Many of the diseases in Western civilization may. Siege of Leningrad Survivors Phenotyping and Biospecimen Collection ORIGINAL ARTICLE

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1 BIO ver9-Rotar_1P.3d 08/06/15 2:17pm Page 1 BIOPRESERVATION AND BIOBANKING Volume 00, Number 00, 2015 ª Mary Ann Liebert, Inc. DOI: /bio BIO ver9-Rotar_1P Type: research-article ORIGINAL ARTICLE Siege of Leningrad Survivors Phenotyping and Biospecimen Collection Oxana P. Rotar, 1 Ekaterina V. Moguchaya, 1 Maria A. Boyarinova, 1 Asilat S. Alieva, 1 Alexander V. Orlov, 1 Elena Y. Vasilieva, 2 Victoria A. Yudina, 2 Sergey V. Anisimov, 3 and Alexandra O. Konradi 1 Background: Poor nutrition during the early stages of human development can lead to rare pathological conditions in adult life. The best-known and most severe historical cases of famine include the Dutch Hunger Winter, the Finnish famine, the Chinese Great famine, and the siege of Leningrad. The siege of Leningrad (now Saint Petersburg) was one of the longest in history, lasting 872 days, from September 8, 1941 to January 27, There were 670,000 registered deaths of the civil population, in which 97% died due to starvation. The aim of the present study is to create a collection of biospecimens from extensively phenotyped siege of Leningrad survivors, who underwent starvation during the early periods of their lives, and from a matched control group. Methods: A total 305 siege survivors and 51 age- and sex- matched control subjects were investigated in of an observational retroprospective cohort study in at a baseline visit. After 3 years of follow-up, 252 siege survivors (182 females and 70 males; mean age years) and 45 controls (32 females and 13 males; mean age years) were examined. All siege survivors were exposed to the extreme dietary restriction and stress associated with the siege in their early childhood. All participants signed informed consent and were subject to questionnaires and physical examination, as well as various laboratory and instrumental tests. Anthropometry, blood measurement, cognitive and physiological testing, and vascular damage assessment were performed. Results: Blood specimens of the extensively phenotyped siege survivors were collected and processed (blood plasma, blood serum, and flash-frozen PBMC); serum and urine were used for laboratory tests. Conclusions: We believe that data obtained from this unique collection of biospecimens can elucidate the mechanisms of healthy aging and emphasize the importance of reproductive health, counseling, and monitoring among people with eating disorders. Introduction Many of the diseases in Western civilization may be the result of programming of the metabolism and function of a tissue, or organ, as a result of diminished supply of certain nutrients during critical stages of development ( thrifty phenotype hypothesis). 1 Poor maternal nutrition has been implicated as a common antecedent for many pathological conditions. World history accounts for numerous cases of famine, providing an opportunity to study the effects of extreme dietary restriction. The best-known and most severe historical cases of famine include the Dutch Hunger Winter ( ), the Finnish famine ( ), the Chinese Great famine from the late 1950s to the early 1960s, and the siege of Leningrad ( ). The increased life expectancy in developed countries led to increased interest in the problem of healthy aging. Public policies aim to promote well-being and ultimately the quality of later life. 2 Numerous studies have assessed the medical consequences of dietary restriction, as discussed extensively below. The aim of the present study is to create a collection of biospecimens from extensively phenotyped siege of Leningrad survivors and a matched control group. This collection can become a unique asset for studies aiming to investigate cardiometabolic health, psychological factors, and markers of cardiovascular aging in survivors of the famine. Methods Institutional BioBank North-West Federal Medical Research Center (Saint Petersburg, Russia) provides medical services to people suffering from cardiovascular, endocrine, neurological (adults and children), hematological, and rheumatoid disorders. The Institutional BioBank (operating since 2012) serves as a 1 Department of Hypertension, 2 Clinical Diagnostic Laboratory, and 3 BioBank, North-West Federal Medical Research Center, Saint Petersburg, Russia. 1

