Characterization of the vaginal microbiota of women with premature ovarian insuciency

Background: Premature ovarian insufficiency (POI) is a complex reproductive endocrine 36 disease that can affect multiple systems. It is highly heterogeneous in etiology and the exact 37 etiology remains unclear. Infectious factors may be related to POI, but researches on the 38 alterations of microbiome in POI patients are scarce. 39 Results: Vaginal swabs were collected from 52 POI patients (29 for sequencing, 23 for 40 validation) and 46 healthy individuals of comparable age (26 for sequencing, 20 for 41 validation). 16S rRNA gene sequencing targeting the V3-V4 hypervariable regions was 42 performed to evaluate the alterations of vaginal microbiota in POI patients. The relative 43 abundance of Actinobacteria (23.34% vs 10.65%, P=0.017), Atopobium (11.11% vs 0.01%, P 44 ﹤ 0.001), and Gardnerella (8.05% vs 3.14%, P=0.002) were significantly increased in POI 45 patients, while Bifidobacterium (3.95% vs 7.44%, P=0.017) was significantly decreased. 46 Cluster analysis of dominant strains showed that the proportion of POI patients whose 47 predominant bacteria were not Lactobacillus was increased than the control group, but the 48 difference was not statistically significant (8/29 vs 2/26, P =0.105). More interestingly, these 49 changes in vaginal microbiota were significantly correlated with declined ovarian function in 50 POI patients, including decreased ovarian reserve, ovarian endocrine disruption, and 51 symptoms of perimenopausal syndrome. Actinobacteria , Atopobium , and Gardnerella 52 appeared to be detrimental to ovarian function, while Bifidobacterium seemed to be benificial. Conclusions: The present study revealed the correlation between vaginal microbiota and POI, fill the gap in the field of microbial and POI research and provide a new research strategy for POI. But the causal relationship is still unclear, and we could not clarify detailed roles of specific constituents of the vaginal microbiota in the rRNA


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Premature ovarian insufficiency (POI) is a clinical syndrome defined by loss of ovarian 77 activity before 40 years old. Clinically, POI is characterized by menstrual disturbance 78 (oligomenorrhea or amenorrhea) with raised gonadotrophins and low estradiol 1 . According to 79 the epidemiological data, the prevalence of POI is approximately 1%, population 80 characteristics such as ethnicity may affect the prevalence 1-3 . Recently, it is believed that the 81 morbidity is increasing, the reported morbidity may be lower than it actually is, and the age of 82 onset tends to be younger. There is no effective method to predict the occurrence of POI now, 83 and when patients reach the diagnostic criteria, their ovarian function is often nearly complete 84 failure.

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POI is a complex disease that can affect multiple systems. The clinical manifestation differs 86 between individuals, mainly including menstrual disturbance, infertility and symptoms of 87 perimenopausal syndrome 1 . Besides, POI patients may experience complications such as 88 cardiovascular disease and osteoporosis, mainly due to the lack of estrogen 4,5 . A diagnosis of 89 POI also has a significant negative impact on psychological wellbeing and quality of life 1,6 . 90 Therefore, early prevention, early detection and early intervention to reduce the risk of 91 long-term complications and other systemically related diseases of POI are very important.

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The clinically recommended treatments are hormone replacement therapy (HRT) and in vitro 93 fertilization-embryo transfer (IVF-ET) with eggs donation based on HRT 1 . However, for those 94 with HRT contraindication or no source of donated eggs, they often suffer from the disease 95 due to the absence of effective alternative therapies. 96 POI is highly heterogeneous in etiology and the exact etiology remains unclear. Although a 97 wide spectrum of causes has been considered, including genetic 2,7-9 , autoimmune 3,10 , 98 iatrogenic 11-13 or environmental pollutants 14,15 . However, as many as half of patients are 99 diagnosed with idiopathic POI because the causal factors are unknown. The complexity of 100 POI, the increasing morbidity, the decreasing age of onset, and the lacking of early warning 101 and treatment methods all indicate that it is of great importance to study POI.

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The vaginal microbiota accounts for about 9% of the total human microbiota and remains 103 under-studied despite its importance for female health and future generations [16][17][18][19]   Menopause is a sign of ovarian aging. As a pathological ovarian aging, the reproductive 121 endocrine status of POI patients is similar to that of postmenopausal women 1,37 , while 122 whether their vaginal microbiota will undergo similar changes is still unknown and remains to 123 be explored. In addition, inflammation and autoimmunity are important factors that affect 124 ovarian function and promote the occurrence and development of POI 3,38 . Moreover, 125 cross-sectional studies have also found that the levels of TNF-α, IL-1α, IL-1β, IL-6 in the 126 serum and (or) follicular fluid of POI patients are significantly higher than those in healthy controls 39,40 . And the levels of these cytokines have significant correlations with sex hormones.

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Considering the important role of vaginal microbiota in maintaining female reproductive 129 health, as well as the potential connection between POI and chronic inflammation caused by 130 changes of vaginal microbiota, it is of great importance to explore the change pattern of 131 vaginal microbiota in POI patients.

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The purpose of this case-control study was to draw the characteristic spectrum of vaginal 133 microbiota in POI patients by comparing with healthy women of comparable age, and to 134 analyze the correlation between changes in vaginal microbiota and the declined ovarian

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Characteristics of the study population

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To identify the specific and key bacterial taxa associated with POI, LEfSe analysis was 299 performed to generate the cladogram of the vaginal microbiota (Fig. 3B). At the phylum level, 300 Actinobacteria was significantly enriched in POI patients (Fig. 3A). At the genus level,

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The potential of key bacterial taxa in the vagina to predict POI

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For key bacterial taxa with significant differences in relative abundance between the two 345 groups, including Actinobacteria, Gardnerella, Atopobium and Bifidobacterium, ROC curves 346 were constructed and AUC values were calculated to assess their potential to predict POI. The 347 accuracies of Gardnerella and Atopobium in predicting POI were considered acceptable, the 348 corresponding AUC values were 0.732 and 0.759, respectively, which were both greater than 349 0.7 49 (Fig. 5). However, the accuracies of Actinobacteria and Bifidobacterium were very low,           ROC curves were constructed and AUC values were calculated to assess the potential of key bacterial taxa to predict POI. A. T he AUC was 0.685 for Actinobact eria , P =0.019. B. T h e AUC was 0. 732 for Gardnerella P =0.0 03 C . T he AUC was 0. 759 for Atopobium , P =0 .0 01 D. T he AUC was 0.6 78 for