Objective Non-Invasive Bio-Parametric Evaluation of Regenerated Skin: A Comparison of Two Acellular Dermal Substitutes.

Several dermal substitutes are available on the market, but there is no precise indication that helps surgeons choose the proper one. Few studies have tried to compare different xenogeneic bioengineered products, but no objective bio-parametric comparison has been made yet. Fifteen patients who underwent skin reconstruction with Integra® or Pelnac® were retrospectively evaluated. After at least 12 months of follow-up, an objective and quantitative assessment of several skin biophysical properties, such as color, texture, elasticity, hydration, glossiness and trans-epidermal water loss, were measured with non-invasive skin measurement devices. The grafted skin showed a reduction of the superficial hydration level and a tendency to lower values of trans-epidermal water loss with both dermal substitutes. Melanic and hemoglobin pigmentation were higher in comparison to the donor site in both groups, while a melanic pigmentation increase versus the surrounding skin was seen just with Integra®. Finally, the skin was found to be more elastic when reconstructed with Integra®. The skin barrier appeared to be intact in both groups. Hence, these substitutes are valuable means of skin regeneration. Integra® seems to be more advantageous for reconstructing areas that need more skin flexibility.

Sex Influence on Autophagy Markers and miRNAs in Basal and Angiotensin II-Treated Human Umbilical Vein Endothelial Cells.

Cardiovascular diseases (CVD) display many sex and gender differences, and endothelial dysfunction, angiotensin II (Ang II), and autophagy represent key factors in the autophagic process Therefore, we studied whether Ang II modulates the mentioned processes in a sex-specific way in HUVECs obtained from healthy male and female newborns. In basal HUVECs, the Parkin gene and protein were higher in FHUVECs than in MHUVECs, while the Beclin-1 protein was more expressed in MHUVECs, and no other significant differences were detected. Ang II significantly increases LAMP-1 and p62 protein expression and decreases the expression of Parkin protein in comparison to basal in MHUVECs. In FHUVECs, Ang II significantly increases the expression of Beclin-1 gene and protein, and Parkin gene. The LC3 II/I ratio and LAMP-1 protein were significantly higher in MHUVECs than in FHUVECs, while Parkin protein was significantly more expressed in Ang II-treated FHUVECs than in male cells. Ang II affects the single miRNA levels: miR-126-3p and miR-133a-3p are downregulated and upregulated in MHUVECs and FHUVECs, respectively. MiR-223 is downregulated in MHUVEC and FHUVECs. Finally, miR-29b-3p and miR-133b are not affected by Ang II. Ang II effects and the relationship between miRNAs and organelles-specific autophagy is sex-dependent in HUVECs. This could lead to a better understanding of the mechanisms underlying sex differences in endothelial dysfunction, providing useful indications for innovative biomarkers and personalized therapeutic approaches.

Stratification of Amniotic Fluid Cells and Amniotic Fluid by Sex Opens Up New Perspectives on Fetal Health.

Amniotic fluid is essential for fetus wellbeing and is used to monitor pregnancy and predict fetal outcomes. Sex affects health and medicine from the beginning of life, but knowledge of its influence on cell-depleted amniotic fluid (AF) and amniotic fluid cells (AFCs) is still neglected. We evaluated sex-related differences in AF and in AFCs to extend personalized medicine to prenatal life. AFCs and AF were obtained from healthy Caucasian pregnant women who underwent amniocentesis at the 16th-18th week of gestation for advanced maternal age. In the AF, inflammation biomarkers (TNFα, IL6, IL8, and IL4), malondialdehyde, nitrites, amino acids, and acylcarnitines were measured. Estrogen receptors and cell fate (autophagy, apoptosis, senescence) were measured in AFCs. TNFα, IL8, and IL4 were higher in female AF, whereas IL6, nitrites, and MDA were similar. Valine was higher in male AF, whereas several acylcarnitines were sexually different, suggesting a mitochondrial involvement in establishing sex differences. Female AFCs displayed higher expression of ERα protein and a higher ERα/ERß ratio. The ratio of LC3II/I, an index of autophagy, was higher in female AFCs, while LC3 gene was similar in both sexes. No significant sex differences were found in the expression of the lysosomal protein LAMP1, while p62 was higher in male AFCs. LAMP1 gene was upregulated in male AFCs, while p62 gene was upregulated in female ones. Finally, caspase 9 activity and senescence linked to telomeres were higher in female AFCs, while caspase 3 and ß-galactosidase activities were similar. This study supports the idea that sex differences start very early in prenatal life and influence specific parameters, suggesting that it may be relevant to appreciate sex differences to cover knowledge gaps. This might lead to improving the diagnosis of risk prediction for pregnancy complications and achieving a more satisfactory monitoring of fetus health, even preventing future diseases in adulthood.