2 BIO ver9-Rotar_1P.3d 08/06/15 2:17pm Page 2 2 ROTAR ET AL. core research facility, collecting, processing, and storing various biospecimens. By early 2015, over 32,072 biospecimen aliquots from 18,575 individual patients were accumulated in the BioBank s storage facilities. Ethics The study was conducted in compliance with current Good Clinical Practice standards and in accordance with the principles set forth under the Declaration of Helsinki (1989). Institutional review board approval of the study protocol was obtained prior to the initiation of study participant enrollment (protocol No. 243, dated December 12, 2012). All participants agreed to and signed informed consent (IC), allowing for the performance of genetic studies in particular. All biospecimens were provided with unique identifiers (BL0001, BL0002, etc.) and a translation table ensured the sample annotation was secured by the coordinator of the study. Baseline medical examination ( ) Data on survivors was provided by the Saint Petersburg Primorski (Seaside) District Society for siege survivors. Candidate study participants were contacted by the coordinator of the study. At the baseline visit (performed at ), a total of 305 survivors and 51 control subjects were investigated in the terms of an observational retroprospective cohort study. The control sex- and age-matched subjects were born in the Soviet Union (present-day Russia) during the same time period without any exposure to extreme famine of the siege, and stayed in Leningrad (presentday St.-Petersburg) after the war. Details of design and results of this stage of the study were described earlier. 3 Briefly, the following evaluation methods were utilized: 1. Questionnaire regarding lifestyle, risk factors, cardiovascular disease, co-morbidities, and medication. 2. Special questionnaire regarding life settings during the siege (breast feeding, number of family members, hospitalizations, duration of presence in the besieged city, time of evacuation from Leningrad (if applicable)). From the intrauterine group out of 46 subjects majority (n = 44) stayed in the city up to the end of the siege and only 2 were evacuated. From the infant group, 120 subjects were evacuated and 140 stayed in Leningrad up to January 1944 (official termination of the siege). Absence of registries in Russia for a long time, and inadequate medical records about the period during and after siege, made the process of siege exposure estimation difficult. 3. Anthropometry (weight, height, waist circumference (WC)) according to standard procedures by medical scales VEM-150 Massa-K (Russia) and medical stadiometer (Russia). The body mass index (BMI) was calculated according to Quetelet formula (weight, kg/ (height, m)2). 4. Blood pressure (BP) and heart rate (HR) measurements (Omron, Japan) in sitting and standing position, after 3 minutes. 5. Electrocardiogram was registered by MAC1200ST, echocardiography and carotid ultrasound were performed on a Vivid-7 (all GE Medical, Germany). 6. Carotid-femoral pulse wave velocity (PWV) was assessed by the SphygmoCor (Atcor, Australia). 7. Fasting blood lipids and glucose were detected by Hitachi- 902 ( Japan) with Roche Diagnostic kits (Switzerland). 8. Relative telomere lengths were measured by quantitative PCR and the ratio of telomere repeat copy number to single gene copy number was calculated for each DNA sample. Follow-up medical examination ( ) Participants were invited for a repeat visit to the Center 3 years following the baseline visit: 252 (81.5%) siege survivors and 45 (83.3%) controls complied, while 25 (8.0%) survivors and 1 (1.8%) controls were reported dead, 28 (9.0%) survivors and 4 (7.4%) controls refused to be examined, and 4 (1.5%) survivors and 4 (7.4%) controls could not be contacted. In the Center facility, the following investigative program was conducted: 1. Anthropometry, BP, HR measurement and ECG according to the same procedures 2. Psychological questionnaires: HADS, 4 SF-36, 5 EQ-5D 6 3. Cognitive function (Mini Mental Score Evaluation (MMSE) test) 4. Central aortic pressure and arterial stiffness (Sphygmocor, AtCor, Australia) 5. Ankle-brachial index (ABI), cardio-ankle vascular index (CAVI) (VaSera, Fukuda, Japan) 6. Albumin/creatinine ratio of urine portion (Cobas Integra 400 plus, Switzerland) 7. Fasting lipids, glucose, creatinine were detected by Abbott Architect 8000 (USA) with Roche diagnostic kits (Switzerland). 