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Breastfeeding and human milk (HM) are critically important to maternal, infant and population health. This paper summarizes the proceedings of a workshop that convened a multidisciplinary panel of researchers to identify key priorities and anticipated breakthroughs in breastfeeding and HM research, discuss perceived barriers and challenges to achieving these breakthroughs and propose a constructive action plan to maximize the impact of future research in this field. Priority research areas identified were as follows: (1) addressing low breastfeeding rates and inequities using mixed methods, community partnerships and implementation science approaches; (2) improving awareness of evidence‐based benefits, challenges and complexities of breastfeeding and HM among health practitioners and the public; (3) identifying differential impacts of alternative modes of HM feeding including expressed/pumped milk, donor milk and shared milk; and (4) developing a mechanistic understanding of the health effects of breastfeeding and the contributors to HM composition and variability. Key barriers and challenges included (1) overcoming methodological limitations of epidemiological breastfeeding research and mechanistic HM research; (2) counteracting ‘breastfeeding denialism’ arising from negative personal breastfeeding experiences; (3) distinguishing and aligning research and advocacy efforts; and (4) managing real and perceived conflicts of interest. To advance research on breastfeeding and HM and maximize the reach and impact of this research, larger investments are needed, interdisciplinary collaboration is essential, and the scientific community must engage families and other stakeholders in research planning and knowledge translation.

When mother's own milk (MOM) is unavailable or insufficient, donor human milk (DHM) is recommended as the next best alternative for low birthweight infants. DHM use for healthy, term infants is increasing, but evidence for growth and tolerability is limited. This retrospective study evaluated growth in term infants in the community who received DHM from a UK milk bank. Mothers of infants receiving DHM between 2017 and 2019 were contacted (n = 49), and 31 (63.2%) agreed to participate. Fourteen infants received DHM as a supplement to other feeds (MOM and/or infant formula) and 17 were exclusively fed DHM where breastfeeding was impossible (range: 3–6 weeks). Growth was assessed by deriving z‐scores using the WHO standard for infant growth and compared with 200 exclusively breastfed infants. Multivariate regression analysis revealed no feeding method‐specific association between z‐score and age, nor between weight and age, suggesting that z‐scores and growth velocity were not affected by feeding exclusive MOM, supplemental DHM or exclusive DHM. DHM was well‐tolerated with no adverse events that led to early cessation. After receiving supplemental DHM group, 63% of infants whose mothers had no physical barrier to breastfeeding (5/8 infants) were exclusively breastfed. This novel study reports adequate growth outcomes of healthy nonhospitalized infants receiving DHM, either as the sole milk source or supplement. Prospective studies are needed to confirm whether DHM is a suitable feeding alternative for term infants in the community, optimal durations, as well as the impact of DHM availability on breastfeeding rates and maternal mental health., DHM recipients were compared with the who infant growth standard (World Health Organization, 2020) by deriving z‐scores. The WHO Anthro Macro (World Health Organization, 2011 ) was used to calculate weight‐for‐age, length‐for‐age and head circumference‐for‐age z‐scores; z‐scores are the standard deviation of an infant's anthropometric measurements when compared with the WHO standard for infant growth and are gender and age specific.

If maternal milk is unavailable, the World Health Organization recommends that the first alternative should be pasteurised donor human milk (DHM). Human milk banks (HMBs) screen and recruit milk donors, and DHM principally feeds very low birth weight babies, reducing the risk of complications and supporting maternal breastfeeding where used alongside optimal lactation support. The COVID‐19 pandemic has presented a range of challenges to HMBs worldwide. This study aimed to understand the impacts of the pandemic on HMB services and develop initial guidance regarding risk limitation. A Virtual Collaborative Network (VCN) comprising over 80 HMB leaders from 36 countries was formed in March 2020 and included academics and nongovernmental organisations. Individual milk banks, national networks and regional associations submitted data regarding the number of HMBs, volume of DHM produced and number of recipients in each global region. Estimates were calculated in the context of missing or incomplete data. Through open‐ended questioning, the experiences of milk banks from each country in the first 2 months of the pandemic were collected and major themes identified. According to data collected from 446 individual HMBs, more than 800,000 infants receive DHM worldwide each year. Seven pandemic‐related specific vulnerabilities to service provision were identified, including sufficient donors, prescreening disruption, DHM availability, logistics, communication, safe handling and contingency planning, which were highly context‐dependent. The VCN now plans a formal consensus approach to the optimal response of HMBs to new pathogens using crowdsourced data, enabling the benchmarking of future strategies to support DHM access and neonatal health in future emergencies.

