Ovarian tissue cryopreservation involves freezing and storing of surgically retrievedovarian tissue in liquid or vapour nitrogen below –190°C. The tissue can be thawed andtransplanted back with the aim of restoring fertility or ovarian endocrine function. Thetechniques for human ovarian tissue freezing and transplantation have evolved over thelast 20 years, particularly in the context of fertility preservation in pre-pubertalcancer patients. Fresh ovarian tissue transplantation, using an autograft or donortissue, is a more recent development; it has the potential to preserve fertility andhormonal function in women who have their ovaries removed for benign gynaecologicalconditions. The techniques of ovarian tissue cryopreservation and transplantation haveprogressed rapidly since inception; however, the evidence on the success of thisintervention is largely based on case reports and case series.The aim of this study was to systematically review the current evidence byincorporating study-level and individual patient-level meta-analyses of women whoreceived ovarian transplants, including frozen–thawed transplant, fresh or donorgraft.The review protocol was registered with PROSPERO (CRD42018115233). A comprehensiveliterature search was performed using MEDLINE, EMBASE, CINAHL and Cochrane CentralRegister of Controlled Trials from database inception to October 2020. Authors were alsocontacted for individual patient data if relevant outcomes were not reported in thepublished manuscripts. Meta-analysis was performed using inverse-variance weighting tocalculate summary estimates using a fixed-effects model.The review included 87 studies (735 women). Twenty studies reported on ≥5 cases ofovarian transplants and were included in the meta-analysis (568 women). Fertilityoutcomes included pregnancy, live birth and miscarriage rates, and endocrine outcomesincluded oestrogen, FSH and LH levels. The pooled rates were 37% (95% CI: 32–43%) forpregnancy, 28% (95% CI: 24–34%) for live birth and 37% (95% CI: 30–46%) for miscarriagefollowing frozen ovarian tissue transplantation. Pooled mean for pre-transplantoestrogen was 101.6 pmol/l (95% CI: 47.9–155.3), which increased post-transplant to522.4 pmol/l . Pooledmean of pre-transplant FSH was 66.4 IU/l (95% CI: 52.8–84), which decreasedpost-transplant to 14.1 IU/l .The median time to return of FSH to a value <25 IU/l was 19 weeks . The median duration of graft function was2.5 years . The analysisdemonstrated that ovarian tissue cryopreservation and transplantation could restorereproductive and hormonal functions in women. Further studies with larger samples ofwell-characterized populations are required to define the optimal retrieval,cryopreservation and transplantation processes.Ovarian tissue cryopreservation and transplantation may not only be effective inrestoring fertility but also the return of reproductive endocrine function. Althoughthis technology was developed as a fertility preservation option, it may have the scopeto be considered for endocrine function preservation. There is an increase in the number of young girls and women diagnosed with cancer globally.In 2020 alone, ∼0.9 million cases of new cancers were diagnosed worldwide in women aged0–39 years (crude and age-standardized incidence rate per 100 000) . Ground-Fresh ovarian transplants have also been used to establish endocrine function and fertilityin recipients with premature ovarian insufficiency (POI). The first successful fresh humanovary transplantation was reported in 2005 in monozygotic twins .The review protocol was registered with PROPERO (CRD42018115233) on 15 November 2018 terms: ovarian, cryopreservation, transplantation, fresh transplantation,pregnancy, live birth and ovarian function. The search strategy for MEDLINE is availableas Inclusion and exclusion criteria for studies were established before the literaturesearch was conducted. Study selection was carried out by two independent reviewers (H.K.and R.M.). Any disagreements about inclusion were resolved by consensus or arbitration bya third reviewer (L.C.).All studies that reported fertility or endocrine outcomes from either fresh orfrozen–thawed ovarian transplants for at least one participant were included. Thesecomprised cohort studies, observational studies, case reports, case series, conferenceabstracts and grey literature . Commentaries, editorials, correspondence and letterswere excluded. When more than one publication originated from the same centre, populationor cohort, reports were individually assessed to identify and remove duplicates. This wasalso double-checked by cross referencing and contacting the authors directly forclarification, and with the most recent or complete publication being selected.For each patient identified and included in the study, the aim was to collect data onovarian reproductive function, such as pregnancy, live births and miscarriages, andendocrine function, such as oestrogen, progesterone, FSH, LH and anti-Müllerian hormone(AMH) levels. The return of hormonal function was defined by an increase in oestrogen, anda decrease in FSH and LH along with return of menstruation. Although the accuracy ofvalues of FSH has not been assessed robustly in the literature, for the purpose of thisreview, the ESHRE guidelines on management of women with POI is used as a reference .Mean and SD, or median, interquartile range (IQR) and range were used to summarize thedata from studies that reported ≥5 cases. All the outcomes were converted to a standardunit for analysis; for FSH and LH, IU/l and for oestrogen, pmol/l. The pooled outcomeswere calculated as mean difference (MD) for FSH, LH and oestrogen using the inversevariance method with 95% CIs and a fixed effects model . Risk Ra2 test was used and significance was set atP < 0.05, where I2 was used forquantifying heterogeneity  were includedin the review. We were able to extract IPD for 355 women and study-level data for 380 women.Studies that reported ≥5 cases of ovarian transplants were included in the statisticalanalysis (568 women). The characteristics of studies included in the meta-analysis arereported as Eighteen studies (547 women) were included in meta-analysis for reproductive outcomesand at least one pregnancy was reported in 184 women . The preSeventeen studies (539 women) were included in the meta-analysis and at least one livebirth was reported in 134 women .The livFifteen studies reported miscarriage rates. The mean age at cryopreservation in womenwho had miscarriages was 27.8 years (SD: 5.8). Miscarriage rate for frozen transplantswas 37% (95% CI: 30–46%) and for fresh transplants was 33% (95% CI: 13–89%) as presentedin Eight studies  reportedI2 = 79% (P = 0.0001). Overall FSH levelspost-transplant were reported for 187 out of 735 women. A decrease in FSH below 25 IU/lwas achieved in 72% (135/187 women). The median time to return of FSH to a value<25 IU/l was 19 weeks .Eleven studies  reportedI2 = 0% (P = 0.64). Overall, LH valuespost-transplantation were described in 69 out of 735 women. A decrease in LH below15 IU/l was achieved in 46 out of 69 women (67%). The median time to return of LH to avalue <15 IU/l was 19.5 weeks .Six studies  reportedAdditionally, we were able to collate reproductive outcomes based on whetherparticipants achieved an FSH of ≤25 IU/l and compared to those who had FSH of>25 IU/l post-transplantation. It was found that an FSH of ≤25 IU/l was reported in128 women, 64 (50%) of whom were able to achieve at least one pregnancy and 44 (34%) atleast one live birth .For reproductive outcomes in relation to levels of oestrogen, we only assessed the dataof participants who were truly menopausal before transplantation (levels of oestrogen< 100 pmol/l) .Data poOnly one study provided enough data for AMH pre- and post-transplantation  women were reported to have resumed menstruation. The mediantime to return of menstrual activity was 18 weeks .The return of menstrual activity coincides with that of hormonal function .z = 4.08;I2 = 0%), as shown in z = 5.91; I2 0%).Through the literature search, we identified 45 fresh transplants, 11 of which used agraft from a donor (twin sister). Fifteen pregnancies and eight live births were reportedfor participants receiving fresh ovarian transplantation. Two studies that included fiveor more participants receiving fresh transplants were included in meta-analysis  and S2. Duration of the ovarian graft function was reported in 19 studies (181 women). In 15studies, the authors reported the exact duration (or median duration if more than onepatient was reported). The median duration of function was 2.5 years (IQR: 1.4–3.4 years),range: 0.7–5 years. The mean age at cryopreservation for this group of women was27.1 years (SD: 6.8). A further three studies, including 26 women with a mean age atcryopreservation 30.3 years (SD: 2.5), reported a range with pooled duration of functionbeing 1.2–7.7 years. A case series of three participants who received fresh ovariantransplants (patient’s own ovary) between the ages of 1–5 years experienced menarche aswell as 13–15 years of duration of function . Return of hormonal function is shown as a decrease in FSH and was lower in thegroup that had ovarian tissue frozen at ≤35 years of age   .Mode of conception was provided clearly in 276 pregnancies. It was noted that 199 (69%)pregnancies were conceived naturally, whereas ART was used for 90 pregnancies .Whether a participant received anti-cancer therapy before retrieval of ovarian tissuewas reported in 122 out of 735 participants. It was found that 56 out of 122 patients(46%) had received anti-cancer treatment before ovarian tissue cryopreservation.Thirty-five pregnancies and 24 live births were reported in these women. In women withlive births, 11 had Hodgkin’s lymphoma, 5 had non-Hodgkin’s lymphoma, 1 had microscopicpolyangiitis, 1 had acute myeloid leukaemia (AML) and 1 had Wilms’ tumour. Mean age atcryopreservation for women who received chemotherapy before tissue freezing and achievedlive birth was 29 years (SD: 6). We were able to perform meta-analysis on data from fivestudies that reported reproductive and endocrine outcomes based on whether the womenreceived anti-cancer treatment before cryopreservation or not. Although the results werenot statistically significant, a decrease in FSH, an increase in oestrogen and increasedpregnancy rates were noted in participants who did not receive anti-cancer therapybefore cryopreservation , and S5.The five most common cancers at participant level included breast cancer, cervicalcancer, non-Hodgkin’s lymphoma, ovarian cancer and sickle cell anaemia. There wasinsufficient IPD across the cohorts to be able to perform a meta-analysis and providepregnancies and live birth rates based on type of cancer. The sums of pregnancies andlive birth rates in women with the five most common cancers based on IPD are describedin There was inconsistency in how the amount of ovarian tissue transplanted was measured.The authors described the amount of the ovarian tissue in volume, strips, fragments,pieces, biopsies or sections. Not all authors reported a 3D measurement of the ovariangraft. Owing to the variation in size of the ovary and follicular count at baseline, itis not possible to estimate the optimal amount of tissue for achieving desirablereproductive and hormonal outcomes.In our review we found 13 cases in whom vitrification was used to cryopreserve ovariantissue  andS8.The surgical approach for retrieval of