Associations Of Earlier Age At Menopause And Occupational Exposures And Mechanism Exploration

Part 1.Associations of occupations and related hazards with age at natural menopause in Chinese female workersBackground Natural menopause occurs around the age of 45 to 55.Menopause is not only the destination of a woman’s reproductive life,but also the entrance of ‘aging highway’.The nature of menopause is the exhaustion of ovarian follicles,which results in low estradiol level in blood tests.Long-term insufficiency of estradiol increased the risk of osteoporosis,cardiovascular disease and cognitive decline.As people’s life rapidly improved,health care and management have been paid attention to a lot.It is particularly important to understand the mechanism of ovarian aging and prevent premature decline of ovarian function.Despite the strong effect of genetic components,age at menopause have been highly variable which cannot be entirely attributed to inheritance.For instance,over 75% of premature ovarian insufficiency(POI)cases are idiopathic,which is characterized by approaching menopause before age 40.Therefore,non-genetic factors,such as environmental and sociopsychological factors,have been regarded as important determinants in menopause timing.The development of modern society is companied by increasing access of women to the labor market.Multiple occupational hazards also arouse wide concerns on female reproductive health,such as desynchronization from shift work,contamination of industrial chemicals,and so on.Objectives To investigate the impacts of occupation types and its related hazards on age at natural menopause;To identify the risk factors of earlier onset of natural menopause.Methods This is a nation-wide cross-sectional study.Based on the national survey on female workers’ reproductive health,which is developed by the Chinese Center for Disease Control and Prevention,information of 17,948 female workers aged >40 collected from2015-2017 was retrieved from database.The information includes socio-demographic characteristics,lifestyles and habits,reproductive history,occupational information,occupational hazards,etc.According to The International Classification of Occupations2008(ISCO-08),the occupation types of participants are coded according to the occupational information section of the questionnaire.The main body of the study is divided into two parts: 1)Cox proportional hazards model was adopted to analyze the relationship between occupations and age at natural menopause.P-values were adjusted using Benjamini & Hochberg method;2)Participants were divided into manual and non-manual workers according to their occupation codes.Self-reported occupational exposures between manual and non-manual workers were compared using chi-square test.Cox proportional hazards model and Kaplan-Meier survival curves were adopted to identify risk factors of earlier onset of natural menopause among multiple occupational hazards.All analyses were conducted using R(3.6.2).Results According to inclusion and exclusion criteria,17948 female workers were included in these analyses.15109(84.18%)participants have been in premenopausal status,meanwhile 2839(15.82%)in postmenopausal status.Kaplan-Meier survival curve showed the median age at natural menopause of Chinese female workers is 51.1)Higher risks of earlier age at natural menopause was encountered among legislators and senior officials(ISCO Minor group:111,HR=2.328,P<0.001),among other health associated professionals(ISCO Minor group: 325,HR=1.477,P=0.003),the workers involved in mining and mineral processing(ISCO Minor group: 811,HR=1.515,P=0.048)and metal processing and finishing(ISCO Minor group: 812,HR=1.722,P<0.001).On the other side,some occupations registered later age at natural menopause,including: business and administration professionals(ISCO Sub-major group: 24,HR=0.752,P=0.014),Manufacturing and Construction Supervisors(ISCO Minor group: 312,HR=0.477,P=0.002),administrative and specialized secretaries(ISCO Minor group: 334,HR=0.788,P=0.045),cleaners and helpers(ISCO Minor group: 911,HR=0.633,P=0.01).2)Menopause-free survival curves between manual and non-manual workers didn’t show significant difference using log-rank test(P=0.800).Prevalence of every kind of working conditions,chemical and physical exposures was significantly different between manual and non-manual workers(all P<0.001).Abnormal workload(model 2,HR=1.125,P=0.014),noise(model 2,HR=1.22,P<0.001)could be two risk factors of earlier age at natural menopause among all participants as a whole.Noise(model 2,HR=1.201,P=0.013)could be a risk factor of earlier age at natural menopause among non-manual workers.Cadmium(model 2,HR=1.695,P=0.041),Noise(model 2,HR=1.201,P=0.013)could be risk factors of earlier age at natural menopause among manual workers.Conclusions Based on the national survey on female workers’ reproductive health,this study first explored the correlation between occupational types and age at natural menopause.The results suggested there are several occupational types of manual and non-manual workers with an increased risk of earlier onset of natural menopause,while exposure to occupational hazards(i.e.noise)may be one of the risk factors to accelerate ovarian aging.This suggests that the female workers engaged in the above-mentioned occupations and those who have been exposed to noise for a long time,should receive occupational education as soon as possible,in order to improve their awareness of protection.Part 2.DNA copy number variations in patients with premature ovarian insufficiencyBackground Premature ovarian insufficiency(POI)refers to a clinical syndrome of ovarian dysfunction in women before the age of 40,with an incidence of 1% and 0.1% under age 40 and 30,respectively.In nature,POI is pathologically advanced senescence of the ovary.More than 75% POI is idiopathic which means arising from unknown causes.Our previous results suggest that certain occupational exposures are associated with earlier onset of menopause.Ovarian aging is slowly progressed physiologically.However,external environmental stressors on cellular senescence has a cumulative impact,which is most likely to be reflected in the change of genetic materials represented by DNA damage.Repeated sequences account for nearly two-thirds of the total DNA of human cells.Ribosomal DNA(r DNA),one of crucial repeated sequences,is present