Sparse data exist regarding the normal range of composition of maternal milk beyond the first postnatal weeks. This single timepoint, observational study in collaboration with the &lsquo, Parenting Science Gang&rsquo, citizen science group evaluated the metabolite and bacterial composition of human milk from 62 participants (infants aged 3&ndash, 48 months), nearly 3 years longer than previous studies. We utilised rapid evaporative ionisation mass spectrometry (REIMS) for metabolic fingerprinting and 16S rRNA gene metataxonomics for microbiome composition analysis. Milk expression volumes were significantly lower beyond 24 months of lactation, but there were no corresponding changes in bacterial load, composition, or whole-scale metabolomic fingerprint. Some individual metabolite features (~14%) showed altered abundances in nursling age groups above 24 months. Neither milk expression method nor nursling sex affected metabolite and metataxonomic fingerprints. Self-reported lifestyle factors, including diet and physical traits, had minimal impact on metabolite and metataxonomic fingerprints. Our findings suggest remarkable consistency in human milk composition over natural-term lactation. The results add to previous studies suggesting that milk donation can continue up to 24 months postnatally. Future longitudinal studies will confirm the inter-individual and temporal nature of compositional variations and the use of donor milk as a personalised therapeutic.

Background Donor human milk is the World Health Organization’s recommendation for infant feeding when the mother’s own breast milk is unavailable. Breast milk has been shown to reduce morbidity and mortality and in low birthweight infants, donor milk reduces the incidence of necrotising enterocolitis, late onset sepsis and improves outcomes. There is a paucity of literature documenting outcomes of using donor human milk in older children who need additional support for a variety of health issues. Case presentation A series of seven case studies is presented of orphaned and abandoned children, many of whom were either HIV exposed or positive. All children were fed with pasteurised donor human milk at a transition home and their progress reported. Conclusions Although detailed medical records were not always available, the case studies provide anecdotal evidence of the protective effects of donor human milk against failure to thrive, diarrhoea, atopic dermatitis, and opportunistic infections.

OBJECTIVES: Aortic stiffness is an emerging risk factor for cardiovascular disease. The predictive value of aortic pulse wave velocity (PWV) for quality of life (QoL) and severity of surgical aortic valve stenosis (AS) have not been examined. METHODS: PWV was measured in patients undergoing aortic valve replacement (AVR) for AS. QoL [SF-36 and European QoL 5-dimensions (EQ-5D) questionnaires] was assessed pre- and postoperatively (409 ± 159 days). PWV was analysed: (i) as a continuous variable and (ii) as a dichotomous variable (PWV-norm and PWV-high groups) according to the published normal reference value. RESULTS: Fifty-six patients (16 females), mean age of 71 ± 8.4 years, were included, and 50 (89%) patients completed follow-up. The two groups were matched for age, gender and classical haemodynamic measurements. There was no significant relation between AS severity and PWV. PWV-norm patients (n= 35) scored significantly better than PWV-high (n= 21) patients in the EQ-5D visual analogue scale and the EQ-5D index pre- (P< 0.001 and P= 0.03, respectively) and postoperatively (P< 0.001 for both). In SF-36, PWV-norm group scored better than PWV-high group in physical health domains preoperatively and in all domains postoperatively. Spearman’s correlation was significant between PWV and QoL component summaries pre- and postoperatively. Among PWV, age and gender, multiple regression analysis demonstrated PWV to be independently related to QoL pre- and postoperatively (P-values ranged from

Background Most biomarkers of exposure tend to have short half-lives. This includes cotinine, a metabolite of nicotine widely used to assess smoke exposure. Cotinine is thus unsuitable as a determinant of past exposure to cigarette smoke. Methods We used bisulphite pyrosequencing of a set of four genomic loci (AHRR, 6p21, and two at 2q37) that had differential DNA methylation levels in peripheral blood DNA dependent on tobacco exposure to create a predictive model of smoking status. Results Combining four gene loci into a single methylation index provided high positive predictive and sensitivity values for predicting former smoking status in both test (n = 81) and validation (n = 180) sample sets. Conclusions This study provides a direct molecular measure of prior exposure to tobacco that can be performed using the quantitative approach of bisulphite pyrosequencing. Epigenetic changes that are detectable in blood may more generally act as molecular biomarkers for other exposures that are also difficult to quantify in epidemiological studies.