Brain perivascular spaces and autism: clinical and pathogenic implications from an innovative volumetric MRI study.

Introduction: Our single-center case-control study aimed to evaluate the unclear glymphatic system alteration in autism spectrum disorder (ASD) through an innovative neuroimaging tool which allows to segment and quantify perivascular spaces in the white matter (WM-PVS) with filtering of non-structured noise and increase of the contrast-ratio between perivascular spaces and the surrounding parenchyma. Methods: Briefly, files of 65 ASD and 71 control patients were studied. We considered: ASD type, diagnosis and severity level and comorbidities (i.e., intellectual disability, attention-deficit hyperactivity disorder, epilepsy, sleep disturbances). We also examined diagnoses other than ASD and their associated comorbidities in the control group. Results: When males and females with ASD are included together, WM-PVS grade and WM-PVS volume do not significantly differ between the ASD group and the control group overall. We found, instead, that WM-PVS volume is significantly associated with male sex: males had higher WM-PVS volume compared to females (p = 0.01). WM-PVS dilation is also non-significantly associated with ASD severity and younger age (< 4 years). In ASD patients, higher WM-PVS volume was related with insomnia whereas no relation was found with epilepsy or IQ. Discussion: We concluded that WM-PVS dilation can be a neuroimaging feature of male ASD patients, particularly the youngest and most severe ones, which may rely on male-specific risk factors acting early during neurodevelopment, such as a transient excess of extra-axial CSF volume. Our findings can corroborate the well-known strong male epidemiological preponderance of autism worldwide.

Smoking and combined oral contraceptives should be considered as an independent variable in sex and gender-oriented studies.

The influence of sex combined with smoking and combined oral contraceptives (COC) use on atherogenic indexes is scarcely studied. Thus, traditional lipid parameters were measured, and non-traditional atherogenic indexes were calculated in a young and healthy population of men, COC-free women, and COC users. Total cholesterol (TChol), low-density lipoprotein (LDL), high-density lipoprotein (HDL), and HDL/LDL ratio were lower in men, while triglycerides (TG)/HDL ratio, atherogenic index of plasma (AIP), Castelli's Risk Index I (CRII) and CRI-II, atherogenic coefficient (AC), creatinine, creatinine clearance, and estimated glomerular filtration rate (eGFR) were higher in men. The use of COC modified TChol, HDL, TG, TG/HDL, and AIP which had significantly higher values in COC users. In addition, TG were also increased in COC users in comparison with men. Smoking reduced sexually divergent parameters: BMI, TG, HDL/LDL, TG/HDL, AIP, CRII, CRI-II, and AC became similar among the three cohorts, losing the reported sex differences. Smoking also reduced differences in TChol, HDL, TG, and AIP between COC-free women and COC users, but it does not affect CRII, CRI-II, creatinine, creatinine clearance, and eGFR, underlining that COC users and COC-free women have to be considered two different populations. Our results represent a complex landscape suggesting that for both sexes smoking should be an independent variable in medical studies. Moreover, in women, the use of COC evidenced two different cohorts. Thus, more variables should be considered during a single study indicating that sex, smoking, and COC should be studied together to get a picture of the real-life context.

Human monocytes respond to lipopolysaccharide (LPS) stimulation in a sex-dependent manner.

Monocytes play a critical role in inflammation and immune response, their activity being sex-dependent. However, the basis of sex differences is not well understood. Therefore, we investigated the lipopolysaccharide (LPS) effects on tumor necrosis factor-α (TNF-α) release, autophagy, and chemotaxis in freshly isolated monocytes from healthy young men and women. In basal conditions, male and female monocytes had similar TNF-α release, chemotaxis, and estrogen receptors (ER-α) and ER-ß expression, while the LC3II/I ratio was significantly higher in males. LPS treatment induced qualitative and quantitative sex differences. It reduced autophagy and increased TNF-α release only in male monocytes, while, chemotaxis was significantly influenced only in female cells. Moreover, it reduced the expression of ER-α only in female cells, while ER-ß expression was reduced in both sexes, but more markedly in female cells. Finally, the interplay between LPS treatment and 17-ß-estradiol (E2 ) was present only in female cells. Globally, these findings expand the concept that sex plays a role in regulating monocytes' functions, being sex differences cell- and parameter-specific.

