fertility preservation services also must be contextualized as it relies on individual , interpersonal , organizational systems , and societal . Thus, fertility preservation interventions must consider multilevel frameworks in developing strategies and evaluating outcomes. While we found that most interventions targeted at least two levels of influence, all analyses were conducted at the patient-level. Future intervention trials should be designed to report hierarchical outcomes. For example, applying innovative implementation science frameworks for intervention development, analysis, and evaluation efforts would be useful in this endeavor.In considering the multilevel complexity of fertility preservation, it was surprising that very few interventions reported engaging stakeholders at any level of influence in guiding intervention development. Among the few that did, some engaged patient focus groups and structured interviews, others targeted health care providers. Further, stakeholder input was often considered prior to intervention implementation not during and after. Without continued stakeholder engagement to assess intervention progress and whether modifications need to be made, this could limit sustainability.To develop interventions that are relevant, feasible, and sustainable, a more integrated and multi-level stakeholder engagement effort will be needed to identify linkages between levels of influence and mechanisms that might be amenable to intervention . For exaThere are some limitations to this review that warrant attention. First, we based our guideline alignment on ASCO clinical practice guidelines. It is notable that there are not yet a single set of unified guidelines for oncologists and fertility specialists for addressing fertility preservation . SeveralBy 2023, the WHO estimates that 1.4 million reproductive-aged women will be diagnosed with cancer annually. As a result of scientific advances, increases in cancer survival rates have also led to the rising recognition for the field of Oncofertility and the need for fertility preservation. As seen in the literature, fertility preservation is still relatively new and evolving clinical area at the intersection of the fields of oncology and reproductive endocrinology. Yet, we can anticipate based on other new treatment implementation that systematic processes for delivering these services equitably will be needed . This reESM 1(DOCX 43 kb)


The liver was identified as a primary target organ for the chemo-radiological effects of uranyl acetate (UA). Although the anti-oxidant and anti-apoptotic properties of gallic acid (GA) make it a promising phytochemical to resist its hazards, there is no available data in this area of research.To address this issue, eighteen rats were randomly and equally divided into three groups. One group was received carboxymethyl cellulose (vehicle of GA) and kept as a control. The UA group was injected intraperitoneally with UA at a single dose of 5 mg/kg body weight. The third group (GA + UA group) was treated with GA orally at a dose of 100 mg/kg body weight for 14 days before UA exposure. UA was injected on the 15th day of the experiment in either the UA group or the GA + UA group. The biochemical, histological, and immunohistochemical findings in the GA + UA group were compared to both control and UA groups.The results showed that UA exposure led to a range of adverse effects. These included elevated plasma levels of aspartate aminotransferase, lactate dehydrogenase, total protein, globulin, glucose, total cholesterol, triglycerides, low-density lipoprotein cholesterol, and very-low-density lipoprotein and decreased plasma levels of high-density lipoprotein cholesterol. The exposure also disrupted the redox balance, evident through decreased plasma total antioxidant capacity and hepatic nitric oxide, superoxide dismutase, reduced glutathione, glutathione-S-transferase, glutathione reductase, and glutathione peroxidase and increased hepatic oxidized glutathione and malondialdehyde. Plasma levels of albumin and alanine aminotransferase did not significantly change in all groups. Histopathological analysis revealed damage to liver tissue, characterized by deteriorations in tissue structure, excessive collagen accumulation, and depletion of glycogen. Furthermore, UA exposure up-regulated the immuno-expression of cleaved caspase-3 and down-regulated the immuno-expression of nuclear factor-erythroid-2-related factor 2 in hepatic tissues, indicating an induction of apoptosis and oxidative stress response. However, the pre-treatment with GA proved to be effective in mitigating these negative effects induced by UA exposure, except for the disturbances in the lipid profile.The study suggests that GA has the potential to act as a protective agent against the adverse effects of UA exposure on the liver. Its