8. Attrition rate of telomere length shortening is planned to be performed Biospecimen collection and processing Biospecimen collection was performed by qualified personnel in the Center facilities. Peripheral blood (PB) samples were collected by standard venopuncture technique into one K 2 -EDTA tube (4 ml) and one SST II tube (6 ml). Following 30 min incubation of SST II tubes at room temperature, blood serum was isolated by centrifugation at 2000 g for 10 min. For DNA isolation, peripheral blood mononuclear cells (PBMC) were isolated from anticoagulated blood using density gradient centrifugation on Ficoll-Paque PLUS (GE Healthcare) and stored for the subsequent use; the plasma side-product was preserved. All biospecimen aliquots (blood plasma, blood serum, and flash-frozen PBMC) were stored at -70 C. Statistical analysis SPSS Statistics 20 (IBM, USA) software was utilized for statistical analysis. The chi-squared statistics were calculated to compare distribution of qualitative variables and relationships between categorical variables. Differences for continuous variables among the subgroups were assessed by either the one-way ANOVA (parametric) or the Kruskal- Wallis test (nonparametric distribution). Results A total of 252 siege of Leningrad survivors were enrolled in the follow-up, including 70 males (27.8%) and 182 females (72.2%). The control group consisted of 13 (28.9%)

3 BIO ver9-Rotar_1P.3d 08/06/15 2:17pm Page 3 PHENOTYPING OF SIEGE OF LENINGRAD SURVIVORS 3 males and 32 (71.1%) females. Comparative characteristics of siege survivors and age- and sex-matched control group T1 c are provided in Table 1. Siege survivors can be divided into three groups according to their age during the siege: intrauterine group (born during the siege November 1, 1941 January 27, 1944) 35 survivors; newborn/infant group (born during January 1, 1941 October 31, 1941) 44 subjects; childhood group (born before January 1, 1941) 173 subjects. The majority of study participants were thus exposed to the extreme dietary restriction and stress associated with the siege in their early childhood. Extensive phenotyping was performed. A comparative analysis failed to identify many significantly different parameters between siege survivors and the age- and sex-matched control group. It was established, however, that siege survivors had significantly lower anthropometric parameters (weight, body mass index, waist circumference) and higher high density cholesterol (HDL) levels, compared to the controls. Discussion We believe that future studies on this exceptional group of survivors will provide invaluable data on the impact of famine exposure, particularly in early childhood, on the prevalence of cardiovascular complications, markers of cardiovascular aging, and metabolic biomarkers. As mentioned above, the most severe cases of famine include the Dutch Hunger Winter, the Finnish famine, the Chinese Great famine, and the siege of Leningrad. All of these serve as examples for important studies aiming to Table 1. Characteristics of Siege Survivors and Controls All Controls P Parameter (n = 252) (n = 45) S vs C Age (years), mean SD Height (cm), mean SD Weight (kg), mean SD BMI (kg/m 2 ), mean SD WC (cm), mean SD WC > 94 in M and >80 cm F, No. (%) 191 (76.7%) 39 (86.7%) 0.13 Obesity (BMI > 30 kg/m 2 ), No. (%) 106 (42.4%) 20 (44.4%) 0.79 Current smokers, No. (%) 18 (7.2%) 1 (2.2%) 0.21 Ex-smokers, No. (%) 46 (18.3%) 8 (17.8%) 0.61 Low income, No. (%) 28 (11.2%) 9 (20.5%) 0.09 Spending for food more than 2/3 of monthly 78 (31.2%) 6 (13.3%) 0.02 average income, No. (%) Drinking alcohol, No. (%) 199 (80.2%) 35 (77.8%) 0.70 Drinking alcohol once a week or less, No. (%) 117 (70.5%) 131 (72.4%) 0.24 SBP (mm Hg), mean SD DBP (mm Hg), mean SD HR, beats/minute, mean SD Orthostatic SBP (mm