ovarian tissue was reported for 237 out of 735women, with 225 women (95% having had laparoscopy and 12 (5%) having had laparotomy). Asfor transplantation, surgical approach was reported for 323 women; 205 (64%) hadlaparoscopy and 95 (29%) had laparotomy. Other sites and techniques are listed in Of the 440 women, 175 had ovarian tissue transplanted onto their remainingpostmenopausal ovary. FSH levels pre- and post-transplantation were reported in 55participants, with mean FSH of 71.5 IU/l (SD 44.6) pre-transplant and 25.3 (SD: 28.5)post-transplantation. Oestrogen at pre-transplantation and post-transplant was reportedin 33 of the 175 participants. Mean oestrogen before transplant was 104.9 pmol/l (SD:143.7) and post-transplant 387.5 pmol/l (SD: 419.6). Fifty-two pregnancies and 34 livebirths have been recorded from transplant onto the remaining ovary.Ovarian tissue was transplanted onto the pelvic side wall or peritoneal pockets in 184women. Oestrogen levels at baseline and post-transplant were described in 22participants. The mean oestrogen level before transplant was 207.5 pmol/l (SD: 245.1)and post-transplant 1204.4 pmol/l (SD: 1164.3). FSH was reported in 25 participants,with mean pre-transplant FSH of 58.8 IU/l (SD: 38.5) and post-transplant 14.4 IU/l (SD:13.2). Forty-one pregnancies and 37 live births were reported.Fifty participants had their ovarian tissue transplanted in the pelvic peritoneum aswell as the remaining ovary. The mean oestrogen level pre-transplantation in theseparticipants was 172 pmol/l (SD: 144) and post-transplant 1922 pmol/l (SD: 3257.9),reported in 14 participants. FSH was reported in 24 participants with meanpre-transplant 84.7 IU/l (SD: 47.6) and post-transplant 20.5 IU/l (SD: 21.1).Twenty-eight pregnancies and 10 live births were reported.Eight participants received ovarian transplantation in three sites during oneoperation. FSH was reported in seven participants with a mean of 62.9 IU/l (SD: 28.1)pre-transplant and 10.8 IU/l (SD 8.5) post-transplant. There was insufficient data toreport on oestrogen post-transplantation. Four pregnancies and one live birth werereported. The rest of the transplant sites are described in the The included studies did not report any specific complications related to ovariantransplantation other than those of gynaecological laparotomy and laparoscopic surgery.The only complications reported thus far were those of skin infection and injury tosurrounding organs . There is evidence to suggest thatunilateral oophorectomy appears to reduce the age of menopause by 1–1.8 years . Although the results were reassuringin terms of decreasing FSH to a level of <25 IU/l and levels of oestrogen increasing to>200 pmol/l, it was not possible to make a comparison with frozen–thawed transplantsowing to such a small sample size.Furthermore, clinical heterogeneity of the studies included in the meta-analysis resultedin weakness in our analysis. To overcome this, we endeavoured to gather IPD from authorsand six centres agreed to provide this and further clarification. Collecting IPD was oneof the significant challenges of this project. In order to help authors who may have alack of time, funding or organizational support, we offered assistance in data collationand also sent them an outcome spreadsheet to assist with data collection . FinallyThrough previous publications and this review, we can conclude that ovarian tissuetransplantation has shown promising results in preserving ovarian reproductive andendocrine function. This procedure should no longer be considered experimental and itshould be offered to women who wish to preserve their fertility out of the researchcontext. The potential use of transplants in preserving hormonal function is an area thatneeds to be explored further. There is currently no robust guidance for clinicians orpatients about this procedure and its potential uses. With its potential use inalleviating menopausal symptoms and improving quality of life, there is an urgent unmetneed for a robust policy and standard. The results of this review will hopefully guideclinicians in advising women about the benefits and shortcomings of this procedure until aformal guideline is produced.Further data with larger studies that include participants with results that were notsuccessful need to be included to give an accurate success rate of this procedure.Furthermore, a robust guideline for the optimal size of the tissue graft and surgicalprocedure for retrieval and transplantation of ovarian tissue needs to be produced toguide centres with this technique. There is also an urgent unmet need for a worldwideregistry to provide a database that records all cases of ovarian transplantation. Finally,predefined outcome sets need to be determined to ensure all randomized control trials,cohort studies or case series report similar outcomes for ovarian transplantation.Human Reproduction Update online.The data underlying this article are available in the article and its online dmac003_Supplementary_DataClick here for additional data file.


The extended semen was chilled slowly to 5 °C within 2 h and equilibrated for 2 h, before frozen on the liquid nitrogen vapor and cryopreserved at −196 °C. The straws were evaluated before freezing and post-thawing for sperm motility, vitality and abnormality besides plasma-membrane and DNA integrities. The moisture, ash, protein, and fatty acid (FA) contents of chicken EY, quail EY and their mixture were analyzed. Sperm vitality, plasma membrane integrity and DNA integrity before freezing were significantly (P < 0.05) higher in quail EY than chicken EY and mixed EYs cryodiluent. The chicken EY extender significantly improved the vitality, plasma membrane and DNA integrities of post-thawed ram semen in comparison with quail EY or mixed EYs extenders. While, the post-thawing sperm abnormalities was lower (P ≤ 0.05) in quail EY than chicken EY and mixed EYs cryodiluent. The post-thawing sperm motion kinetics parameters were higher in quail EY than chicken EY and mixed EYs cryodiluent. The highest percentages of moisture, ash, saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) were detected in quail EY had. While, the highest percentages of fat, protein and polyunsaturated fatty acids (PUFAs) were detected in chicken EY. In conclusion, using of chicken EY can improve total motility, vitality, plasma membrane integrity and DNA integrity of cryopreserved ram semen. While, using of quail EY can improve sperm abnormalities and kinetic motion parameters of cryopreserved ram semen. Mixing chicken and quail EYs added no value for post-thawing ram semen parameters.The effects of adding mixed chicken and Japanese quail egg yolks (EYs) to the cryodiluent on the quality of ram semen before freezing and post-thawing were evaluated. Additionally, the composition of chicken and quail egg EYs and their mixture were analyzed for results explanation. The semen was collected from rams ( Cryopreservation of semen is one of the essential technologies in animal research for enhancing productive and reproductive efficiencies . Semen cDiluents, dilution, cooling, freezing, and thawing procedures all have a vital role in the success of ram semen cryopreservation , 7. The Egg yolk (EY) has a good effect as a non-permeable cryoprotectant at both cooling and sub-zero temperatures , 12. It The mechanism by which EY protects sperm cells during cooling and freezing remains un fully understood. In 1939, the protective effects of EY on sperm during freezing were discovered for the first time . HoweverThe chemical composition of the EYs of various avian species differs, particularly in terms of their cholesterol, fatty acid, and phospholipid contents , 24, whivia semen evaluation before freezing and post-thawing. Additionally, the composition of chicken and quail egg EYs and their mixture were analyzed for results explanation.Comparison among the efficacy of EYs of different avian species has started gaining momentum. Chicken and quail EYs have been reported to be better among many other avian species for cryopreservation of ram semen. Because of the relatively lower saturated fatty acids (SFA) ratio to polyunsaturated fatty acids (PUSFAs) in quail yolk , quail E−1 dry matter, respectively). All rams were healthy and passed the breeding soundness evaluation test including semen evaluation using computer-assisted sperm analysis (CASA) . The King Saud University Research Ethics Committee (REC) authorized the current project . This authorization is based on the advice of the Research Ethics Sub-Committee (minute number 7 and date 22/04/2021), as well as a suitable risk-to-benefit ratio and a study design that minimizes risks.This experiment was performed in the autumn on seven mature, fertile Najdi rams aged 2–4 years with an average body condition score of 3 . At the experimental farm, Department of Animal Production, King Saud University, Riyadh, Saudi Arabia (latitude 24° 48′ N and longitude 46° 31′ E), rams were sheltered in a covered yard within an open-sided barn. The daily energy and protein requirements of rams were met with commercial mixed pellets  was added. Three different diluents were prepared based on the type of egg yolk (EY): Tris chicken EY (C), Tris Japanese quail EY (Q), Tris chicken and Japanese quail EYs mixture (1:1) (CQ).Unless otherwise stated, all materials were obtained from Sigma . Tris-based buffer was prepared by dissolving 250 mM (3.028 g) Tris, 88.5 mM (1.78 g) citric acid, and 69.38 mM (1.25 g) fructose in 100 mL of distilled water . The buf® . The yellow and milky ejaculates that met the conventional parameters of 1.5 mL volume, 4 mass activity score, 80% sperm progressive motility, and 2 × 109 sperm mL−1 concentration were selected for further processing. The ejaculates were pooled to eliminate individual ram variations. Seven pooled ejaculates were utilized in this study.Semen samples were collected twice weekly from each ram using an artificial vagina. The color, density, volume, mass activity, sperm motility, and sperm cell concentration of each ejaculate were evaluated macroscopically and microscopically. Sperm motility and sperm cell concentration were automatically determined using a Sperm Class Analyzervia submerging in liquid nitrogen.In a water bath, various types of diluents were used to dilute the semen gradually in a dilution rate 1:4 . The diluted semen was gradually chilled within 2 h from 37 to 5 °C. The diluted cooled semen was loaded into 0.25 mL straws using a semiautomatic filling and sealing machine  and left at 5 °C for 2 h to allow for glycerol equilibration. After achieving equilibrium, the straws were frozen in liquid nitrogen vapor (5 cm above liquid nitrogen surface) for 15 min. The straws were then preserved at 196 °C ®  which is computer-assisted sperm analysis (CASA) system for semen analysis allows the accurate, repetitive and automatic assessment of the following sperm parameters: motility, concentration, morphology, DNA fragmentation and vitality  and post-thawing. The frozen semen was examined after at least 2 weeks of cryopreservation. Thirty seconds were spent thawing the frozen straws in a 37 °C water bath. The semen evaluation was done mainly using SCAvitality , 40. The® was used to evaluate sperm velocity in three ways  for each sperm. The average pathway velocity (VAP), straight-line velocity (VSL), and curvilinear velocity (VCL) of sperm movement were measured. For evaluating progressiveness on a relative scale, the linearity (LIN = VSL/VCL), straightness (STR = VSL/VAP), and wobble (WOB = VAP/VCL) metrics were utilized and reported as a percentage. The amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) were measured. The results were categorized according to velocity as rapid (VCL >75 μm/s), medium (45 < VCL < 75 μm/s), slow (10 < VCL < 45 μm/s) or static (VCL < 10 μm/s). The sperm presenting movement with a STR index ≥80% was considered progressive motile. The results were categorized as rapid progressive, slow progressive, non-progressive or static according to WHO 4th edition. The results were categorized as total progressive (summation of rapid and slow progressive), non-progressive or immotile according to WHO 5th edition.SCAUsing a FluoVit kit and protocol , the live/dead sperms were determined. Briefly, 10 μL of sperm sample was placed in a 1.5-ml Eppendorf tube, and 1 μL of warmed (37 °C) Hoechst trihydrochloride trihydrate stain was added. Five min were spent incubating this mixture at 37 °C. Then, 1 μL of warmed (37 °C) propidium iodide was added and stirred gently using a micropipette. Five min later, 10 μL of the stained sample was placed on a standard microscope slide with a cover glass and examined using fluorescence microscopy. A dead sperm displayed a fluorescent red hue, while a live sperm displayed a luminous blue hue. SCA vitality software was used to perform an automatic analysis. Each sample was tested with at least 200 sperms.