ability to restore redox balance and inhibit apoptosis makes it a promising candidate for countering the harmful effects of chemo-radiological agents such as UA. The enduring presence of depleted uranium (DU) in bio-ecological systems, its diverse entry routes, magnification through the food chain, and the compounded effects of both metallic and radiation toxicities , 2 estabThese challenges are driving a new wave of research focused on natural biological approaches to mitigate chemo-radiological risks posed by UA. Our laboratory demonstrated the effectiveness of thymoquinone and N-acetylcysteine against UA-induced testicular damage in rats, primarily through their anti-apoptotic and cytoprotective mechanisms rather than their antioxidant properties . SubstanUA dihydrate (purity ≥ 98% and molecular weight 424.15 g/mol) was purchased from Sigma-Aldrich Company . GA (purity ≥ 99%) was obtained from Sd Fine Chem. Limited Company, India. Carboxymethyl cellulose (CMC) 98% CAS: 9005-64-5 was obtained from Alpha Global Search Company, New York, USA.ad libitum.A total of 18 adult male Wistar rats were used for this study. The rats were purchased from the Egyptian Company for Production of Vaccines, Sera, and Drugs, Egypt, and housed under natural light/dark cycles, at a temperature of 20–25 °C, and relative humidity of 55.0 ± 5.0%. They were provided with commercial pelleted feed and water After a one-week acclimatization period, the rats were randomly divided into three groups, each consisting of six animals. The control group received carboxymethyl cellulose (CMC); the vehicle of GA. The second group (UA group) received an intraperitoneal injection of UA at a single dose of 5 mg/kg body weight . The thiAt the end of the experimental period, blood samples were collected from the retro-orbital sinus after overnight fasting. Plasma was separated by centrifugation at 3000 rpm for 10 min and stored at -20 °C for subsequent biochemical analyses. Rats were euthanized by cervical dislocation under anesthesia induced by intraperitoneal injection of sodium thiopental. Half of the liver was promptly excised, homogenized in 1 ml of 0.1 M phosphate buffer (pH 7.4), and centrifuged at 10,000 rpm for 15 min using IKA Yellow line DI homogenizer  to give 10% w/v homogenate. The homogenates were centrifuged at 10.000 rpm for 15 min, and the resulting supernatants were frozen at -20 °C for measurement of oxidant/antioxidant parameters. The other half of liver was fixed in 10% neutral buffered formalin for histopathological evaluation. The experimental procedure was represented in Fig. Plasma alanine aminotransferase (ALT) , aspartate aminotransferase (AST) , albumin level , total protein , glucose , total cholesterol (TC) , triglyceride (TG) , and high-density lipoprotein cholesterol (HDL-C)  were assessed according to the manufacturer’s instructions using commercial kits provided by Egyptian Company for Biotechnology Company, Egypt. Plasma low-density lipoprotein cholesterol (LDL-C) was determined using the Friedewald formula: LDL-C = TC – HDL-C – [TG/5] . Plasma Total globulins were calculated by subtracting the obtained albumin level from the obtained total proteins level . Plasma Liver sections were fixed in 10% neutral buffered formalin, processed using the paraffin-embedding technique, and then sectioned for staining. Hematoxylin and eosin stain was used for general histological examination , Picrosi2O2 for 10 min. Each section was put in a blocking solution at room temperature for one hour. The primary cleaved caspase-3 antibody (1:1000)  and anti-Nrf2 antibody (1:500)  were then added for 24 h, followed by the secondary antibodies (1:5000) for two hours. After establishing the reaction with 3,3′-diaminobenzidine for 2–3 min, the sections were stained with hematoxylin for 2–5 min [Formalin-fixed liver tissues were put in 10% neutral buffered (pH 7.2). Paraffin-embedded tissues were sectioned, cleared, and rehydrated in a grade of ethanol solutions (100% − 70%) and rinsed in water. Extraction of antigens was done by boiling the slides in 1 mM ethylenediaminetetraacetic acid for 10 min, and emerging sections in 3% H 2–5 min .p < 0.05 were considered to be statistically significant.Data were represented as mean ± standard error of the mean (SEM). The results were analyzed by one-way analysis of variance (ANOVA) followed by Duncan post-test using SPSS program version 16 . Differences of Exposure to UA resulted in a significant increase in AST, LDH, total protein, globulin, and glucose, with no significant change in ALT and albumin. Supplementation with GA before UA intoxication normalized the AST, LDH, and glucose, although total protein and globulin became higher than the control group. There was no significant change when comparing ALT and albumin in the UA group with the GA + UA group Table . UA-chalA