Hg), mean SD Orthostatic DBP (mm Hg), mean SD Orthostatic HR, beats/minute, mean SD Hypertension, No. (%) 217 (86.1%) 41 (91.1%) 0.36 Total cholesterol (mmol/l), mean SD Total cholesterol >4.9 mmol/l, No. (%) 205 (83.7%) 36 (81.8%) 0.76 HDL (mmol/l), mean SD HDL <1.03 in M and 1.29 mmol/l F, No. (%) 92 (37.7%) 20 (45.5%) 0.33 Triglycerides (mmol/l), mean SD Triglycerides >1.7 mmol/l, No. (%) 48 (19.7%) 11 (25.0%) 0.42 Glucose (mmol/l), median [25%;75%] 5.4 [5.0;6.0] 5.6 [5.2;6.0] 0.74 Glucose >5.6 mmol/l, No. (%) 111 (45.5%) 24 (54.5%) 0.26 Diabetes, No. (%) 44 (17.5%) 7 (15.6%) 0.73 GFR (ml/kg/1,73), mean SD GFR <60 ml/kg/1.73, No. (%) 29 (11.6%) 4 (8.9%) 0.59 Albumin/creatinine ratio (mg/mmol), median [25%;75%] 0.97 [0.57;1.68] 0.91 [0.63;1.66] 0.61 Albumin/creatinine ratio >3 mg/mmol, No. (%) 29 (11.6%) 8 (17.8%) 0.25 MMSE score, mean SD MMSE <25, No. (%) 14 (5.9%) 4 (9.1%) 0.43 PWV (m/s), mean SD PWV >10 m/s, No. (%) 141 (60.8%) 24 (60.0%) 0.90 CAVI, mean SD CAVI >9, No. (%) 126 (68.5%) 31 (70.5%) 0.79 ABI, mean SD ABI <0.9, No. (%) 26 (14.1%) 6 (13.6%) 0.93 ABI, ankle-brachial index; BMI, body mass index; C, controls; CAVI, cardio-ankle vascular index; DBP, diastolic blood pressure; F, females; GRF, glomerular filtration rate; HDL, high density cholesterol; HR, heart rate; M, males; MMSE, mini mental score estimation; PWV, pulse wave velocity; S, siege survivors; SBP, systolic blood pressure; SD, standard deviation; WC, waist circumference.

4 BIO ver9-Rotar_1P.3d 08/06/15 2:17pm Page 4 4 ROTAR ET AL. dissect pathophysiological mechanisms of aging and disorders of many systems. The Dutch famine was a 6-month period of severe starvation occurring in the urban western part of the Netherlands at the end of World War II, causing about 22,000 deaths. Results published by Dutch scientists showed that exposure to maternal malnutrition is linked with metabolic disturbances (impaired glucose tolerance, atherogenic lipid profile, and higher levels of obesity) and contributes to negative consequences in their offspring. 7 Later, the authors studied 7,837 women from Prospect-EPIC (European Prospective Investigation Into Cancer and Nutrition) and established that a short period of moderate to severe malnutrition during postnatal development increases the risk of type 2 diabetes in adulthood. 8 It is worth mentioning that the Dutch population rapidly developed into a wealthy and rich population after the famine, but other starving cohorts remained relatively poor. The Chinese Great famine lasted from the late 1950s to the early 1960s and caused millions of excess deaths. Authors analyzed the data from 7874 adults and established that fetal exposure to severe famine increases the risk of hyperglycemia in adulthood. 9 These findings were not supported by Kannisto et al., 10 who analyzed the survival of the cohorts born in Finland during the severe famine from , and concluded that severe famine survivors did not experience any after effects that influenced their survival in later life. The siege of Leningrad (later renamed to its historical name Saint Petersburg) was one of the longest and most destructive sieges in history, causing considerable devastation to the city. This siege lasted for 872 days, from September 8, 1941 to January 27, The Soviet government reported about 670,000 registered deaths of the civil population from 1941 to January 1944, although some independent studies suggest a much higher death toll of between 700,000 and 1.5 million. Of the total civil population death toll, approximately 3% were killed by bombing and shelling, while 97% died due to starvation. 11 Most of these deaths occurred during the hunger winter of November 1941 to February 1942, when the siege was in full force and the bread ration was reduced to 250 g for workers and as low as 125 g for others. The average daily ration for most of the citizens of Leningrad during this time was around 300 calories and contained virtually no protein. From December 1941, the bread ration increased to 300 g and 200 g, respectively. 12 The evacuation of the Leningrad citizens was enforced via the water route of Ladoga Lake during the summer period, over the ice during the winter period (so-called Road of Life ), and by airlift (absolute numbers were minimal). Approximately 1.5 million Leningrad citizens were evacuated altogether. 