in the human genome in the format of tandem repeats.In mammalian cells,r RNA transcribed by r DNA accounts for 35% of all RNA.The high transcription activity of r DNA and GC-rich sequence characteristics make it the most vulnerable part of the genome,prone to DNA double-stranded fractures and copy number variations.These all lead to genome instability.In addition to ribosomes,mitochondria are also extremely rich organelles in cells.Mitochondria generate energy in the form of ATP through oxidative phosphorylation.Mitochondrial DNA(mt DNA)is its unique genetic material.Due to the lack of histone protection and effective DNA repair mechanisms,mt DNA is particularly vulnerable to reactive oxygen microenvironment.Thus,mt DNA copy number can indirectly reflect mitochondrial function,indicating the number of mitochondria per cell and the number of genomes per mitochondria.Telomeres are repeated DNA sequences and its binding proteins located at the end of chromosomes.Unlike the instability of r DNA and mt DNA,the main function of telomeres is to maintain genomic stability and protect the ends of chromosomes from being shortened by cell replication.In every cell division,telomeres can still be shortened by 50-150 bp.Due to the abundance of guanine and the easy formation of secondary structure,Environmental stressors can accelerate telomere loss through genotoxicity and oxidative stress,which can promote genomic instability.Objectives To explore r DNA and mt DNA copy number variations and telomere length in POI and investigate their relevant factors.Methods This study included 61 patients with POI in the clinics of gynecological endocrinology in Women’s hospital,Zhejiang University School of Medicine between June 2020 and February 2021.73 age-matched healthy controls were recruited.Basic demographic characteristics were collected and sex hormones were measured.An additional 5m L peripheral blood samples were collected from participants.After extracting DNA from peripheral blood,r DNA and mt DNA copy number and telomere length were measured by real-time quantitative PCR.Statistical analyses,such as Mann-Whitney test,Kruskal-Wallis H test,Spearman’s rank correlation coefficient,were used to compare r DNA and mt DNA copy number variation and telomere length between patients with POI and healthy control.Their correlations with demographic characteristics,sex hormones and Hormone replacement therapy(HRT)were investigated.Results The study included 61 patients with POI and 73 health control with ages of 35.81(27.94-38.33),34.23(26.28-38.73),with BMI of 21.23(19.47-23.71),20.90(19.71-23.44)kg/m2,respectively,which both had no significant difference.Of 61 patients with POI,12 had never received HRT and 49 had received HRT treatment over1 month.Part 1:Peripheral blood r DNA copy number was measured by RT-PCR.28 S,18S,5.8S r DNA copy number in between is highly positive correlation(rs=0.67-0.81,P<0.001)but with no significant correlation with 5S r DNA(P>0.05).28 S,18S and 5.8S r DNA copy numbers in the POI group was 1.85(1.6-2.22),1.26(1.06-1.55),1.01(0.86-1.18),which is significantly lower than 2.11(1.8-2.37),1.43(1.19-1.65),1.22(1.05-1.39)in the healthy control(all P-values <0.05).18 S r DNA copy number of POI with HRT is higher than that of those who have never received HRT(never HRT: 1.17(0.95-1.3)vs.HRT: 1.35(1.11-1.57),P=0.027).5.8S r DNA also has this trend but with no statistical significance(never HRT treatment: 0.91(0.81-1.04)vs HRT treatment: 1.03(0.89-1.25),P=0.052).However,none of these four r DNA fragments’ copy numbers was not significantly correlated with HRT duration or FSH,LH,E2 after HRT in patients with POI(all P-values >0.05).However,in the healthy population,5S r DNA copy numbers have a weak positive correlation with baseline FSH level(rs=0.283,P=0.015).In either POI group or healthy control group,28 S r DNA copy numbers were positively correlated with age(POI: rs =0.390,P=0.002;healthy controls: rs=0.238,P=0.042).5S r DNA copy numbers in the POI group was moderately negatively correlated with age at menarche(rs =0.464,P<0.001),while there was a negative correlation trend with age(rs=-0.208,P=0.108).In the healthy control group,5S r DNA copy numbers were significantly positively correlated with age(rs =0.335,P=0.004).Part 2:Mitochondrial gene COX1 and ND1 were quantified to represent peripheral blood mt DNA copy numbers.It was verified that COX1 and ND1 copy numbers have a very high correlation(rs>0.9,P<0.001).COX1 copy numbers in the POI group and healthy control group was respectively 233.71(208.49-299.88),235.57(203.42-268.81),while ND1 copy numbers were 299.9(257.11-378.22),295.24(257.64-361.09).No significant differences were observed between two groups(both P>0.05).As well,no significant difference of mt DNA copy numbers between patients with POI receiving HRT and those never treated with HRT.Part 3:Telomere length in the healthy control group was significantly higher than that of POI group(healthy control: 9.44(8.47-10.63)vs.POI group: 8.18(4.56-10.18)).Additionally,relatively high degree of discreteness of telomere length data in the POI group,respectively.There was no significant correlation between telomere length and age or whether HRT or not in the POI group(age: rs=0.165,P=0.205;HRT: rs=0.023,P=0.858),but was weakly positively correlated with age at menarche(rs=0.319,P=0.012).In the healthy control group,there was a weak negative correlation between telomere length and age(rs=-0.234,P=0.046),but with no significant correlation with age at menarche(rs=0.036,P=0.765).Conclusions This is the first study to report the difference between peripheral blood r DNA copy numbers in POI patients and healthy population.45 S r DNA component copy numbers are significantly lower than healthy control,while HRT may partially reverse this trend;Peripheral blood telomere length of POI patients was found to be significantly shorter,which is not related to age however.It suggests that patients with POI may have increased telomere depletion,which makes it difficult to maintain genomic stability.Among patients with POI,telomere length could not indicate senescence,providing new evidence for the controversy of telomere length in POI.Follow-ups of patients with POI in different stages were more helpful to clarify the patterns of r DNA copy numbers and telomere length in the course of accelerated ovarian aging and the effects of HRT.