significant reduction was observed in plasma TAC in the UA group compared to the control group. Liver tissues from UA-exposed rats demonstrated a significant increase in MDA and GSSG and a significant decrease in NO, SOD, GSH, GST, GR, and GPx. GA supplementation effectively returned plasma TAC and hepatic MDA, SOD, GSH, and GST to the control levels. The hepatic GR and GPx in the UA + GA group were significantly improved compared to the UA group, but they were still significantly lower than the UA group. The hepatic NO in the GA + UA group was significantly higher than the control group. The hepatic GSSG was significantly reduced in the GA + UA group but still significantly higher than the control group Table .Hematoxylin and Eosin staining of liver tissue from the control group revealed normal structure Fig. a,b. The Examination of collagen fibers by Picrosirius red stain in the control group showed a tiny amount around the central vein Fig. a. Huge aGlycogen content examination by PAS stain in the control group showed a great amount of glycogen content Fig. a. NoticeImmunohistochemical analysis of cleaved caspase-3 revealed a negative immunoreaction in the control group Fig. a, contraPositive immunoreaction of Nrf2 was observed in the control group Fig. a, while 2+ bursts that activate caspases [The significant increase in plasma AST and LDH activities mirrors observations from a previous study . In our caspases . Converscaspases . The hepcaspases –31.The UA-associated hyperglobulinemia in our experimental irradiated rats aligns with increased serum immunoglobulin levels observed in orally supplemented mice . This suSimilar to gamma-irradiated rats , our expde novo lipid biosynthesis pathways, and excessive lipids are secreted in lipoproteins or stored in lipid droplets [The disturbances in lipid profile observed in the UA group are similar to the findings in gamma-irradiated rats . Elevatedroplets . Hypergldroplets , 57. Perdroplets . Hyperlidroplets . The curdroplets . Factorsdroplets .The substantial increase in lipid peroxidation end products after UA intoxication concurs with findings by Yuan et al. . UA stimMatched with the depletion of NO in the testicular tissues of UA-exposed rats , our finSimilar to the findings of Hao et al.  and PourTotal antioxidant capacity (TAC) provides a holistic view, accounting not only for the sum of individual antioxidants but also for their complex interactions . The norvia Fenton redox reactions, and directly through covalent binding to DNA [The hepatic histoarchitectural deteriorations induced by UA exposure are consistent with other reports , 91. Theg to DNA . GA excrg to DNA . The antg to DNA . Scavengg to DNA  underliePre-treatment of UA-exposed rats with GA efficiently restored the liver’s redox stability and cyto-functionality by inhibiting lipid peroxidation, up-regulating Nrf2, and suppressing the apoptotic cascade. This discovery holds significant value in guiding the scientific community toward recognizing the beneficial role of natural phytochemicals in mitigating the health risks associated with DU exposure. Further studies are highly recommended to highlight the molecular mechanisms underlying the protective effects of GA against UA intoxication.


Lactiplantibacillus plantarum, Lentilactobacillus buchneri, Lacticaseibacillus paracasei, and Pediococcus acidilactici) and LI2 (containing Lactiplantibacillus plantarum and Lentilactobacillus buchneri). The microbial community, LAB synergy, and cleaner fermentation were analyzed at 1, 3, 6, 15, 35, and 90 days of ensiling. The LAB inoculant improved fermentation quality, with significantly (P < 0.05) lower pH, ammonia nitrogen content, and gas production and higher lactic acid and acetic acid contents than those of the control. Enterobacteriaceae was the main bacterial community in early stage of fermentation, which utilizes sugar to produce CO2 gas, causing dry matter (DM) and energy loss. As fermentation progressed, the microbial diversity decreased, and the microbial community shifted from Gram-negative to Gram-positive bacteria. The inoculation of multispecies LAB displayed community synergy; Pediococcus acidilactici formed a dominant community in the early stage of fermentation, which produced an acid and anaerobic environment for the subsequent growth of Lentilactobacillus and Lacticaseibacillus species, thus forming a LAB-dominated microbial community. The predicted functional profile indicated that the silage inoculated with LI1 enhanced the carbohydrate metabolism pathway but inhibited the amino acid metabolism pathway, which played a role in promoting faster lactic acid production, reducing the decomposition of protein to ammonia nitrogen, and improving the fermentation quality of silage. Therefore, oat silage can be processed to high-quality and cleaner fermented feed by