Epigenetics is the study of all mechanisms that regulate gene transcription and genome stability that are maintained throughout the cell division, but do not include the DNA sequence itself. The best-studied epigenetic mechanism to date is DNA methylation, where methyl groups are added to the cytosine base within cytosine–guanine dinucleotides (CpG sites). CpGs are frequently clustered in high density (CpG islands (CGIs)) at the promoter of over half of all genes. Current knowledge of transcriptional regulation by DNA methylation centres on its role at the promoter where unmethylated CGIs are present at most actively transcribed genes, whereas hypermethylation of the promoter results in gene repression. Over the last 5 years, research has gradually incorporated a broader understanding that methylation patterns across the gene (so-called intragenic or gene body methylation) may have a role in transcriptional regulation and efficiency. Numerous genome-wide DNA methylation profiling studies now support this notion, although whether DNA methylation patterns are a cause or consequence of other regulatory mechanisms is not yet clear. This review will examine the evidence for the function of intragenic methylation in gene transcription, and discuss the significance of this in carcinogenesis and for the future use of therapies targeted against DNA methylation.

Exomphalos is a midline defect, with a viable sac composed of amnion and peritoneum containing herniated abdominal contents with an incidence of about 1 in 4,000 live births. Associated major abnormalities can be karyotypic, syndromic or structural in up to 70% of cases. The aim of this study is to determine the factors that influence survival of antenatally diagnosed exomphalos. All antenatally diagnosed and postnatally confirmed exomphalos registered with our fetal medicine unit, during 2002–2007, were reviewed. Both prenatal and postnatal outcomes were analysed. Of 88 cases identified with exomphalos, 85 were prenatally diagnosed. Fifty-five of them died in utero (45 terminations, 5 spontaneous abortions and 5 still births). There were 33 live births (37.5%), 7 of which were premature (30–35/40 gestation). Five babies died before coming to surgery (all with major exomphalos as well as abnormal karyotype) while 28 were operated upon. Fourteen cases with minor exomphalos, all isolated, were primarily closed and all survived to discharge. Of 14 babies with major exomphalos, 4 were closed primarily. Nine required silo formation and six successfully underwent secondary closure (one of which had a prenatal diagnosis of giant ruptured exomphalos). Three died before closure, two from sepsis and multi-organ failure, and one from an undiagnosed tracheo-oesophalgeal cleft. All three deaths had antenatally diagnosed giant ruptured exomphalos and were less than 34/40 weeks gestation. One baby was managed conservatively with antiseptic solution applied to the sac and left to heal by secondary intention. There were 17 cases of isolated exomphalos (with no other structural abnormalities), all of which survived. Antenatal diagnosis of exomphalos is 96% sensitive. Severe karyotypic and structural abnormalities were present in all intra-uterine and early postnatal deaths. Overall survival to discharge was 28%. Both minor and isolated exomphalos carried a good prognosis. Isolated exomphalos was a better prognostic factor than severity of the exomphalos itself. Ruptured giant exomphalos were associated with a poorer outcome especially in premature babies.

Germline pathogenic mutations in BRCA1 increase risk of developing breast cancer. Screening for mutations in BRCA1 frequently identifies sequence variants of unknown pathogenicity and recent work has aimed to develop methods for determining pathogenicity. We previously observed that tumor DNA methylation can differentiate BRCA1-mutated from BRCA1-wild type tumors. We hypothesized that we could predict pathogenicity of variants based on DNA methylation profiles of tumors that had arisen in carriers of unclassified variants. We selected 150 FFPE breast tumor DNA samples [47 BRCA1 pathogenic mutation carriers, 65 BRCAx (BRCA1-wild type), 38 BRCA1 test variants] and analyzed a subset (n=54) using the Illumina 450K methylation platform, using the remaining samples for bisulphite pyrosequencing validation. Three validated markers (BACH2, C8orf31, and LOC654342) were combined with sequence bioinformatics in a model to predict pathogenicity of 27 variants (independent test set). Predictions were compared with standard multifactorial likelihood analysis. Prediction was consistent for c.5194-12G>A (IVS 19-12 G>A) (P>0.99); 13 variants were considered not pathogenic or likely not pathogenic using both approaches. We conclude that tumor DNA methylation data alone has potential to be used in prediction of BRCA1 variant pathogenicity but is not independent of estrogen receptor status and grade, which are used in current multifactorial models to predict pathogenicity.