Low back pain prevalence and risk factors in Italian adolescent male soccer players: results from an online survey.

BACKGROUND: The purpose of this retrospective survey was to analyze the prevalence of low back pain (LBP) among Italian adolescent soccer players, and to identify potential risk factors. METHODS: Participants were requested to answer an online survey based on the standardized Nordic questionnaires for musculoskeletal symptoms. RESULTS: Data were obtained from 204 male soccer players aged 14-17 years competing at the national and regional level. More than half of the players had experienced LBP in their lives. One-way ANOVA revealed that the players with LBP were taller, heavier and with a higher BMI (all P values<0.00001). When considering the playing position, ANOVA revealed that 14-15 years-old strikers displayed higher LBP scores than all other roles (all P values<0.05). Accordingly, strikers were exposed to a higher risk of LBP than midfielders (RR=1.48; 95% CI: 1.10-2.01; P=0.01) and goalkeepers (RR=1.48; 95% CI: 1.02-2.971; P=0.04), but not defenders (RR=1.23; 95% CI: 0.93-1.63; P=0.15). Within the 14-15 age-class, strikers were, again, those most exposed to LBP risk (all P values<0.05). CONCLUSIONS: Anthropometric and soccer-related features should be monitored to ensure early identification of potential risk factors for LBP. This information should be considered along with the specific playing position as strikers emerged as the roles most exposed to LBP risk.

Sex Differences in X-ray-Induced Endothelial Damage: Effect of Taurine and N-Acetylcysteine.

Ionizing radiation (IR) can induce some associated pathological conditions due to numerous cell damages. The influence of sex is scarcely known, and even less known is whether the effect of antioxidants is sex-dependent. Given the increased use of IR, we investigated whether male human umbilical vein endothelial cells (MHUVECs) and female human umbilical vein endothelial cells (FHUVECs) respond differently to IR exposure and whether the antioxidants 10 mM taurine (TAU) and 5 mM N-acetylcysteine (NAC) can prevent IR-induced damage in a sex-dependent way. In untreated cells, sex differences were observed only during autophagy, which was higher in FHUVECs. In non-irradiated cells, preincubation with TAU and NAC did not modify viability, lactate dehydrogenase (LDH) release, migration, or autophagy, whereas only NAC increased malondialdehyde (MDA) levels in FHUVECs. X-ray irradiation increased LDH release and reduced viability and migration in a sex-independent manner. TAU and NAC did not affect viability while reduced LDH release in irradiated cells: they have the same protective effect in FHUVECs, while, TAU was more protective than NAC in male cells.. Moreover, TAU and NAC significantly promoted the closure of wounds in both sexes in irradiated cells, but NAC was more effective at doing this in FHUVECs. In irradiated cells, TAU did not change autophagy, while NAC attenuated the differences between the sexes. Finally, NAC significantly decreased MDA in MHUVECs and increased MDA in FHUVECs. In conclusion, FHUVECs appear to be more susceptible to IR damage, and the effects of the two antioxidants present some sex differences, suggesting the need to study the influence of sex in radiation mitigators.

The Person's Care Requires a Sex and Gender Approach.

There is an urgent need to optimize pharmacology therapy with a consideration of high interindividual variability and economic costs. A sex-gender approach (which considers men, women, and people of diverse gender identities) and the assessment of differences in sex and gender promote global health, avoiding systematic errors that generate results with low validity. Care for people should consider the single individual and his or her past and present life experiences, as well as his or her relationship with care providers. Therefore, intersectoral and interdisciplinary studies are urgently required. It is desirable to create teams made up of men and women to meet the needs of both. Finally, it is also necessary to build an alliance among regulatory and ethic authorities, statistics, informatics, the healthcare system and providers, researchers, the pharmaceutical and diagnostic industries, decision makers, and patients to overcome the gender gap in medicine and to take real care of a person in an appropriate manner.