using an LAB inoculant, and LI1 showed better efficiency than LI2.To investigate community synergy of lactic acid bacteria (LAB) and cleaner fermentation of oat silage, oat silages were prepared with or without (control) commercial LAB inoculants LI1 (containing IMPORTANCE Oat natural silage is rich in Enterobacteriaceae, increasing gas production and fermentation loss. Lactic acid bacteria interact synergistically to form a dominant community during ensiling. Pediococci grow vigorously in the early stage of fermentation and create an anaerobic environment. Lactobacilli inhibit the harmful microorganisms and result in cleaner fermentation of oat silage. Avena sativa) is a dual-purpose crop, widely planted in Europe, North America, and northern China for food and feed production , resulting in greenhouse gas emissions and losses of DM and energy from the ensiled material  by the inoculants, the ensiling duration, or their interaction. DM loss increased significantly (P < 0.05) with ensiling duration. Higher DM loss was detected in LI2-treated silage on average in comparison with the control silage, whereas DM loss in LI1- and LI2-treated silages had no significant difference. The inoculant, ensiling duration, and their interaction altered (P < 0.05) lactate buffer capacity (LBC), water-soluble-carbohydrate (WSC) content, and LAB and aerobic-bacterium counts in silages. The LBC of fresh oat was 250.84 meq/kg DM. Overall, a lower level (P < 0.05) of LBC was observed for LI2-inoculated silage than for LI1- and control-treated silages. The level of LBC markedly varied during the ensiling process. The LI1-treated silage had higher (P < 0.05) contents of WSC, while the control and LI2-inoculated silages showed no difference in WSC contents. The WSC content was significantly (P < 0.05) influenced by the ensiling duration. However, at 1, 3, 6, and 90 days of fermentation, the WSC content of LI1-inoculated silage was significantly (P < 0.05) greater than that of the control and LI2 silages. The inoculant and ensiling duration did not alter the content of crude protein (CP) in silages. However, the contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) altered due to ensiling duration. In particular, the NDF content in the LI1-inoculated silage significantly increased at 90 days compared to 1, 3, 6, and 15 days of ensiling. The numbers of yeast and aerobic bacteria were lower (P < 0.05) in LI1-inoculated silage than control and LI2-inoculated silage. Moreover, the numbers of yeast and aerobic bacteria decreased (P < 0.05) with increase in ensiling duration. Coliform bacteria in fresh oats exceeded 7 log10 CFU/g FM, declined to <5 log10 CFU/g FM in inoculated silage on the 1st day of ensiling, and disappeared after the 3rd day of fermentation, while coliform bacteria of control silage were detected continuously on the 3rd day of fermentation.Data on the nutrient composition, microbial community, and diversity index of pre-ensiled oat are summarized in P < 0.05) influenced pH and acetic acid, ammonia N, and gas production of oat silages (P < 0.05) pH than control and LI2-inoculated silages in this investigation. The pH values in control and LI2-inoculated silages gradually decreased, reaching their lowest levels at 15 days of ensiling, while they significantly increased from 15 to 90 days. In contrast, the pH of LI1 silages rapidly decreased to below 4.0 at 3 days of ensiling and remained at their lowest level (<4.0) until 15 days. Moreover, the pH values of LI1 silages at 35 and 90 days were lower (P < 0.05) than those of LI2 silages. Ensiling duration and interaction of ensiling duration and inoculant significantly (P < 0.05) affected lactic acid concentration. Unexpectedly, lactic acid and acetic acid contents did not vary between LI1- and LI2-inoculated silages. However, the lactic acid and acetic acid contents were significantly (P < 0.05) higher in the inoculated silages than in the control. The lactic acid content in LI1-inoculated silages remained high (>51 g/kg DM) at 1 to 6 days of fermentation and then significantly (P < 0.05) declined, reaching its lowest level at 90 days of ensiling. Consistently, on 1 and 3 days of ensiling, lactic acid content in the LI2 treatment was significantly (P < 0.05) lower than that in LI1 treatment; nevertheless, lactic acid content between LI1and LI2 treatments had no significant difference (P > 0.05) on 6 and 15 days of ensiling. The acetic acid contents in all treatments consistently increased during ensiling. Propionic acid content ranging from 2 to 9 g/kg DM was detected in LI1-inoculated silage at 35 and 90 days of ensiling. Inoculations of the LAB decreased the ammonia N concentrations of oat silages. On average, the ammonia N concentration gradually increased during ensiling. At 1, 3, 15, and 35 days of ensiling, the LI1- and LI2-inoculated silages