® fixative/stain , and the excess stain was drained. The slides were then dipped once in distilled water for approximately 3 sec, put at an angle of 60–80 degrees to remove excess fluid, and allowed to air dry. All of the sperm components  were stained with varying blue intensities. Sperm abnormalities were automatically determined and recorded as head length (m), head width (m), head area (m2), head perimeter (m), acrosome (percent), elongation, ellipticity, regularity, rugosity, midpiece width (m), midpiece area (m2), distance (m), and angle (°). The stained smears were examined with SCA® abnormality software under a microscope at 600x magnification.The semen samples were placed on glass slides, and a conventional sperm smear was performed in duplicate and left to dry in the air. The slides containing sperm smears were placed vertically for 1–2 min on a staining tray with New Rapid SpermBlueA hypo-osmotic swelling test (HOST) was utilized as a supplemental test to the viability assessment technique to determine the functional integrity of the sperm plasma membrane. The experiment was carried out by incubating 10 μL of sperm with 100 μL of a 190 mOsm hypo-osmotic solution for 40 min at 37 °C in a 1.5-ml Eppendorf tube. On a warm slide, 100 μL of the sample was distributed with a cover slip. At least five distinct fields were utilized to count 200 sperms. The percentages of sperm with inflated and coiled tails were determined.® kit  was an in vitro diagnostic solution that allows the measurement of sperm DNA fragmentation for ram (Ovis aries). Halomax® offers a better evaluation of semen quality compared to a traditional method which ignore the most important valuable parameter for a breeder, which is the quality of the genetic material that the sperm cell will transmit to the oocyte. Elevated sperm DNA fragmentation has a negative impact on pregnancy rates and litter size. The evaluation was done according to the manufacturer's instructions. Briefly, the samples of sperm were diluted to a concentration between 5 and 10 × 106 mL1. The agarose gel from the kit was incubated at 90–100 °C for 5 min to fuse the agarose and then at 37 °C for 5 min in a water bath. Twenty-five microliters of the semen sample were added to the Eppendorf tube along with the gel and thoroughly mixed. Twenty microliters of the solution were deposited on a super-coated slide, then placed on a cool surface and covered with a 22x22-mm coverslip. Slides were refrigerated for 5 min at 4 °C to produce a micro-gel containing an implanted sperm. Carefully removing the coverslips, the slides were immersed for 7 min in the previously prepared acid solution (80 μL HCl in 10 mL of distilled water). The slides were then moved to a tray containing the lysing solution and incubated for 25 min, followed by rinsing with distilled water and dehydration in increasing ethanol concentrations for 2 min . The slides were stained with Giemsa dye or Wright stain, washed with tap water, and dried at 25 °C. Each slide was examined at 100 magnifications with a light microscope, and 200 sperms were scored. A sperm without fragmented DNA was placed in an agarose matrix and treated with lysing solutions to deproteinize the nucleus and generate the halos of dispersed DNA. The halos relate to the DNA loops loosely connected to the remaining nuclear structure (core). The sperm nuclei with fragmented DNA produce tiny or no halos of dispersed DNA, whereas the nuclei without fragmented DNA release DNA, forming large halos.Halomax−1, nitrogen was used as a carrier gas. The pressure of hydrogen and air was established at 0.5 kg cm−1. The injector and detector were both kept at 250 degrees Celsius. A comparison of retention durations and standards was used to identify the FA methyl esters. The fatty acid composition was expressed as a percentage of the total FAs  of EY were quantified using gas chromatography. FA methyl esters were separated utilizing a GC-9A equipped with a flame ionization detector and a silica capillary column. At 35 mL minotal FAs . For traotal FAs .jk = μ + Ak + ejk” where Yjki is the target variable, μ is the overall mean, Ak is the fixed effect of the treatment (avian egg yolk extender), and ejki is the residual error. Duncan's multiple range test was used to compare between the means of treatment to identify the statistically significant differences. The data was represented as mean ± standard error. Differences were considered significant at P < 0.05.All data were subjected to one-way analysis of variance (ANOVA) to compare the various types of extenders. The following model was used: “YP > 0.05) difference was observed between the three groups regarding percentages of oleic and palmitic FAs. Margaric and heneicosylic FAs were detected in chicken EY and were not detected in quail EY  lower in CQ diluent than in the C and Q diluents. However, slow progressive motility before freezing was significantly (P < 0.05) lower in the CQ diluent than in C and Q diluents (The rapid motility before freezing was significantly (diluents .P < 0.05) lower in the CQ diluent than in the Q diluent. Vitality, plasma membrane integrity and DNA integrity before freezing were significantly (P < 0.05) higher in Q diluents than in C and CQ diluents. Vitality and DNA integrity before freezing were significantly (P < 0.05) higher in C diluent than in CQ diluent. There was not significantly (P > 0.05) different between CQ and C diluents regarding plasma membrane integrity before freezing. However, the sperm abnormality before freezing was significantly (P < 0.05) lower in Q diluent than in C and CQ diluents.Total progressive motilities before freezing were significantly lower in CQ than C and Q diluents . Total mP < 0.05) higher in C diluent than in Q and CQ diluents. The sperm VCL and ALH were significantly (P < 0.05) higher in Q diluent than in CQ diluent. While, the sperm VSL and WOB were significantly (P < 0.05) higher in CQ diluent than in Q diluent. LIN of sperm in C and CQ diluents was significantly (P < 0.05) higher than Q diluent. The STR of sperm was not significantly differed between three diluents. The BCF in Q diluent was significantly (P < 0.05) higher than C and CQ  higher in diluent CQ than C and Q. The percentage of sperm acrosome before freezing was significantly (P < 0.05) higher in C diluent than in Q diluent. No significant differences were observed between three diluents regarding head length, head perimeter, elongation, ellipticity, regularity, distance and angle (The sperm head width in diluent CQ was higher than Q. The sperm head area and midpiece area before freezing were significantly (nd angle .P > 0.05) different after freezing. There was no significant (P > 0.05) difference between the three diluents regarding medium and slow sperm motility. The static motility sperm in diluent C was significantly (P < 0.05) lower than in diluents in Q and CQ (The fast and rapid sperm motilities in the three diluents were not significantly (Q and CQ .P < 0.05) higher in C diluent than in Q and CQ diluents. The percentage of sperm abnormality was significantly (P < 0.05) lower in diluent Q than in C and CQ diluents. All sperm kinetic parameters were significantly higher in Q diluent than in C and CQ diluents  on bull semen cryopreservation, the pigeon EY was suggested as a good alternative to chicken EY because it increased post-thawed vitality and progressive motility . For cryIn another study comparing usage of quail and chicken EYs for cryopreservation of Spanish Ibex sperm, quail EY offered no advantages over chicken EY . The autin vitro post-thaw semen quality and in vivo buffalo fertility.In buffalo bulls, duck EY extender has superior post-thaw sperm quality than chicken EY . In a siIn the current study, the significant differences in evaluating parameters between quail and chicken EYs diluents and their mixture can be explained by the results of EY analysis which showed that quail EY had more SFAs and MUFAs and less PUFAs than chicken EY. Quail EY had more myristic, palmitoleic and stearic FAs and less linoleic, linolenic, gondoic, and lignoceric FAs than chicken EY. This explanation is in agreement with the finding of other researches who reported that the difference in post-thaw semen quality is due to the difference in the biochemical composition of EYs , 31. VarThe lipid concentration and composition of the sperm plasma membrane determine its cryotolerance. Due to its lipid and FA contents, ovine sperm is particularly vulnerable to cold shock , 50. RamOur results revealed that all evaluating parameters significantly affected by freezing when comparing these parameters before and after freezing. Generally, post-thaw sperm motility, mitochondrial function, membrane integrity, and vitality decreased after cryopreservation and thawing. This drop in the post thawing parameters can be explained by the effect of cryopreservation on lipids. Cryopreservation of ram sperm alters choline glycerophospholipids with or without EY , 56. Aft® extender improved chilled and frozen bull sperm  authorized the current project . This authorization is based on the advice of the Research Ethics Sub-Committee (minute number 7 and date 22/04/2021), as well as a suitable risk-to-benefit ratio and a study design that minimizes risks.AS contributed to conceptualization, data curation, formal analysis, investigation, methodology, writing—original draft, review and editing. HB-A contributed to investigation and methodology. IO and IS contributed to writing—review and editing. AA contributed to funding acquisition, resources, software, supervision, validation, and visualization. All authors contributed to the article and approved the submitted version.This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH), King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (13-BIO2462-02).