The Italian law on body donation: A position paper of the Italian College of Anatomists.

In Italy, recent legislation (Law No. 10/2020) has tuned regulations concerning the donation of one's postmortem body and tissues for study, training, and scientific research purposes. This study discusses several specific issues to optimise the applicability and effectiveness of such an important, novel regulatory setting. Critical issues arise concerning the learners, the type of training and teaching activities that can be planned, the position of academic anatomy institutes, the role of family members in the donation process, the time frame of the donation process, the eligibility of partial donation, or the simultaneous donation of organs and tissues to patients awaiting transplantation. In particular, a universal time limit for donations (i.e., one year) makes it impossible to plan the long-term use of specific body parts, which could be effectively preserved for the advanced teaching and training of medical students and surgeons. The abovementioned conditions lead to the limited use of corpses, thus resulting in the inefficiency of the whole system of body donation. Overall, the donors' scope for the donation of their body could be best honoured by a more flexible and tuneable approach that can be used on a case-by-case basis. Furthermore, it is deemed necessary to closely monitor the events scheduled for corpses in public nonacademic institutions or private enterprises. This paper presents useful insights from Italian anatomists with the hope of providing inspiration for drafting the regulations. In conclusion, this paper focuses on the critical issues derived from the recently introduced Italian law on the donation and use of the body after death and provides suggestions to lawmakers for future implementations.

Sex Affects Human Premature Neonates' Blood Metabolome According to Gestational Age, Parenteral Nutrition, and Caffeine Treatment.

Prematurity is the leading cause of neonatal deaths and high economic costs; it depends on numerous biological and social factors, and is highly prevalent in males. Several factors can affect the metabolome of premature infants. Accordingly, the aim of the present study was to analyze the role played by gestational age (GA), parenteral nutrition (PN), and caffeine treatment in sex-related differences of blood metabolome of premature neonates through a MS/MS-based targeted metabolomic approach for the detection of amino acids and acylcarnitines in dried blood spots. GA affected the blood metabolome of premature neonates: male and female very premature infants (VPI) diverged in amino acids but not in acylcarnitines, whereas the opposite was observed in moderate or late preterm infants (MLPI). Moreover, an important reduction of metabolites was observed in female VPI fed with PN, suggesting that PN might not satisfy an infant's nutritional needs. Caffeine showed the highest significant impact on metabolite levels of male MLPI. This study proves the presence of a sex-dependent metabolome in premature infants, which is affected by GA and pharmacological treatment (e.g., caffeine). Furthermore, it describes an integrated relationship among several features of physiology and health.

Human Umbilical Cord: Information Mine in Sex-Specific Medicine.

Biological differences between sexes should be considered in all stages of research, as sexual dimorphism starts in utero leading to sex-specific fetal programming. In numerous biomedical fields, there is still a lack of stratification by sex despite primary cultured cells retaining memory of the sex and of the donor. The sex of donors in biological research must be known because variations in cells and cellular components can be used as endpoints, biomarkers and/or targets of pharmacological studies. This selective review focuses on the current findings regarding sex differences observed in the umbilical cord, a widely used source of research samples, both in the blood and in the circulating cells, as well as in the different cellular models obtainable from it. Moreover, an overview on sex differences in fetal programming is reported. As it emerges that the sex variable is still often forgotten in experimental models, we suggest that it should be mandatory to adopt sex-oriented research, because only awareness of these issues can lead to innovative research.

Combined oral contraceptives modify the effect of smoking on inflammatory cellular indexes and endothelial function in healthy subjects.