Background Extrapolation of rat testicular descent studies to humans has been criticized because of anatomical differences of the cremaster muscle. Human cremaster is described as a thin strip rather than a large, complete sac as in rats, which is proposed to be more important in propelling the testis during descent. This study investigated cremaster muscle anatomy and ontogeny in both normal and cryptorchid rat models. Methods Gubernacula from 4 groups of neonatal rats were sectioned longitudinally and transversely: normal Sprague-Dawley, capsaicin pretreated, flutamide pretreated, and congenital cryptorchid rats. Gubernacula were stained with hematoxylin-eosin, Masson trichrome, and desmin immunohistochemistry to study muscle development. Results Myoblasts are more numerous at the gubernacular tip, whereas the most differentiated muscle is proximal. Rat cremaster develops as an elongated strip rather than a complete sac derived from abdominal wall muscles. Flutamide and capsaicin pretreatment disrupts development. Conclusion Rat cremaster muscle develops as a strip, bearing close resemblance to human cremaster muscle, permitting extrapolation of cremaster function to human testicular descent. The cremaster muscle appears to differentiate from the gubernacular tip during elongation to the scrotum, and requires intact sensory innervation and androgen.

John Hilton was the foremost anatomist of his day. From only humble beginnings he became an anatomy demonstrator at Guy's Hospital. When appointed Surgeon to Guy's Hospital, his meticulous clinical skills, arising from his depth of anatomical knowledge, led him to develop many anatomical principles culminating in a series of lectures on ‘Rest and Pain’. For the first time the clinical importance of each was highlighted in surgical practice. By public demand the lectures were published as a book, still in print today, which brought a new emphasis to clinical anatomy that would permeate surgery thereafter. He became President of the Royal College of Surgeons and was Surgeon Extraordinary to Queen Victoria. A substantial review of his life has not been published; with the present decline in anatomical teaching, we can learn much from understanding a surgeon who dedicated his life to anatomy.

Background DNA methylation variability regions (MVRs) across the oestrogen receptor alpha (ESR1) gene have been identified in peripheral blood cells from breast cancer patients and healthy individuals. In contrast to promoter methylation, gene body methylation may be important in maintaining active transcription. This study aimed to assess MVRs in ESR1 in breast cancer cell lines, tumour biopsies and exfoliated epithelial cells from expressed breast milk (EBM), to determine their significance for ESR1 transcription. Methods DNA methylation levels in eight MVRs across ESR1 were assessed by pyrosequencing bisulphite-converted DNA from three oestrogen receptor (ER)-positive and three ER-negative breast cancer cell lines. DNA methylation and expression were assessed following treatment with DAC (1 μM), or DMSO (controls). ESR1 methylation levels were also assayed in DNA from 155 invasive ductal carcinoma biopsies provided by the Breast Cancer Campaign Tissue Bank, and validated with DNA methylation profiles from the TCGA breast tumours (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450 K (n = 36) and pyrosequencing in a further 53 donor samples. ESR1 mRNA levels were measured by qRT-PCR. Results We show that ER-positive cell lines had unmethylated ESR1 promoter regions and highly methylated intragenic regions (median, 80.45%) while ER-negative cells had methylated promoters and lower intragenic methylation levels (median, 38.62%). DAC treatment increased ESR1 expression in ER-negative cells, but significantly reduced methylation and expression of ESR1 in ER-positive cells. The ESR1 promoter was unmethylated in breast tumour biopsies with high levels of intragenic methylation, independent of ER status. However, ESR1 methylation in the strongly ER-positive EBM DNA samples were very similar to ER-positive tumour cell lines. Conclusion DAC treatment inhibited ESR1 transcription in cells with an unmethylated ESR1 promoter and reduced intragenic DNA methylation. Intragenic methylation levels correlated with ESR1 expression in homogenous cell populations (cell lines and exfoliated primary breast epithelial cells), but not in heterogeneous tumour biopsies, highlighting the significant differences between the in vivo tumour microenvironment and individual homogenous cell types. These findings emphasise the need for care when choosing material for epigenetic research and highlights the presence of aberrant intragenic methylation levels in tumour tissue. Electronic supplementary material The online version of this article (doi:10.1186/s12885-015-1335-5) contains supplementary material, which is available to authorized users.