13 Throughout the siege, immediately subsequent to it, as well as after the war, an increased level of hypertension and cardiovascular mortality was detected in siege survivors. 14 Lang and Chernorutskii independently described the conditions of alimentary dystrophy and stress hypertension in siege survivors, leading to accelerated cardiovascular mortality. 15 Afew decades later, the problem of remote consequences of starvation in the intrauterine period and the early childhood period were revealed. A few large studies were conducted addressing the association between starvation in childhood and cardiometabolic disturbances in adult life. Two research studies addressing medical consequences of the siege of Leningrad were performed to date. In the 1990s, such a study was organized jointly by the University College London Medical School (UK) and Otto Institute of Obstetrics and Gynecology, Russian Academy of Medical Science. The study revealed no correlation between intrauterine malnutrition and metabolic disturbances, or cardiovascular diseases in adulthood. 16 Another large study of siege survivors was performed jointly by the Karolinska Institute (Sweden) and the Institute of Experimental Medicine, Russian Academy of Medical Sciences. Those siege survivors who experienced famine around the age of puberty had increased blood pressure, as well as a higher level of mortality from ischemic heart disease and stroke. 17 Another important investigation was performed by Khoroshinina, who confirmed that insulin-independent diabetes without obesity develops more often and earlier in women who went through the siege of Leningrad in their childhood. 18 It was also observed that children conceived during the famine were at increased risk of schizophrenia and depression, had a more atherogenic plasma lipid profile, were more responsive to stress, and the rate of coronary heart disease doubled. People exposed during any period of gestation had a higher prevalence of type 2 diabetes. 19 However, despite circumstantial evidence showing that childhood famine may lead to a similar catch up fat phenotype as underweight newborns, the mechanism by which food deprivation in these periods increases cardiovascular disease risk remains obscure. It is possible that epigenetic mechanisms are involved. It is thus essential that future investigations should address the effects of prenatal famine exposure across generations. In our current study, we have performed extensive phenotyping and laboratory evaluation of a moderate group of siege survivors and controls. Clinical and laboratory data, as well as biospecimens collected, are now available for comparative studies of various design. Interested parties should contact the corresponding author of this article. At the present time, hunger and prenatal malnutrition are major problems in many African and Asian countries, 20 with one in seven inhabitants of our planet suffering from lack of food. Adequately feeding women before and during pregnancy may be a promising strategy in preventing chronic diseases worldwide. Furthermore, eating disorders (for example, anorexia nervosa) are currently considered common psychiatric disorders of women of childbearing age in developed countries. Reproductive health outcomes are compromised in women with a history of eating disorders, among all types. Our findings in siege survivors emphasizes the importance of reproductive health counseling and monitoring for women with eating disorders. 21 While much information related to cardiovascular and metabolic consequences caused by famine was accumulated in these studies as well as others, further investigations are essential to discover the underlying mechanisms. The siege of Leningrad represents one of the major examples of studies addressing famine consequences. We aim to further expand our collection of biospecimens (DNA, blood serum, and blood plasma) of the extensively phenotyped siege survivors. North-West Federal Medical Research Center (Saint Petersburg) is open for collaboration with both academic and industrial research institutions.