Little information is available on the influence of sex in combination with smoking habits and combined oral contraceptives (COC) use on cellular inflammatory indexes such as neutrophil/lymphocyte ratio (NLR), derived NRL (dNLR), platelet/lymphocyte ratio (PLR), monocyte/lymphocyte ratio (MLR), mean platelet volume/platelet count (MPV/PLT), aggregate inflammation systemic index (AISI), and systemic inflammation response index (SIRI), which are cost-effective biomarkers to assessing inflammation. Therefore, the effect of COC was studied alone or in association with smoking and compared with results from healthy COC-free women and men. Furthermore, the association of cellular inflammatory indexes with endothelial function (arginine (Arg), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and lipid peroxidation (malondialdehyde MDA) biomarkers was evaluated. Blood was collected for hematological and biochemical analysis, which were used to calculate PLR, NLR, dNLR, MLR, MPV/PLT, AISI, and SIRI. Serum samples were assayed for Arg, ADMA, SDMA, and MDA. Monocytes, MLR, SIRI, and MPV/PLT were higher in men, while PLT count was higher in women. COC use increased lymphocytes and lowered PLR and MLR. Smoking reduced sexually divergent parameters, especially in COC users: smoking and non-smoking COC-free women displayed six divergent parameters, while COC users displayed only two (monocytes and MPV). In addition, COC affected endothelial function, reducing ADMA and Arg. Moreover, COC-free women had lower Arg levels than men. In conclusion, COC use strongly influence the effects of tobacco smoking, which are sex and parameter specific. Further, these data stress that COC use and smoking attitude select different cohorts indicating that sex and gender studies need intersectionality.

Deregulation of cancer-stem-cell-associated miRNAs in tissues and sera of colorectal cancer patients.

Colorectal cancer (CRC) is a deadly tumour in Western countries characterized by high cellular/molecular heterogeneity. Cancer stem cells (CSC) act in cancer recurrence, drug-resistance and in metastatic epithelial-to-mesenchymal transition. microRNAs (miRNAs) contribute to cancer is increasing, and miRNA roles in CSC phenotype and fate and their utility as CRC biomarkers have also been reported. Here, we investigated miR-21, miR-221, miR-18a, miR-210, miR-31, miR-34a, miR-10b and miR-16 expression in experimental ALDH+ and CD44+/CD326+ colorectal CSCs obtained from the human CRC cell lines HCT-116, HT-29 and T-84. Then, we moved our analysis in cancer tissue (CT), healthy tissue (HT) and serum (S) of adult CRC patients (n=12), determining relationships with clinical parameters (age, sex, metastasis, biochemical serum markers). Specific miRNA patterns were evident in vitro (normal, monolayers and CSCs) and in patients' samples stratified by TNM stage (LOW vs HIGH) or metastasis (Met vs no-Met). miR-21, miR-210, miR-34a upregulation ad miR-16 dowregulation associated with the CSCs phenotype. miR-31b robustly overexpressed in monolayers and CSCs, and in CT ad S of HIGH grade and Met patients, suggesting a role as marker of CRC progression and metastasis. miR-18a upregulated in all cancer models and associated to CSC phenotype, and to metastasis and age in patients. miR-10b downregulated in CT and S of LOW/HIGH grade and no-Met patients. Our results identify miRNAs useful as colorectal CSC biomarker and that miR-21, miR-210, miR-10b and miR-31b are promising markers of CRC. A specific role of miR-18a as metastatic CRC serum biomarker in adult patients was also highlighted.

Neuroanatomy, the Achille's Heel of Medical Students. A Systematic Analysis of Educational Strategies for the Teaching of Neuroanatomy.

Neuroanatomy has been deemed crucial for clinical neurosciences. It has been one of the most challenging parts of the anatomical curriculum and is one of the causes of "neurophobia," whose main implication is a negative influence on the choice of neurology in the near future. In the last decades, several educational strategies have been identified to improve the skills of students and to promote a deep learning. The aim of this study was to systematically review the literature to identify the most effective method/s to teach human neuroanatomy. The search was restricted to publications written in English language and to articles describing teaching tools in undergraduate medical courses from January 2006 through December 2017. The primary outcome was the observation of improvement of anatomical knowledge in undergraduate medical students. Secondary outcomes were the amelioration of long-term retention knowledge and the grade of satisfaction of students. Among 18 selected studies, 44.4% have used three-dimensional (3D) teaching tools, 16.6% near peer teaching tool, 5.55% flipped classroom tool, 5.55% applied neuroanatomy elective course, 5.55% equivalence-based instruction-rote learning, 5.55% mobile augmented reality, 5.55% inquiry-based clinical case, 5.55% cadaver dissection, and 5.55% Twitter. The high in-between study heterogeneity was the main issue to identify the most helpful teaching tool to improve neuroanatomical knowledge among medical students. Data from this study suggest that a combination of multiple pedagogical resources seems to be the more advantageous for teaching neuroanatomy.

Epigenetics, Stem Cells, and Autophagy: Exploring a Path Involving miRNA.