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nutritional status, health risk behaviors, eating habits, and other comorbidities (such as diabetes) may be associated with recommended amounts of physical activity (PA) and exercise (EX) in healthy older adults. However, these associations are still unclear for older hypertensive adults, who require greater care from health professionals. The purpose of this study was to associate the nutritional status, health risk behaviors, eating habits, and the presence of diabetes with recommended amounts of physical activity and exercise practice of older hypertensive adults.Ten thousand seven hundred eighty-nine older hypertensive adults (70.9 ± 7.4 years) from the VIGITEL telephone survey were classified according to PA levels (insufficiently active/sufficiently active) and EX practice (non-practitioners/practitioners). Binary logistic regression was used to observe the odds ratio (OR) between independent variables  with recommended amounts of PA/EX (dependent variable).PA = 0.975 [95%-CI: 0.965 – 0.985]; OREX = 0.981[95%-CI: 0.972 – 0.991]), age , screen time (ORPA = 0.909[95%-CI: 0.835 – 0.990]), alcohol consumption , tobacco  ultra-processed foods consumption score  and having diabetes  reduced the odds of being sufficiently active/practicing exercise (p < 0.05). Male sex , years of study , and minimally processed foods consumption score increased the odds of being sufficiently active/practicing exercise .Highest nutritional status (ORNutritional status, health risk behaviors, eating habits, and the presence of diabetes were associated with the odds of older hypertensive adults complying with PA and EX recommendations. The results may help health professionals understand how these factors are associated with the changes of older hypertensive adults participating in physical activity and exercise.The online version contains supplementary material available at 10.1186/s12889-022-14873-4. Aging is characterized by the progressive loss of adaptive capacity, which leads to a subsequent reduction in functionality . Over thThe first line of treatment for arterial hypertension, regardless of severity, focuses on the adoption of healthy lifestyles , 6. PhysAlthough many older adults exercise regularly, a significant amount does not seem to meet the 150 min of moderate to vigorous physical activity recommended by the World Health Organization . AccordiSome behavior factors can increase the odds of older hypertensive adults adhering to exercise programs and being sufficiently active –22. ThesThus, the purpose of this study was to verify the association of nutritional status, health risk behaviors, eating habits, and the presence of diabetes on recommended amounts of physical activity and exercise of older hypertensive adults. We hypothesized that nutritional status, health risk behaviors eating habits, and the presence of diabetes were associated with the odds of older hypertensive adults engaging in sufficient levels of physical activity and exercise.The study design was observational and cross-sectional. The data originated from the epidemiological project called Estudo de Vigilância de Fatores de Risco e Proteção para Doenças Crônicas por Inquérito Telefônico [Surveillance of Risk and Protection Factors for Chronic Diseases by Telephone Survey] (VIGITEL). VIGITEL is conducted every year between January and December by the Ministry of Health in partnership with the Group of Studies, Research and Practices in Food and Health Environment of the Universidade Federal de Minas Gerais (GEPPAAS/UFMG) and the Nucleus for Epidemiological Research in Nutrition and Public Health of Universidade de São Paulo (NUPENS/USP). For this study, data from 2019 was used since this was the last year before the COVID-19 pandemic.VIGITEL data collection is conducted through telephone survey systems with voluntary subjects between 18 and 106 years old, residents in the 26 Brazilian state capitals, and the Federal District. All subjects who have at least one telephone landline  are eligible to participate in the survey. In short, participants  were called (recruited) randomly and those who completed the survey were selected as participants.The inclusion criteria for participants in the study were: a) ages between 60 and 90 years old; b) arterial hypertension diagnosis; c) recorded body mass (kg) and height (m); and d) available information on physical activity and exercise, education level, screen time, alcohol and tobacco consumption, minimally and ultra-processed foods consumption, and the presence (or not) of diabetes. We only selected participants between 60 and 90 years old because nonagenarians and the oldest are in a situation of extreme longevity , 24. SomIn this sense, from a random sample , all participants that fit the inclusion criteria mentioned above were selected for our study.Sociodemographic variables considered for the study were chronologic age, sex, and education level (years of study), self-reported at the time of the interview. Years of education were included with the question “Up to what grade did you study?”, and the responses were reported in years  (p. 117 of ).2. Nutritional status was classified according to Lipschitz (1994) into three categories  ), each year of schooling (OR: 1.050[95% CI: 1.041 – 1.058]) and minimally processed food consumption score (OR: 1.167 [95% CI: 1.145 – 1.191]) increase the OR to be exercise practitioner (p < 0.001).All factors were associated with the OR of older hypertensive adults being exercise practitioners, except for screen time Table . For eacp < 0.05). The factors of male sex (OR: 1.633) and minimally processed food consumption score (OR: 1.132 [95% CI: 1.109 – 1.155]) increase the OR to be sufficiently active, respectively (p < 0.001).All factors were associated with the OR of older hypertensive adults being sufficiently physically active. For each higher classification of BMI (OR: 0.975 [95% CI: 0.965 – 0.985]), screen time (OR: 0.909[95% CI: 0.835 – 0.990]), alcohol (OR: 0.683[95% CI: 0.621 – 0.758) and tobacco (OR: 0.601 [95% CI: 0.492 – 0.736]) consumption ultra-processed food consumption score (OR: 0.896[95% CI: 0.871 – 0.921]), and presence of diabetes (OR: 0.780[95% CI: 0.708 – 0.859]), there is a decrease of OR on older adults to be sufficiently active , health risk behaviors, eating habits, and the presence of diabetes. The results showed that BMI, age, screen time , alcohol and tobacco consumption, ultra-processed food consumption scores, and having diabetes decreased the odds of being exercise practitioners or sufficiently active. On the other hand, male sex, years of study, and minimally processed foods increased the odds of older hypertensive adults being exercise practitioners or sufficiently active. Therefore, this study advances the investigation of hypertension and behavioral factors that are associated with exercise and physical activity participation of older adults in a region so far not investigated (Brazil).Previous findings indicated that higher nutritional status (overweight/obesity) is associated with lower odds of compliance with exercise and physical activity , 22, 31.n = 404). Even older non-practitioners of exercise can be sufficiently active through occupational and transportation activities. Leisure activities  are not the only way in which older people can be sufficiently active. Therefore, these sufficiently active older adults may benefit from a more active lifestyle compared to insufficiently active older adults, as regular physical activity reduces the risk of coronary heart disease, obesity, and some cancers  line), interview time (longer-interviews tend to have more errors in some questions), and duration of the survey (reduced time to collect more data). Despite being comprehensive and representative, the cross-sectional design did not allow the establishment of cause-effect relationships. The extrapolation of these findings to populations from other countries should be considered with caution, considering other differences that can impact the odds of older adults being exercise practitioners and sufficiently active. The lack of cognitive assessment before the interview is a limitation because some answers could be misunderstood by the participants. The use of BMI to determine nutritional status also limited the study due to BMI not distinguishing body composition components. However, it is the most used epidemiological index in the world, being adequate and feasible for representative samples . Anothernd 0.47) . Future nd 0.47) .As practical implications of this study’s findings, health professionals responsible for the treatment and control of hypertension in older adults can educate older adults about the associated factors of exercise and physical activity participation. The magnitude of positive and negative associations of each factor  may help change the lifestyle of older hypertensive adults. Future research should explore the underlying reasons for the frequency of health risk habits in insufficiently active older hypertensive adults to help the development of public health interventions. In the public health field, helping older hypertensive adults reach the physical activity recommendations will contribute to the reduction of healthcare services expenses . One posFrom a multifactorial approach, many reasons could be associated with a reduced chance of being an exercise practitioner. Beyond what is explored in our study, other factors including accessibility to exercise (adequate space to exercise practice), monthly income , and public policies promoted by the government  could beAdditionally, we suggest the longitudinal analysis (cohort or intervention studies) of the associated factors and levels of exercise and physical activity of older hypertensive older adults to establish the causal relationships between the factors analyzed in our study and recommended amounts of physical activity and exercise. We also suggest reproducing the study in other countries for greater external validity, and as a result, generalization across other populations.Nutritional status, sociodemographic characteristics, health risk behaviors, eating habits, and the presence of diabetes are associated with recommended amounts of physical activity and exercise in older hypertensive adults. Higher BMI, age, screen time , and alcohol, tobacco, ultra-processed food consumption score, and having diabetes are negatively associated with exercise and physical activity suitability in older hypertensive adults. Being a male, level of education and minimally processed food consumption score are positively associated with exercise and sufficient levels of physical activity. The findings from this study may help health professionals understand how these factors are associated with the increase/decrease of the odds of older hypertensive adults participating in exercise and being sufficiently active. In this sense, best strategies can be elicited for treatment and arterial hypertension control.Additional file 1: Supplementary Table 1. Percentage values (%) of exercise types practiced by hypertensive older adults’ from VIGITEL project (n=5.535).Additional file 2: Supplementary Table 2. Binary logistic regression model  with the association of nutritional status (BMI) and other factors on exercise participation (be or not a practitioner) and recommended amount of physical activity (sufficiently active).Additional file 3: Supplementary Chart 1. Summary of main benefits of exercise and physical activity and recommendations of corresponding intensity, duration, and frequency for older hypertensive adults.