Cell proliferation at the gubernacular tip increases in response to exogenous calcitonin gene-related peptide (CGRP) during migration into the scrotum. Calcitonin gene-related peptide is contained in the masculinized sensory branches of the genitofemoral nerve. We tested the independent effects of chemical sensory nerve disruption and prenatal androgen blockade on the in vitro gubernacular proliferative response to CGRP.Neonatal Sprague-Dawley rats were injected with capsaicin, a sensory nerve toxin, and gubernacula dissected 2 days later (D2). Sprague-Dawley dams were injected with flutamide, an androgen receptor antagonist, between days 15 and 19 of gestation. Flutamide pretreated males, and normal neonatal rats, were dissected at D0 and D2. Gubernacula were cultured for 24 hours +/- CGRP, pulse-labelled for the last 4 hours of culture with bromodeoxyuridine, a thymidine analogue marker for DNA replication, sectioned, and stained using immunohistochemistry. The percentage of positively staining cells in the gubernacular tip was calculated from three separate counts by a blinded observer and compared using analysis of variance.Normal D0 gubernacular tips showed a significant response of cell proliferation to exogenous CGRP (34% vs 9% in controls, P.001), which resolved by day 2 (16% vs 12%, P.05). Calcitonin gene-related peptide markedly increased cell proliferation in D2 capsaicin pretreated gubernacula compared with controls (25% vs 14%, P.01) and normal D2 gubernacula cultured with CGRP (P.01). D0 flutamide pretreated cultured with CGRP showed no increase in cell proliferation compared with controls (16% vs 11%), but a small response was seen by D2 (19% vs 9%, P.05). There was no significant difference between proliferation rates in the control groups.Sensory innervation interruption sensitises the gubernaculum to exogenous CGRP, suggesting upregulation of CGRP receptors. In contrast, androgen blockade abolishes the increased rate of cell proliferation within the gubernacular tip. We conclude that androgens are necessary to "preprogramme" the proliferative response of the gubernaculum to CGRP.

A single cytosine-guanine dinucleotide (CpG) site within coagulation factor II (thrombin) receptor-like 3 (F2RL3) was recently found to be hypomethylated in peripheral blood genomic DNA from smokers compared with former and non-smokers. We performed two epigenome-wide association studies (EWAS) nested in a prospective healthy cohort using the Illumina 450K Methylation Beadchip. The two populations consisted of matched pairs of healthy individuals (n = 374), of which half went on to develop breast or colon cancer. The association was analysed between methylation and smoking status, as well as cancer risk. In addition to the same locus in F2RL3, we report several loci that are hypomethylated in smokers compared with former and non-smokers, including an intragenic region of the aryl hydrocarbon receptor repressor gene (AHRR; cg05575921, P = 2.31 × 10(-15); effect size = 14-17%), an intergenic CpG island on 2q37.1 (cg21566642, P = 3.73 × 10(-13); effect size = 12%) and a further intergenic region at 6p21.33 (cg06126421, P = 4.96 × 10(-11), effect size = 7-8%). Bisulphite pyrosequencing validated six loci in a further independent population of healthy individuals (n = 180). Methylation levels in AHRR were also significantly decreased (P < 0.001) and expression increased (P = 0.0047) in the lung tissue of current smokers compared with non-smokers. This was further validated in a mouse model of smoke exposure. We observed an association with breast cancer risk for the 2q37.1 locus (P = 0.003, adjusted for the smoking status), but not for the other loci associated with smoking. These data show that smoking has a direct effect on the epigenome in lung tissue, which is also detectable in peripheral blood DNA and may contribute to cancer risk.