MiRNAs, a small family of non-coding RNA, are now emerging as regulators of stem cell pluripotency, differentiation, and autophagy, thus controlling stem cell behavior. Stem cells are undifferentiated elements capable to acquire specific phenotype under different kind of stimuli, being a main tool for regenerative medicine. Within this context, we have previously shown that stem cells isolated from Wharton jelly multipotent stem cells (WJ-MSCs) exhibit gender differences in the expression of the stemness related gene OCT4 and the epigenetic modulator gene DNA-Methyltransferase (DNMT1). Here, we further analyze this gender difference, evaluating adipogenic and osteogenic differentiation potential, autophagic process, and expression of miR-145, miR-148a, and miR-185 in WJ-MSCs derived from males and females. These miRNAs were selected since they are involved in OCT4 and DNMT1 gene expression, and in stem cell differentiation. Our results indicate a difference in the regulatory circuit involving miR-148a/DNMT1/OCT4 autophagy in male WJ-MSCs as compared to female cells. Moreover, no difference was detected in the expression of the two-differentiation regulating miRNA (miR-145 and miR-185). Taken together, our results highlight a different behavior of WJ-MSCs from males and females, disclosing the chance to better understand cellular processes as autophagy and stemness, usable for future clinical applications.

Orchestrating stem cell fate: Novel tools for regenerative medicine.

Mesenchymal stem cells are undifferentiated cells able to acquire different phenotypes under specific stimuli. In vitro manipulation of these cells is focused on understanding stem cell behavior, proliferation and pluripotency. Latest advances in the field of stem cells concern epigenetics and its role in maintaining self-renewal and differentiation capabilities. Chemical and physical stimuli can modulate cell commitment, acting on gene expression of Oct-4, Sox-2 and Nanog, the main stemness markers, and tissue-lineage specific genes. This activation or repression is related to the activity of chromatin-remodeling factors and epigenetic regulators, new targets of many cell therapies. The aim of this review is to afford a view of the current state of in vitro and in vivo stem cell applications, highlighting the strategies used to influence stem cell commitment for current and future cell therapies. Identifying the molecular mechanisms controlling stem cell fate could open up novel strategies for tissue repairing processes and other clinical applications.

Association of Klippel-Feil syndrome, Dandy-Walker malformation, spina bifida: A case report.

Klippel-Feil syndrome is a congenital malformation characterized by the fusion of at least 2 cervical vertebrae. It may occur in association with other clinical syndromes and disorders. We describe a case of prenatal diagnosis of a Klippel-Feil syndrome with Dandy-Walker malformation, and spina bifida, proved by ultrasound examination. A postmortem x-ray and autopsy were performed in a female fetus of 16 + 6 weeks of gestation: several malformations have been discovered. To the best of our knowledge, no similar cases have been reported in the medical literature. This case report underscores the importance of a careful ultrasound screening during pregnancy for an adequate diagnostic and therapeutic management.

Posterior tibial perforators relationship with superficial nerves and veins: A cadaver study.

BACKGROUND: Most authors have evaluated the location of lower leg arterial perforators, but little is still known about the relationship between the arterial network and great saphenous vein (GSV) and saphenous nerve (SN). The aim of this study is to evaluate the relationship between the arterial network of the posterior tibial artery perforators, the cutaneous nerves, and the superficial venous system in the lower one third of the leg. METHODS: Eighteen lower limbs from cadavers were used for this study. The arterial and venous compartment were selectively injected with a mixture of barium sulfate and epoxy. The specimen were CT scanned and the superficial veins, nerves, and the arterial perforators were dissected. RESULTS: A large perforator of the posterior tibial artery was found at a mean distance of 6.23 cm ± 0.88, with a 95% CI: 5.79-6.67, from the medial malleolus. The average diameter was 0.9 mm ± 0.17, with a 95% CI: 0.81-0.99. In 67% the connection of the venae comitantes to the superficial venous system was established with the GSV, in the other cases, with Leonardo's vein. Both dissection and imaging studies showed perineural interperforator connections along the branches of SN in all the specimens examined. CONCLUSIONS: The distribution pattern of posterior tibial artery perforators followed the superficial nerves in this region. There is an interperforator anastomotic network along the SN. The various patterns of the venous drainage system, in relationship to the distribution of the branches of posterior tibial artery perforators, have been clarified.