To compare the clinical outcomes of the different treatments for acute primary angle closure (APAC).  We retrospectively reviewed the clinical charts of 87 eyes of 87 patients undergoing treatment for APAC. We investigated the best spectacle-corrected visual acuity (BSCVA), intraocular pressure (IOP), corneal endothelial cell density (ECD), and secondary interventions after each treatment. U test, p=0.149), the IOP was 12.8 ± 2.6, and 12.6 ± 2.9 mmHg (p=0.860), and the ECD was 2295.9 ± 658.2 and 2244.1 ± 622.0 cells/mm2 (p=0.735) in the LI and lensectomy groups, respectively.  The pretreated IOP was 56.4 ± 9.0 mmHg. As the first treatment for APAC, all eyes underwent topical 2% pilocarpine and systemic mannitol administration. Subsequent laser iridotomy (LI) and lensectomy were necessary in 29 eyes (33%) and 35 eyes (40%), respectively. Bullous keratopathy developed in 1 eye (1%), and following glaucoma surgery was required in 7 eyes (8%). The BSCVA at the final follow-up was 0.16 ± 0.53 and 0.01 ± 0.20 logMAR (Mann–Whitney  Approximately 26% of eyes with APAC were resolved after the initial medical treatment, and subsequent surgical treatments, such as LI and lensectomy, were required in 33% and 40% of eyes, respectively. We found no significant differences in the BSCVA, the IOP, or the ECD among LI and lensectomy treatment groups. Acute primary angle closure (APAC) has been widely acknowledged as one of the significant ophthalmic diseases requiring emergency treatment. As the initial treatment, patients developing APAC usually undergo topical pilocarpine and systemic mannitol administration. When the initial medical treatment does not dissolve the acute attack, the surgical interventions, including laser iridotomy (LI) or lensectomy, are necessary to resolve the pupillary block and subsequent intraocular pressure (IOP) rise. Individual ophthalmologists habitually select the emergency treatment option based on their skills and experiences with such treatments.Until now, there have been several studies on the treatment outcomes of APAC –10. Howe000048942). This retrospective study comprised a total of 87 eyes of 87 consecutive patients , who developed APAC between January 2006 and June 2020 at Kitasato University Hospital and who completed at least a 3-month follow-up. Eyes with any history of ocular surgery, ocular trauma, or other concomitant eye diseases except for glaucoma were excluded from the study. Only one eye was randomly chosen from each patient for statistical analysis when bilateral APAC occurred. This retrospective review of the clinical charts was approved by the Institutional Review Board at Kitasato University (B20-123) and followed the tenets of the Declaration of Helsinki. Our Institutional Review Board waived the requirement for informed consent for this retrospective study.The study protocol was registered with the University Hospital Medical Information Network Clinical Trial Registry  and the Nd : YAG laser . For lensectomy, experienced surgeons performed standard phacoemulsification through a 2.8 mm temporal corneal incision. The surgical technique consisted of capsulorhexis, nucleus, and cortex extraction. If possible, we also performed subsequent nontoric intraocular lens (IOL)  implantation. The presence or absence of IOL implantation was determined by each surgeon based on the intactness of the capsular bag.As the initial treatment, all eyes underwent topical 2% pilocarpine and systemic mannitol administration. After that, we performed a surgical treatment, such as laser iridotomy (LI) or lensectomy, when APAC was not resolved after the first treatment. We mainly selected lensectomy when there were experienced surgeons in our institution and when the corneal edema was mild. Otherwise, we selected LI when there were no experienced surgeons or when the corneal edema was moderate to severe. In addition, we conducted prophylactic LI or lensectomy after that, even when the first treatment resolved APAC. For LI, laser iridotomy was performed in the superior peripheral region using a sequential argon laser and an Nd : YAG laser –14. We aWe divided the subjects into two treatment groups (LI and lensectomy groups), based on the APAC treatment except for those who did not require the secondary surgical interventions after the initial treatment. We assessed the patient demographics, the selected treatment method, best spectacle-corrected visual acuity (BSCVA), intraocular pressure (IOP) measured with a Goldmann applanation tonometry, and corneal endothelial cell density (ECD) measured with a noncontact specular microscope  at the final follow-up after each treatment.U test was used to compare the BSCVA, the IOP, and the ECD of the two groups. The results are expressed as mean ± standard deviation, and a value of p < 0.05 was considered statistically significant.We conducted statistical analyses using commercially available statistical software . The Mann–Whitney 2, respectively. After lensectomy, IOL implantation was simultaneously performed in 17 of 35 eyes (49%); 15 eyes underwent in-the-bag fixation, and 2 eyes underwent out-of-the-bag fixation. IOL implantation was secondarily performed in 17 eyes of the remaining 18 eyes , and only one eye was left as an aphakia, since this patient did not wish to undergo secondary IOL implantation due to optic nerve atrophy. We found zonular weakness during cataract surgery in 9 eyes but found no significant postoperative complications.U test, p=0.149), the IOP (p=0.860), or the ECD (p=0.735) between the two treatment groups.Bullous keratopathy developed in 1 eye (1%) after LI, and glaucoma surgery was required in 7 eyes (8%) . Our results showed no significant differences in visual acuity, IOP, or ECD among the two treatment groups in the present study. We assume that this information is simple but clinically meaningful for better understanding of the real-world prognosis of post-APAC patients in a clinical setting.There are at least four limitations to this study. Firstly, we did not assess the ECD in all eyes since this study was performed in a retrospective fashion. Secondly, the minimum follow-up time was set at 3 months, and the observation period was 15.5 ± 19.3 months. A prospective study with a more extended observation period is still necessary to clarify this point. Thirdly, we did not confirm the exact etiology of APAC, including pupillary block, plateau iris, lens factor, and ciliary body factor, since we did not obtain images using anterior segment optical coherence tomography or ultrasound biomicroscopy in all eyes. Fourthly, the study was conducted in a retrospective, uncontrolled fashion. A prospective controlled study in a large cohort of another population would be ideal for confirming the authenticity of our results.In summary, our results may support the view that approximately 30% of eyes with APAC were resolved after the initial medical treatment and that subsequent surgical therapies, such as LI and lensectomy, were necessary for 33% and 40% of eyes, respectively. In addition, there were no significant differences in the BSCVA, the IOP, or the ECD among the LI and lensectomy treatment groups in our case series. We believe that this information will be clinically helpful for understanding the overall prognosis of post-APAC patients.


CCND1, EGFR, ENTPD5, HOXA10, IGF1R, MYC, and SNAI2) related to metastatic disease. A significant association was found between increased MMP1 expression and tumor size, grade, pathological T stage, and perineural invasion. Other genes were also associated with clinicopathological variables, CSS and OS. Finally, we found changes in mRNA–miRNA regulation that contribute to understanding the mechanisms involved in tumor progression. Therefore, we identified miRNA and mRNA expression profiles as potential biomarkers associated with lymph node metastasis and prognosis in PeC, in addition to disruption in mRNA–miRNA regulation during disease progression.Penile cancer (PeC) is a rare disease, and no prognostic biomarkers have been adopted in clinical practice yet. The objective of the present study was to identify differentially expressed miRNAs (DEmiRs) and genes (DEGs) as potential biomarkers for lymph node metastasis and other prognostic factors in PeC. Tumor samples were prospectively obtained from 24 patients with squamous cell carcinoma of the penis. miRNA microarray analysis was performed comparing tumors from patients with inguinal lymph node metastatic and localized disease, and the results were validated by qRT-PCR. Eighty-three gene expression levels were also compared between groups through qRT-PCR. Moreover, DEmiRs and DEGs expression levels were correlated with clinicopathological variables, cancer-specific (CSS), and overall survival (OS). TAC software, TM4 MeV 4.9 software, SPSS v.25.0, and R software v.4.0.2 were used for statistical analyses. We identified 21 DEmiRs in microarray analysis, and seven were selected for validation. miR-744-5p and miR-421 were overexpressed in tissue samples of metastatic patients, and high expression of miR-421 was also associated with lower OS. We found seven DEGs ( Penile cancer (PeC) is a rare disease, with 36,000 new cases estimated worldwide for 2020 . Its treSome molecular biomarkers were described as prognostic factors, but none have been applied to clinical practice yet . The disRecently, epigenetic alterations have emerged to provide insights into the carcinogenesis of PeC, with a special focus on microRNAs (miRNAs) ,12,13,14This study aimed to identify miRNA and mRNA expression profiles as potential biomarkers for lymph node metastasis and other prognostic factors in a prospective PeC cohort. We also investigated the deregulation of mRNA–miRNA pairs associated with disease progression. Among 41 cases of PeC diagnosed at ICESP between 2015 and 2018, 24 were included in this study. The exclusion criteria for patients are detailed in n = 11) and in an independent (n = 13) cohort. The mean age at diagnosis ± Standard Deviation (SD) was 61.8 ± 16.1 years old, and the median follow-up was 39.8 months. Partial penectomy was performed in 17 patients (70.8%), and all specimens were classified as the usual histological subtype of squamous cell carcinoma (SCC). Eleven patients had inguinal metastasis confirmed by pathology, and one was classified as cN3. At the analysis time, ten patients (41.7%) had died from any cause, and seven (29.2%) due to PeC. HPV DNA was detected in tumors from eight patients (33.3%) as described in our previous study [us study . n = 6) and localized (n = 5) disease. n = 24)  . Both mi n = 24) . CCND1, EGFR, ENTPD5, HOXA10, IGF1R, MYC, and SNAI2) in the tumors of patients with metastatic  and localized disease (n = 12) . Next, we performed a ROC analysis and found ten mRNA–miRNA pairs with good accuracy for discriminating tumors of patients with metastatic or localized disease . We observed that CD274 appeared twice in this analysis (related to both miRNAs). Seven out of ten pairs  presented disruption in their regulation patterns, changing from no correlation or positive correlation in tumors of patients with the localized disease to a negative correlation in metastatic disease  A, and we tissues B and sel disease .p > 0.05).The means of OS and CSS were 40.9 and 45.3 months, respectively. The maximum follow-up time was 62 months. Clinicopathological characteristics and their association with OS and CSS are summarized in CCND1, EGFR, MYC, and SNAI2 genes were associated with shorter CSS   and gastFew studies have investigated the role of miRNA as a prognostic factor of PeC. To the best of our knowledge, only four studies have described miRNAs associated with metastasis in PeC ,11,12,13CCND1, EGFR, and MYC were shown to be altered in PeC, with DNA amplification or