5 BIO ver9-Rotar_1P.3d 08/06/15 2:17pm Page 5 PHENOTYPING OF SIEGE OF LENINGRAD SURVIVORS 5 Acknowledgments The authors are grateful to Saint Petersburg Primorski (Seaside) District Society for Siege Survivors, for granting us an access to the up-to-date database of the siege of Leningrad survivors. Author Disclosure Statement No competing financial interests exist. References 1. Hales C, Barker D. Type 2 (non-insulin-dependent) diabetes mellitus: The thrifty phenotype hypothesis. Diabetologia 1992;35: Bowling A, Iliffe S. Psychological approach to successful ageing predicts future quality of life in older adults. Health Qual Life Outcomes 2011;9: Rotar O, Moguchaia E, Boyarinova M, et al. Seventy years after the Siege of Leningrad: Does early life famine still affect cardiovascular risk and aging? J Hypertens 2015; in press. 4. Zigmond A, Snaith R. The hospital anxiety and depression scale. Acta Psychiatr Scand 1983;67: Syddall H, Martin H, Harwood R, et al. The SF-36: A simple, effective measure of mobility-disability for epidemiological studies J Nutr Health Aging 2009;13: Sprange K, Mountain G, Brazier J, et al. Lifestyle matters for maintenance of health and wellbeing in people aged 65 years and over: Study protocol for a randomised controlled trial. Trials 2013;14: Roseboom T, van der Meulen J, Ravelli A, et al. Effects of prenatal exposure to the Dutch famine on adult disease in later life: An overview. Mol Cell Endocrinol 2001;185: van Abeelen A, Elias S, Bossuyt P, et al. Famine exposure in the young and the risk of type 2 diabetes in adulthood. Diabetes 2012;61: Li Y, He Y, Qi L, et al. Exposure to the Chinese famine in early life and the risk of hyperglycemia and type 2 diabetes in adulthood. Diabetes 2010;59: Kannisto V, Christensen K, Vaupel J. No increased mortality in later life for cohorts born during famine. Am J Epidemiol 1997;145: Veselov A. Fight against famine during siege of Leningrad. Home History 2002;3: Pavlov D. Leningrad 1941: The Blockade. Chicago: University of Chicago Press, Potemkina MN. Evacuation and national relation in the Soviet home front during Great Patriotic War. Home History 2002;3: Summarizing of War Experience, 5th Edition: Medical Service Experience of Baltic Fleet; Voenmorizdat, Leningrad; 1945; p Chernorutskii MV. About hypertension in the Leningrad during In: Proceedings of Evacuation Hospitals of Front Evacuation Center No. 50 and Army medical institutions. Hypertension. Narcomzdrav USSR, Medical Literature Publishing House, Leningrad; 1944;14: Stanner S, Bulmer K, Andres C, et al. Does malnutrition in utero determine diabetes and coronary heart disease in adulthood? Results from the Leningrad siege study, a cross sectional study. BMJ 1997;315: Sparén P, Vågerö D, Shestov DB. Long-term mortality after severe starvation during the siege of Leningrad: Prospective cohort study. BMJ 2004;328: Khoroshinina LP. Starvation during childhood can be reason of diseases during old age (example of children surviving the siege of Leningrad). Publishing House MAPO; Saint-Petersburg, 2002: p Roseboom T, Painter R, van Abeelen A, et al. Hungry in the womb: What are the consequences? Lessons from the Dutch famine. Maturitas 2011;70: Kim J, Guha-Sapir D. Famines in Africa: Is early warning early enough? Glob Health Action 2012; Linna MS, Raevuori A, Haukka J, et al. Reproductive health outcomes in eating disorders. Int J Eat Disord 2013;46: Address correspondence to: Dr. Oxana P. Rotar Laboratory of Epidemiology of Hypertension Department of Hypertension North-West Federal Medical Research Center Akkuratova Str. 2 Saint Petersburg, Russia rotar@almazovcentre.ru