had significantly lower (P < 0.05) ammonia N concentrations than the control. However, at 6 and 90 days of ensiling, the LI1-inoculated silage had significantly lower (P < 0.05) ammonia N concentrations than control and LI2-inoculated silages. The gas production was lower (P < 0.05) in LI1-inoculated silage than in control and LI2-inoculated silages. The gas production of each treatment increased rapidly in the initial phase of fermentation but gradually decreased with the prolongation of fermentation.The inoculants, ensiling duration, and their interaction significantly ( silages . The LI1P < 0.05) influenced numbers of operational taxonomic units (OTUs) and Chao1, Shannon, and Simpson indexes of silages. In general, lower numbers of OTUs and lower Chao1 and Shannon indexes were detected in the LI2 silages than control and LI1 silages. The average numbers of OTUs and Chao1 indexes gradually decreased until the 35th day but increased significantly after 90 days of ensiling. Higher Shannon indexes were observed in LI1 silages than in control and LI2 silages at 15 days of fermentation. After 90 days of ensiling, control silages had the highest Shannon index compared to LAB-inoculated silages. At 6 and 15 days of fermentation, lower Simpson indexes were observed in the LI1 silages. High Good’s coverage (>99.70%) was detected in all samples.Proteobacteria and unclassified bacteria were both dominant in the FM group, whereas Proteobacteria and Firmicutes played a predominant role in silages. The relative abundance of Firmicutes increased and Proteobacteria declined during the ensiling process. Moreover, the LI1 inoculant decreased the relative abundance of Proteobacteria and increased that of Firmicutes after 1 and 3 days of ensiling. However, the LI2-inoculated silage showed a low relative abundance of Proteobacteria and a high relative abundance of Firmicutes at 6 and 15 days of ensiling compared to control and LI1 silages. At the later stages of ensiling (35 and 90 days), the relative abundances of Firmicutes in LI1 and LI2 treatments reached more than 85%, and there was no significant difference between them.Pantoea dominated the fresh oat, with a relative abundance of more than 57% and 27%, respectively. The relative abundance of Lactobacillus in the fresh oat was less than 1%. Control silage had a higher (P < 0.05) percentage of members of the family Enterobacteriaceae than the LAB-inoculated silages at 1 and 3 days of ensiling. Enterobacteriaceae were gradually replaced by Lactiplantibacillus and Lentilactobacillus in the control group after 6 days of ensiling. Lentilactobacillus and Lactiplantibacillus play a predominant role at 90 days of ensiling in control group. Relative abundances of Enterobacteriaceae also decreased during ensiling in inoculated silages. The relative abundance of Enterobacteriaceae in control treatment at 1, 3, and 90 days of ensiling was significantly (P < 0.01) greater than that in the LAB-inoculated treatments (Enterobacteriaceae in the LI1 treatment was the same as in the control but higher (P < 0.01) than in the LI2 treatment at 6, 15, and 35 days of ensiling. At the 1st day of fermentation, Enterobacteriaceae and Pediococcus were dominant in the LI1 group, while only Pediococcus dominated at 3 days of fermentation and inhibited the growth of Enterobacteriaceae. Much more interestingly, Lactiplantibacillus was just starting to become the predominant genus, followed by Enterobacteriaceae and Pediococcus, at 6 days of ensiling in the LI1 group, and during the end period of ensiling, Lentilactobacillus was the most predominant, followed by Lactiplantibacillus. Lacticaseibacillus showed significantly higher (P < 0.001) relative abundance at only the late stage of ensiling  in the LI1 group than in the LI2 and control groups. Interestingly, for the LI2 group, the silage was dominated by Enterobacteriaceae at 1 day of ensiling. However, Lactiplantibacillus became the most predominant in LI2 at 3, 6, and 15 days of ensiling and then was replaced by Lentilactobacillus at 35 and 90 days of ensiling.eatments . NeverthEnterobacteriaceae had a positive correlation with gas production, and Enterobacter had negative correlation with DM loss; Lentilactobacillus had a positive correlation (P < 0.05) with pH value, acetic acid, ammonia N, and DM loss and a negative correlation (P < 0.05) with lactic acid, WSC, and gas production in all groups. This result was the same as that for Lacticaseibacillus from LI1 and LI2 treatments and Pediococcus only from the LI2 treatment and the opposite of that for Pediococcus from the LI1 treatment. Lactiplantibacillus was negatively correlated with LBC and gas production of the LI1 treatment and pH and acetic acid of the LI2 treatment. In addition, P < 0.05) in the LI2-inoculated silage at 3, 6, and 35 days of ensiling. In the LI1 