copy number variations being associated with a poor prognosis [SNAI2 gene encodes a family member of zinc finger transcriptional factors involved in the epithelial–mesenchymal transition process by inhibiting the transcription of the E-cadherin gene [SNAI2 mRNA expression level is associated with lymph node metastasis in tongue SCC [We also detected seven upregulated genes in PeC patients with metastatic disease tumors. rognosis ,20. Furtrognosis . The SNArin gene . Althougngue SCC , this isngue SCC .MMP1 is associated with several worse prognostic factors in our study, no association with lymph node metastasis was found as described in the study of Kuasne et al. in PeC [Although . in PeC .CD274, miR-421/MYD88, miR-744-5p/CD274, and miR-744-5p/TLR4. The CD274 gene encodes the PD-L1 protein, which binds to its receptor in the T Cell (PD1), shutting it off and reducing the immunological response [MYD88 and TLR4) have been implicated in the innate immune response; part of this response is due to the activation of TLR4 by MYD88, which releases pro-inflammatory cytokines [MYD88 has never been related to carcinogenesis or prognosis in PeC. However, the low expression of the TLR4 protein was observed in patients with HPV infection [In addition to the analysis that showed the alterations of miRNA and mRNA profiles in PeC progression separately, we also investigated mRNA–miRNA pairs that were deregulated during tumor progression. We highlighted four mRNA–miRNA pairs that include genes related to the immune response: miR-421/response . PD-L1 eresponse . The othytokines . The MYDRegarding survival, we found that miR-421 overexpression was associated with shorter OS, similar to the reports in NSCLC  and gastCCND1, EGFR, MYC, and SNAI2 genes were also associated with shorter CSS and overexpression of MYC with shorter OS. Those genes have already been associated with shorter CSS or OS in PeC or other types of SCC [GADD45A overexpression was associated with shorter CSS and OS, with the highest HR values . GADD45A has never been investigated in PeC and is related to DNA damage response [Moreover, overexpression of s of SCC ,23. We aresponse . It actsresponse .One limitation of the study was the small sample size due to the rarity of PeC. This could explain why some DEmiRs detected by microarray analysis were not validated by qRT-PCR. It is also important to mention that, because ICESP is a tertiary referral center, we had a small proportion of patients with pT1 disease. Nevertheless, the presence of lymph node metastasis was balanced in our sample. Further studies with larger PeC cohorts are required to confirm our findings and functionally validate the detected mRNAs–miRNAs interactions.Overall, this study has several strengths. We prospectively followed a PeC cohort for a long period and uniformly treated patients according to guidelines, making clinical endpoints stronger. Another relevant factor was using a microarray technique to identify DEmiRs instead of studying a small panel chosen based on a literature search. Using an innovative analysis, we demonstrated changes in the regulation of mRNA–miRNA pairs during disease progression, enlarging the horizon for future research in PeC. From July 2015 to January 2018, patients with pathologically confirmed SCC of the penis were prospectively enrolled in our study. They were all newly diagnosed localized or locally advanced cases referred for surgical treatment with curative intent. The exclusion criteria were the presence of other malignancies at diagnosis, previous treatment for PeC, previous pelvic radiotherapy, palliative surgery without curative intent, patients who underwent treatment for the primary tumor in another institution, Human Immunodeficiency Virus (HIV), Hepatitis B or C infections. Fresh frozen PeC tumoral tissues were collected and stored in the Academic Biobank for Research on Cancer at the University of Sao Paulo (USP), Instituto do Cancer do Estado de Sao Paulo (ICESP), Sao Paulo, Brazil.This study was approved by the Local Ethics Committee (Protocol 1.016.980) and carried out under the terms of the Helsinki Declaration. The study and Biobank’s written informed consent and epidemiological questionnaire were obtained from all participants.Medical history and physical examination data were obtained during hospital admission. Pathological tumor data such as tumor size, histology, grade, staging, lymphovascular and perineural invasions, margins, and lymph node metastasis, were collected from the patients’ anatomopathological reports and medical records. Metastatic disease was defined as metastasis to inguinal or pelvic lymph nodes, either clinically or pathologically confirmed, or distant metastasis.The disease was staged by computed tomography imaging of the chest, abdomen, and pelvis. Patients underwent partial or total penectomy according to the extent of the disease and were prospectively followed from the time of enrollment until April 2021 according to the European Association of Urology (EAU) Guidelines on PeC [TM miRNA Isolation kit , according to the manufacturer’s instructions. RNA concentration and purity were determined, and only samples with an A260/A280 ratio between 1.8 and 2.1 and an RNA Integrity Number above seven were included.PeC frozen tissues (30 mg) were used for total RNA extraction using the miRVanan = 5) using the GeneChip miRNA 4.0 Array (Thermo Fisher Scientific) following the manufacturer’s protocol. The samples were hybridized on the array, stained in the Affymetrix Fluidics Station 450, and scanned with the Affymetrix GeneChip Scanner 3000 7G. Expression Console software (Thermo Fisher Scientific) was used for the quality assessment of microarray data. Transcriptome Analysis Console (TAC) software version 4.0.2 (Thermo Fisher Scientific) was used for background correction, normalization, and summarization of raw data (CEL files) by Robust Multichip Average plus Detection Above Background (RMA + DABG). We used the Linear Models for Microarrays (LIMMA) test with at least 1.5-fold changes (FC > 1.5) and p < 0.05 to identify differentially expressed miRNAs (DEmiRs) between samples from patients with metastatic (n = 6) or localized disease (n = 5). The TAC software constructed hierarchical clustering graphs with the DEmiRs selected for further validation. Microarray datasets are available on the Gene Expression Omnibus database (GSE172095).The miRNA expression profile was investigated in samples from patients with metastatic (n = 6) or localized disease (n = 13) and whole cohort (n = 24). Total RNA was reverse transcribed to cDNA using the TaqMan® Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific). The miRNA levels were quantified using Taqman® Advanced miRNA Assays (−ΔΔCT) [t-test and p < 0.05 in TM4 MultiExperiment Viewer (MeV) 4.9 software. Boxplots were constructed using SPSS v.25.0.The DEmiRs identified from microarray analysis were selected for validation by qRT-PCR in an independent set of PeC samples (A Assays  (Thermo  (−ΔΔCT) . We compn = 12) or localized disease (n = 12) using a panel containing 90 genes , followed by 10 cycles of pre-amplification reaction, which was performed using a pool of 90 Delta Gene™ assays (Fluidigm) representing all investigated genes at a final concentration of 500 nM each. Pre-amplified cDNAs were treated with Exonuclease I  and diluted 1:10.® dye method  and were prepared according to the manufacturer’s instructions. Delta Gene™ Assays  and solutions were pipetted into the 96.96 Dynamic Array Integrated Fluidic Circuit (IFC) according to the manufacturer’s recommended pipetting map and then placed on the Juno IFC controller (Fluidigm) to load samples and assays into the 96.96 Dynamic Array IFC. Next, qRT-PCRs were conducted using the nanofluidic platform Biomark™ HD System Real-Time PCR (Fluidigm) according to the established protocol. Results were extracted from the Biomark Data Collection version 4.5.1 software and were analyzed using the Fluidigm Real-Time PCR Analysis version 4.3.1 software (Fluidigm). Obtained values were plotted individually for each gene, and only samples with CT values lower than 24 were considered for analysis.Gene expression levels were quantified by qRT-PCR using the EvaGreen® Excel add-in [ACTB, B2M, GAPDH, GUSB, HPRT1, RPLP0, and TFRC) used as candidates to normalize qRT-PCR data. Based on each gene’s intra- and inter-group variations, this program could automatically determine the most stably expressed candidate reference genes and gene pairs in a sample. ACTB and RPL0 were the most stable endogenous genes in our samples. FC was calculated using the comparative CT method (2−ΔΔCT) [n = 12) or localized disease (n = 12) in the total sample of PeC patients by using Student’s t-test and p < 0.05.NormFinder, a Microsoftl add-in , was use(2−ΔΔCT) . The TM4After paraffin removal using xylene, PeC samples were digested with proteinase K/SDS 0.1% for 24 h. DNA was obtained by phenol:chloroform extraction, and samples were diluted to 50 ng/µL. The quality of the DNA was determined by amplifying a 110 bp fragment of the human β-globin gene with PCO3/PCO4 primers, followed by analysis on an 8% acrylamide gel electrophoresis . The Innp < 0.05 as significant in SPSS version 25.0. The association of miRNAs and DEGs expression levels with clinicopathological variables was accessed using Student’s t-test in TM4 MeV 4.9 software, considering p < 0.05 as significant.To analyze differences between samples used in the microarray analysis and the independent or expanded validation, we used Chi-square or Fisher’s Exact, Mann–Whitney tests, and The ROC curves were constructed using SPSS version 25.0. The AUC values were calculated to analyze the accuracy of each DEmiR and DEG for distinguishing tumors of patients with metastatic and localized diseases with specificity and sensitivity. The same analysis was performed for all the other clinicopathological variables of the study. For the mRNA–miRNA pairs identified in the correlation analyses, ROC curves analysis was performed to evaluate the prognostic testability of these pairs .p-values (≤0.1) for at least one of the tissues. We estimated these selected pairs’ mRNA/miRNA ratio using the “PRROC” package to calculate AUC values. Subsequently, the “corrplot” and “ggplot2” packages were used to plot correlation values. Finally, we used the “ggrepel” package to tag mRNA–miRNA pairs with AUC values higher than 0.8.Pearson’s correlation coefficients for tumors of patients with metastatic or localized disease were performed in the R software v. 4.0.2 (“Hmisc” package) using expression data for seven miRNAs and 83 genes. The “tidyr” package was loaded to select mRNA–miRNA pairs with significant p < 0.05 value as significant. We used the Kaplan–Meier method, and the statistical differences were analyzed using the log-rank test. We performed a Cox regression model to estimate the Hazard Ratio (HR) and 95% confidence intervals (CI) values. High and low miRNA and transcript expression levels in tumor samples were defined by the median of -dCT values.Cancer-specific (CSS) and overall survival (OS) analyses were performed using SPSS v.25.0 and We detected two miRNAs and seven genes overexpressed in tumors of patients with metastatic disease in a prospective cohort of PeC patients. We also demonstrated that these miRNAs and genes showed good accuracy in identifying patients with metastasis and that they are possible potential biomarkers to be used in PeC. Additionally, we found that the upregulation of miR-421 was associated with shorter OS, and the expression of 19 genes was associated with other clinicopathological variables, ten with CSS and seven with OS in PeC. Finally, we detected changes in mRNA–miRNA regulation that could help to explain the tumor progression process.