Recent studies of testicular descent suggest not only that the gubernaculum does not initially attach to the scrotum, but also that it must migrate from the groin. Two findings suggest that the gubernaculum may behave like an embryonic limb bud during this phase. First, the active growth centre is at the distal tip of the gubernaculum. Secondly, the gubernaculum is loose in the subcutaneous tissues beneath Scarpa's fascia. The free protrusion of the gubernaculum from the abdominal wall was so reminiscent of a developing embryonic limb bud, we thought that the biological controls of both processus may be similar. This review examines what is known about vertebrate limb bud development, and compares the mechanisms to what has recently been discovered in the gubernaculum. The hypothesis that both processes may be similar is initially consistent with the current facts, encouraging us to investigate this further experimentally.

Introduction: DNA methylation variability regions (MVRs) across the estrogen receptor alpha (ESR1) gene have been identified in peripheral blood cells from breast cancer patients and healthy individuals. In contrast to promoter methylation, gene body methylation may be important in maintaining active transcription. This study aimed to assess MVRs in ESR1 in breast cancer cell lines, tumor biopsies and exfoliated epithelial cells from expressed breast milk (EBM) to determine their significance for ESR1 transcription. Methods: DNA methylation levels in eight MVRs across ESR1 were assessed in pyrosequencing bisulphite-converted DNA from three oestrogen receptor (ER)-positive and three ER-negative breast cancer cell lines. DNA methylation and expression were assessed following treatment with the demethylating agent, decitabine (DAC, 1 μM), or DMSO (controls). ESR1 methylation levels were also assayed in DNA from 155 invasive ductal carcinoma biopsies provided by the Breast Cancer Campaign Tissue Bank, and validated with DNA methylation profiles from the TCGA breast tumors (n = 356 ER-pos, n = 109 ER-neg). DNA methylation was profiled in exfoliated breast epithelial cells from EBM using the Illumina 450K (n = 36) and pyrosequencing in a further 53 donor samples. ESR1 mRNA levels were measured by qRT-PCR. Results: We show that ER-positive cell lines had unmethylated ESR1 promoter regions and highly methylated intragenic regions (median, 80.45%) while ER-negative cells had methylated promoters and lower intragenic methylation levels (median, 38.62%). DAC treatment increased ESR1 expression in ER-negative cells, but significantly reduced methylation and expression of ESR1 in ER-positive cells. The ESR1 promoter was unmethylated in breast tumor biopsies with high levels of intragenic methylation, independent of ER status. However, ESR1 methylation in the strongly ER-positive EBM DNA samples were very similar to ER-positive tumor cell lines. Conclusion: Intragenic methylation levels correlated with ESR1 expression in vitro, but with markedly different methylation patterns between homogenous cell populations and heterogeneous tumour biopsies. DAC treatment inhibited ESR1 transcription in cells with an unmethylated ESR1 promoter along with reduced levels of intragenic DNA methylation. These findings emphasize the need for care when choosing tissue types for epigenetic research and interpreting results from heterogenous tissue. Note: This abstract was not presented at the meeting. Citation Format: NATALIE S. SHENKER, Kirsty J. Flower, Charlotte Wilhelm-Benartzi, Wei Dai, Emma Bell, Mona El Bahrawy, Gillian Weaver, James M. Flanagan, Robert Brown. Transcriptional implications of intragenic DNA methylation in the estrogen receptor alpha gene in breast cancer cells and tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1063. doi:10.1158/1538-7445.AM2015-1063

A major manifestation of chronic allograft failure (CAF) is the accelerated onset of atherosclerotic lesions within the graft. Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene have been implicated in the pathogenesis of native atherosclerosis. This study tested the hypothesis that polymorphisms in eNOS are associated with susceptibility to CAF after cadaveric renal transplantation. The patient cohort comprised 140 renal transplant recipients who had received their transplants between 1985 and 1997 at the Oxford Transplant Centre and included 61 patients with biopsy-proven CAF and 79 with stable graft function for at least 10 years (long-term survivors, LTS). Genotyping for one polymorphism in the promoter region and two polymorphisms in the coding regions of the eNOS gene was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP). No association was found between any genetic variant and the development of CAF, even after stratification for other known risk factors. Statistical analysis revealed that all three polymorphisms were closely linked. We conclude that recipient eNOS gene polymorphisms do not alter the risk of CAF after renal transplantation.