treatment, the amino acid metabolism was significantly lower (P < 0.05) than that in control and LI2 treatments at 1, 3, and 6 days of ensiling. The carbohydrate metabolism pathway was more (P < 0.05) dominant in the LI1-treated silages at 3 days than in LI2 silages, while the opposite result was found for LI1 at 6 days of ensiling. In general, at the early stage of ensiling (before 6 days), there were at least 6 metabolic pathways with significant differences at each ensiling day, but there were few metabolic pathways with significant differences in the late period of ensiling.Lentilactobacillus buchneri, resulting in higher fermentative losses  for preferred quality of silage preparations , which have been reported to speed up the fermentation during initial periods of fermentation by more efficiently shifting WSC into lactic acid, resulting in a rapid drop in pH . Chen et al.  during ensiling not only is bad for the environment but also causes dry matter loss in silage. A previous study found that approximately 6.0 L/kg FM of gas was produced after 60 days of ensiling  and mainoduction . The hig with GP . It coulLacticaseibacillus paracasei, Pediococcus acidilactici, Lentilactobacillus buchneri and Lactiplantibacillus plantarum) improved the fermentation quality of silages. Generally, silages with good fermentation quality have low alpha diversity  in early stage of ensiling. Our study indicated that Pediococcus had a negative correlation with DM loss but a positive correlation with LA in LI1. Inoculation of Pediococcus acidilactici enhanced lactate production and DM recovery. Therefore, LAB exert microbial synergy to form a dominant community, effectively inhibiting the growth of undesirable microbes, gas production, and fermentation loss.All the results above verify that LI1 improved the fermentation quality of oat silage. Similar findings were showed by Li et al.  and Na eiversity . In the bacteria . This rebacteria  to 3. Robacteria  reportedme cases . In the me cases  suggesteme cases  and Ogunme cases  also fouion. Cai  reportedEnterobacter in the LI1-inoculated silage  and LI2 , were utilized for the silage preparation of oat. The viable counts of LAB in inoculants were ≥1.0 × 1011 CFU/g for LI1 and ≥1.3 × 1010 CFU/g for LI2.Two commercial multispecies inoculants, LI1 (Bonsilage containing Avena sativa L. cultivar Qinghai444) was cultivated in an experimental field of the Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences , located at the foot of Yinshan Mountain on the Tumochuan plain in Inner Mongolia, China. The crop was reaped at the milk stage with approximately 300 g/kg DM on 24 July 2021 and immediately chopped into 1- to 2-cm lengths. Silages were prepared with a laboratory fermentation system  for 10 min and then filtered through 4 layers of cheesecloth. Extracts of sample were used for pH measurement with a pH meter . Then, the extract sample was filtered through a 0.22-mm filter for analysis of organic acids and ammonium nitrogen by high-pressure liquid chromatography (HPLC) and the Kjeltech autodistillation system  as described by Cai . For mici et al. . The coui et al. . LBC wasi et al. . The toti et al. .The gas volume in silage bags was measured in a 5,000-mL measuring cup kept in a constant-temperature water bath (25°C) as described by Chen et al. . The difDNA extraction and 16S rRNA gene sequencing data processing were performed by LC-Bio Technology Co., Ltd. . Briefly, the library quantification kit for Illumina  was used to assess the size and quantity of the amplicon. Then, the NovaSeq PE250 platform was used to sequence the library. Alpha diversity was analyzed by QIIME (version 1.9.1), and the relative abundances of microbial communities were measured at the phylum and genus levels using the NT-16S with a confidence threshold of 70%. Bacterial function prediction was proof checked from the KEGG database using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt2 v2.3.0_b).P value was lower than 0.05. One-way ANOVA bar plots were made to demonstrate which genera contributed to the changes in the bacterial community. The relationships between the bacterial taxonomic profile and silage quality variables were determined by calculating the Pearson correlation coefficients and were plotted by using the online Majorbio Cloud platform (www.majorbio.com).All the reported results are the means from four replicates. Chemical composition, fermentation quality, alpha diversity, and microbial count data were analyzed using two-way analysis of variance (ANOVA) with Duncan’s multiple-range test and with silage inoculant and ensiling time as the main variables by SAS version 9.1 . Differences between results were considered statistically significant when the SRP410438).Raw sequencing files and associated metadata have been deposited in NCBI’s Sequence Read Archive (