The tumor suppression rate of aucubin could reach 51.31 ± 4.07%. Organ histopathology was evaluated by tissue staining, which demonstrated that aucubin could alleviate the organ inflammatory damage caused by breast cancer without visible side effects. Moreover, aucubin could increase the expression of colonic tight junction protein occluding and adjust the gut microbiome to normal level according to 16S rDNA high-throughput sequencing. Herein, our results provide evidence for developing aucubin as an alternative and safe therapeutic for breast cancer treatment.Aucubin, a natural compound isolated from herbal medicine, has been reported to possess multiple beneficial properties. In this study, we aimed to verify the anticancer effect of aucubin on breast cancer and investigate the effect of cancer on the intestinal flora and whether aucubin has a therapeutic effect on intestinal problems caused by cancer. We established the breast cancer model with mouse 4T1 cell line and BALB/c mice. Aucubin was given once a day by gavage for 14 days. The results showed that aucubin suppress the growth of tumor  Breast cancer is the most common cancer diagnosed among women, and ranks among the most three common cancers worldwide, along with colon and lung cancers . Due to β-diversity, p=0.006) [A research of gut microbiota difference between postmenopausal breast cancer case patients and control patients showed that statistically significantly differences exist in composition p=0.006) . Microbip=0.006) . The mic . Microbp=0.006) . Moreovep=0.006) . In somep=0.006) . TherefoPlantago asiatica, Eucommia ulmoides, and Aucuba japonica. A series of studies have demonstrated that aucubin possess multiple beneficial effects including antioxidation [Aucubin is an iridoid glycoside compound  that is xidation , 11, antxidation , anti-fixidation , hepatopxidation , neuroprxidation , osteoprxidation , antitumxidation , antiendxidation , and antxidation . Aucubinxidation . It has Aucubin with purity over 95% was supplied by Chengdu Must Bio-Technology Co. Ltd. . Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), antibiotic-antimycotic solution, penicillin/streptomycin, phosphate-buffered saline (PBS), and 0.25% (w/v) trypsin/1 mM EDTA were procured by Gibco . Annexin V-FITC Apoptosis Detection Kit was purchased from Elabscience Biotechnology Co. Ltd. .2.Murine metastatic breast cancer 4T1 cell line was obtained from the American Type Culture Collection . Cells were cultured in RPMI 1640 medium supplemented with 1% penicillin/streptomycin and 10% FBS, and maintained in incubators at 37°C under an atmosphere of 5% CO6) were suspended in 60 μL PBS and inoculated into the left flank of mice. When the palpable tumor volume increased to 50–100 mm3, mice were randomly divided into four groups and received different treatments. The schematic depicting of the animal model is shown in V) was calculated based on equation (The specific pathogen-free (SPF) female BALB/c mice  were purchased from the Guangdong Medical Laboratory Animal Center (China). Mice were housed in pathogen-free conditions  with food and water freely to acclimatize for 7 days. All experiments involving animals were approved by Institutional Animal Care and Use Committee, Zunyi Medical University. To obtain the xenograft animal model, 4T1 cells (6 × 10a) and smallest diameter (b) of the tumors were measured with vernier caliper. Wc and Wx represent tumor weight of the model group and treatment group, respectively. The mice were sacrificed by cervical dislocation. The tumors and organs were excised and weighed, and quickly fixed in 4% paraformaldehyde for hematoxylin and eosin stains (H&E).The largest diameter .The tumor tissue was digested into a single cell suspension, and the cells were rinsed with PBS twice. 5 × 10The tumor was embedded in paraffin. The sample was sliced and then deparaffinized. The sample was immersed in citric acid antigen retrieval buffer for antigen retrieval. The slices were put in 3% hydrogen peroxide solution and incubated at room temperature without light for 25 min to block endogenous peroxidase. After washing with PBS, BSA was added dropwise on chamber slides and incubated for 30 min, and the primary antibody was added to incubate overnight at 4°C. Then, it was washed three times with PBS, and the secondary antibody was added and incubated for 50 min at room temperature. After washing with PBS, enhanced DAB solution was added dropwise. After staining the nucleus with Harris hematoxylin, samples were observed under microscope and images were collected.The intestinal tissue was embedded in paraffin. The sample was sliced and then deparaffinized. Then, the sample was immersed in EDTA antigen retrieval buffer for antigen retrieval. BSA was added dropwise on chamber slides and incubated for 30 min, and the primary antibody was added to incubate overnight at 4°C. After washing three times with PBS, the secondary antibody was added and incubated for 50 min at room temperature without light. After washing with PBS, antifluorescence quenching solution was added dropwise. Samples were observed under a fluorescence microscope, and images were collected.The mice blood was collected in a sterile clean, enzyme-free EP tube by orbital blood collection, left it at room temperature for 20 minutes, and then centrifuged at 3000 r/min for 5 minutes. The upper serum was collected in a new sterile clean, enzyme-free EP tube and BCA method was used for protein quantification. The content of aspartate aminotransferase (AST) and alanine transaminase (ALT) in the serum was detected by using the automatic biochemical analyzer .About 100 mg of intestinal contents were taken from each mouse, and microbial community genomic DNA was extracted with the E.Z.N.A.® soil DNA Kit . After PCR amplification, the pure DNA product is harvested by using the AxyPrep DNA Gel Extraction Kit . Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina NovaSeq PE250 platform  according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. . Raw 16S rRNA gene sequencing reads were demultiplexed, quality-filtered by fastp version 0.20.0  and mergp < 0.05 was considered as a statistical difference.The GraphPad Prism 6.0 statistical software  was used for statistical analysis. The results were shown as mean ± standard deviation (SD). Statistical significance was assessed by one-way ANOVA followed by Tukey's multiple comparison, and 3, mice were randomly divided into four groups (n = 6): (1) water, (2) 50 mg/kg of aucubin, (3) 100 mg/kg of aucubin, and (4) 200 mg/kg of aucubin. Drugs were given once a day by gavage.The mouse 4T1 cell line and BALB/c mice were used to establish the breast cancer model. When the palpable tumor volume increased to 50–100 mmThe BALB/c mice without receiving inoculation of 4T1 cells were set as the control group treat with water. Tumor volume and body weight were measured every day to evaluate the therapeutic efficacy. There was no significant difference in the body weight of the mice in all groups after 14 days of continuous monitoring .3 at day 14 . 18 samples of fecal from three groups  were collected, and we compared the gut microbiota of mice in different groups using 16S ribosomal RNA (rRNA) gene amplicon sequencing. The sequences were grouped into provisional clusters as ASV and analyzed on the Illumina NovaSeq PE250 platform. The Lactobacillaceae and Bacillaceae in the model group are significantly decreased. On the contrary, Staphylococcaceae, Bacteroidaceae, and Muribaculaceae exhibited higher abundance. The aucubin group shows the ability to adjust the gut microbiome to normal level. Aucubin could increase the reduction in the proportion of microflora due to tumors, such as Lachnospiraceae, Lactobacillaceae, Bacillaceae, and Ruminococcaceae. It could also reduce the increase in the proportion of microflora, such as Muribaculaceae, Staphylococcaceae, Oscillospiraceae, Bacteroidaceae, Marinifilaceae, Precotellaceae, and Tannerellaceae. The KEGG ortholog functional profile level of the microbial community is predicted by PICRUSt based on 16S rRNA sequencing data, which is shown in According to the one-way ANOVA bar plot at the family level , Lactobain vivo by inducing tumor cell apoptosis. The tumor suppression rate of aucubin could reach 51.31 ± 4.07%.Breast cancer without detectable distant metastases is considered curable in the early stages. The selection of ideal drugs in the process of tumor treatment is still being explored. Aucubin is a safe natural compound which is isolated from herbal medicine. A long-term toxicity study shown that oral administration of 200–800 mg/kg of aucubin had no significant effect in normal rats . Accordiβ1 through suppressing the activity of NOX4. The antioxidative activity of aucubin contributed much to its hepatoprotection [Hepatotoxicity is one of the main side effects of cancer chemotherapy, including steatosis, pseudocirrhosis, long-term hepatic damage, cirrhosis, and even hepatic necrosis. It is previously reported that aucubin was mainly distributed to the kidney and liver . It coulotection . In the A complication of cancer is that the intestinal flora disorder causes body weight loss and decreased immunity. Breast cancer has been widely reported to disrupt intestinal flora, which promotes tumor cell dissemination, metastatic seeding, and significant early inflammation through signaling in the gut , 27. AccIn conclusion, our study clearly verified the anticancer effect of aucubin on breast cancer in mouse model. We also found that aucubin has a therapeutic effect on intestinal problems by regulating intestinal microbiota. This research suggests that aucubin could be a promising anticancer compound in the breast cancer treatment and other diseases related to intestinal dysbacteriosis.


To analyze the demographic data, surgical and adjuvant treatment data and the survival outcomes in adult patients affected by acinic cell carcinoma of the parotid gland (AciCC).A retrospective multicenter analysis of patients treated for AciCC of the parotid gland from 2000 to 2021 was performed. Exclusion criteria were pediatric (0–18 years) patients, the absence of follow-up and patients with secondary metastatic disease to the parotid gland. Multivariable logistic regression was used to determine factors associated with survival.F = 48, M = 33). The mean follow-up was 77.7 months . The 5 years overall survival (OS) was 97.5%. The 5 years disease-free survival (DFS) was 60%. No statistical differences have been found in prognosis for age (< 65 or ≥ 65 years), sex, surgery type , radicality (R0 vs R1 + Rclose), neck dissection, early pathologic T and N stages and adjuvant therapy (p > 0.05).The study included 81 adult patients with AciCC of the parotid gland. The median age was 46.3 years , with a gender female prevalence (This study did not find prognostic factor for poorest outcome. In contrast with the existing literature, our results showed how also high-grade tumours cannot be considered predictive of recurrence or aggressive behaviour.The online version contains supplementary material available at 10.1007/s00405-022-07481-w. Acinic cell carcinoma (AciCC) is the fourth most common epithelial tumour of salivary glands after mucoepidermoid carcinoma, adenocarcinoma  and adenoid cystic carcinoma ; it can The aim of this retrospective study is to present a multicenter experience in the treatment of AciCC, focusing on the demographic, surgical and adjuvant treatment data and the survival outcomes in patients affected by AciCC. To the best of our knowledge, this is the largest European case series and those with the longest follow-up reporting experience about this rare parotid tumour.The primary endpoint was to assess the survival outcomes  of patients treated with curative intent for AciCC of parotid gland. The secondary endpoints were to evaluate the presence of prognostic factors and to describe the progression sites.Associazione Ospedaliera Italia Centromeridionale Otorinolaringoiatrica . Exclusion criteria were pediatric (0–18 years) patients, the absence of follow-up and patients with secondary metastatic disease to the parotid gland.For this multicenter analysis, patients with biopsy proven AciCC of the parotid gland were retrospectively included, treated between January 2000 and September 2021. Patients received surgery with curative intent at the Departments of Otolaryngology-Head and Neck Surgery of San Giovanni-Addolorata Hospital, Rome, Italy; San Giovanni Di Dio e Ruggi D’Aragona Hospital, Salerno, Italy; University of Modena, Modena, Italy; University of Bologna, Bologna, Italy; IRCCS Regina Elena National Cancer Institute, Rome, Italy; AORN Cardarelli, Napoli, Italy; IRCCS Fondazione G. Pascale, Napoli, Italy; University of Foggia, Foggia, Italy; ASL Napoli 3 Sud, Napoli, Italy; AORN Garibaldi, Catania, Italy; San Giovanni Calibita-Fatebenefratelli General Hospital, Rome, Italy; IRCCS “Casa sollievo della sofferenza” San Giovanni Rotondo, Foggia, Italy; San Carlo di Nancy Hospital, Rome, Italy; University of Pisa; IRCCS Fondazione G. Pascale, Naples, Italy. All the Departments involved in this study are part of Data concerning gender, age at the time of diagnosis, fine-needle aspiration biopsy (FNAB) diagnosis, type of surgery upfront , lymph node or distant metastasis at the time of diagnosis, post-operative resection margins and pTNM, adjuvant radiochemotherapy, locoregional or distant recurrences and the status at the follow-up  were systematically collected. The data concerning survival and recurrence outcomes were retrieved from mortality registries, outpatient visits and radiological follow-up. All histologic slides were reviewed from pathologists of each department to confirm the diagnosis of AciCC of the parotid gland. The data about histologic grading were obtained only from recurrent or metastatic AciCC.Ethical approval was waived by the local ethics committee in view of the retrospective nature of the study. The study was conducted in compliance with the Helsinki Declaration. All the clinical data needed for the study were recorded in a computerized database. Patients who were alive at study time were informed about the study and none expressed opposition to inclusion.p value less than or equal to 0.05 was considered statistically significant. To identify factors independently associated with decreased OS, a univariate then multivariable Cox regression analysis was performed. Measures of precision of point estimates are presented as odds ratios or hazard ratios with 95% CI.Quantitative variables were described with median and range [min–max], while qualitative variables were described with numbers and percentages. Chi square or Fisher exact tests were used to compare categorical variables. PFS and OS were assessed using the Kaplan–Meier estimator and compared using a log-rank test. For all tests, a two-tailed n = 7) or because of loss to follow-up (n = 6). Therefore, the study group included 81 adult patients with AciCC. The median age was 46.3 years , with a gender female prevalence . The incidence showed a peak in the fifth decade , with the most of the cases  occurring in the fourth, fifth and sixth decades. Pre-operative evidence of cervical node involvement (cN +) was identified in 7.4% (n = 6) of cases. Detailed patient demographics and oncologic data are summarized in Table Patients with diagnosis of AciCC of the parotid gland in the period study have been 94. Thirteen patients were excluded because of the age  of cases and resulted positive for AciCC only in 14.3% of the cases (n = 11). FNAB was not diagnostic in 55.8% of the patients (n = 43) and other parotid malignancies were shown in 23 cases (29.9%). In this study, none of the patients showed distant metastasis at the time of initial diagnosis of AciCC.Pre-operative FNAB was performed in 95.1% (n = 41) were treated with superficial parotidectomy (I–II), 41.9% (n = 34) with total parotidectomy (I–IV), 6.2% (n = 5) with superficial parotidectomy extended to inferior deep lobe (I–II–III) and 1.2% (n = 1) with total parotidectomy with facial nerve resection (I–IV [VII]). Neck dissection (ND) was not planned in 81.5% (n = 66) of the cases, while 18.5% (n = 15) received ND; in this group of patients, 73.3% (n = 11) received selective ND (levels II–IV).All patients were treated with surgery; the performed surgical procedure depended on the tumour location and the extent of disease. According to European Salivary Glands Society (ESGS) classification of parotidectomies , the 50.n = 15), underwent adjuvant radiotherapy. In this study, none of the patients received chemotherapy as adjuvant post-operative therapy.Among the 81 patients included, 21 (25.9%) received adjuvant radiotherapy (RT) after surgery; among these 21 patients, 14 (66.7%) received locoregional irradiation despite not having positive cervical lymph nodes. In addition, 9 patients (60%) among those received ND ; other pN + (pN2a or pN2b) were rarely observed . No pN2c  nor pN3a category  were observed in this cohort of patients. Histologic grade was recovered only for recurrent or metastatic AciCC; seven of those patients showed low-grade tumour (77.8%), while only two subjects showed high-grade tumour (22.2%).The definitive staging of disease is shown in Table Follow-up data were available for all the patients included in this study. Mean follow-up was 77.7 months ]. The 5 years overall survival (OS) was 97.5% (95% CI 96.9–98)  were alive without any evidence of disease, 7.4% (n = 6) were alive, after recurrence, without any evidence of disease and 2.5% (n = 2) were alive with recurrence. Disease recurrence was found in 11.1% of the patients (n = 9), with a median interval to first recurrence of 53.1 months . Distant recurrences were observed only in one patient (1.2%) and lung was the only organ involved. More patients died because of other cause  than disease . Survival data and univariate analysis of most relevant prognostic factors are shown in Fig. At the end of the study October 2021), 88.9% patients . Although the analysis of the Kaplan–Meier curves of DFS seems to suggest better results in T1 than T3 and T4, no significant difference has been found. The same for analysis of margin (R), where R0 seems to have better prognosis than R1and Rclose, and for age, where young patients seem to have better prognosis , sex, surgery type , radicality (R0 vs R1 + Rclose), neck dissection and adjuvant therapy , so this could suggest that elective neck dissection (END) should not be performed routinely; this is in accordance with the results from van Weert et al. , who fouThe indications for adjuvant RT in AciCC of the parotid gland are still debated; positive surgical margins, T3/T4 disease, high-grade tumours and lymphovascular/perineural invasion haven been considered as indications for post-operative RT , with noThe interpretation of this data is not easy due to impossibility to recover the specific indications for adjuvant therapy of each patient because of the retrospective nature of the study; however, our results seem to confirm the evidence of the previous works, showing no statistical significative difference between patients treated with post-operative RT and those not treated.Different chemotherapy regimen has been proposed as adjuvant therapy, especially for metastatic disease , but nonRecurrent and metastatic AciCC of the parotid gland are not uncommon, and there is no still consensus about the risk factors; in our work, nine patients experienced local recurrence, while only one patient experienced lung metastases. In our analysis, no risk factor can be identified as responsible of the progression of the disease.AciCC with high-grade transformation is considered to be more aggressive and predictive of recurrence and distant metastases; despite a histologic grading system is still lacking, frequent mitosis, ki-67 proliferation > 5%, perineural and vascular invasion, infiltration and tumour necrosis are regarded as high-grade features, explaining the aggressive behaviour of these dedifferentiated tumours , 22. TheThis study shows several limitations. First, this work included data prior to 2010, when mammary analogue secretory carcinoma (MASC), which exhibits a clinical behaviour similar to AciCC, was recognized as a distinct entity; anyhow, all histologic slides were reviewed from pathologists of each department to confirm the diagnosis of AciCC of the parotid gland. Second, we provided the data on a local level, from the Chief of each Head and Neck Department; however, to ensure a good level of quality control, the data were separately analyzed from two investigators . Third, this is a retrospective study and the data lack about treatment decisions . Fourth, perivascular and perineurial invasion were not included in the evaluation; however, according to the last World Health Organization (WHO) Classification of Tumours, these two variables are currently not considered as independent prognostic factors. Despite these limits, this study represents the largest European cohort of patient affected by AciCC of the parotid gland and surgically treated with curative intent, and offers a notable analysis about the outcomes and the prognostic factors of this rare tumour. Furthermore, this study examined DSS and all follow-up information; in addition, this study analyzed the longest follow-up regarding AciCC of the parotid gland.We present here one of the largest case series ever reported about AciCC of the parotid gland, which represents the fourth parotid gland malignancy. AciCC commonly has a good prognosis, except for a little cohort of patients; in the present study univariate Cox analysis did not find any prognostic factor for poorest outcome. In contrast with the existing literature, our results showed how also high-grade tumours cannot be considered predictive of recurrence or aggressive behaviour. According to multilinear regression analysis, recurrences or metastases and age > 65 years can be independent prognosticators. The future goal of this study group will be the proposal of a new histologic grading to further stratify the patients, attempting to identify shared prognostic factors.Supplementary file1 (DOCX 5209 KB)Below is the link to the electronic supplementary material.