Effects of environmental, social and surgical factors on ovarian reserve: Implications for age‐relative female fertility

Abstract Objective To investigate new risk factors for female fertility by analyzing the effects of environmental, social, and surgical factors on antral follicle counts (AFC) and anti‐Müllerian hormone (AMH) levels. Methods A total of 1513 women aged 20–47 years who underwent in vitro fertilization/intracytoplasmic injection treatment in Southwest Hospital from December 2017 to December 2019 were included. Women were assessed for AFC and AMH levels, and completed a questionnaire. Ordinal logistic regression analyses with generalized linear mixed models were used to calculate the adjusted odds ratio (OR) for diminished ovarian reserve. Results Adnexal surgery was the only risk factor associated with low AFC in women aged 20–30 years. Younger age at menarche, alcohol drinking, and adnexal surgery are three independent risk factors for AMH decline in women aged 20–30 years. Intense exercise, sleep quality, and adnexal surgery are three independent risk factors for a low AFC in women aged 31–36 years. Alcohol drinking and adnexal surgery are two independent risk factors for AMH decline in women aged 31–36 years. Conclusion With age, female fertility becomes sensitive to high‐intensity exercise and poor sleep quality. Adnexal surgery and alcohol drinking are two important risk factors for female fertility in women under age 37 years.

luteinizing hormone, and estradiol, are typically used to determine the relative condition of the ovarian reserve in clinical practice, especially the combinations of age, AFC, and AMH. 2 A number of factors affecting AFC or AMH levels have been reported, some are considered controversial or have been reported in limited studies. A committee opinion indicated that smoking has some potential deleterious effects on female fertility by delaying conception, impairing ovarian follicular dynamics, inducing gamete mutations, and increasing the risk of early miscarriages and adverse assisted reproductive technology outcomes. 3 Alcohol was considered to be a risk factor of female fertility through altering endogenous hormone concentrations, hindering ovum maturation, and disturbing ovulation, early blastocyst development, and implantation. 4 The relationships between smoking, alcohol and AFC or AMH levels are ambiguous. Peck et al. 5 provided the first morphologic evidence that smoking decreased human ovarian follicle number, but subsequent studies reported no such association. 6 Similarly, several studies have demonstrated that alcohol and smoking are not related to AMH levels, [6][7][8] but other studies have reported negative associations. 9,10 The effects of age at menarche on AMH levels remains controversial 6,8 ; and associations among additional factors, such as pesticides 9 -which may disturb the hormonal function, 11 and drinking coffee, 7 and AFC or AMH levels have been analyzed in limited studies.
In our previous study, we preliminarily analyzed the risk factors for female fertility in 794 women aged under 37 years and found that adnexal surgery, which may disturb the blood supply of female ovaries and impair the normal microenvironment of antral follicle development, was associated with decreased AFC and AMH levels. We found no associations among intense exercise and AFC, body mass index (BMI, calculated as weight in kilograms divided by the square of height in meters) and AMH levels, which was not consistent with previous studies. 10,12 The present study analyzed the risk factors affecting female fertility with an expanded sample size and with more age groups. This work will provide more reliable evidence for female fertility protection for different female age populations. A questionnaire was designed based on the literature [13][14][15] and included basic information (e.g., demographic data, medical history, reproductive history, and physiologic cycle), living habits and environmental contact (e.g., exercise, smoking and drinking, sleep and rest, and pesticide and organic contact), and dietary habits and emotional states (e.g., liquid intake, food intake, dietary supplement intake, and work stress). Specifically, alcohol drinking referred to whether or not women had consumed alcohol in the past half year, smoking referred to whether or not women had consumed at least one cigarette per day in the past half year, and sleep quality referred to whether or not women had good sleep quality in the past month.

| MATERIAL S AND ME THODS
Representative variables associated with AFC and AMH levels were selected from the questionnaire based on interests, the availability of data, and previous studies. 9,10 Two stimulation protocols were used in this study, the gonadotropin-releasing hormone long agonist protocol and the gonadotropin-releasing hormone antagonist protocol. The total number of 2-to 9-mm follicles in both ovaries in the early follicular phase was counted as AFC using ultrasound guidance by the same experienced gynecologist. Blood samples were collected on a random day of the menstrual cycle, and serum AMH was measured with an AMH ELISA kit (Kangrun Biotech) using an Access II Immunoassay System (Beckman Coulter) by the same experienced laboratory technician. The standard range of detection for the AMH assay was 0.06-18 ng/ml. Double cut-off values of AFC for predicting ovarian reserve were defined as five and ten (bilateral ovaries) based on previous reports. 16 values. For continuous predictors, OR > 1 indicates that poor ovarian reserve and relatively good ovarian reserve are more likely as the predictor increases, and OR < 1 indicates that good ovarian reserve and relatively good ovarian reserve are more likely as the predictor increases. For categorical predictors, OR > 1 indicates that poor ovarian reserve and relatively good ovarian reserve are more likely at the level of the predictor than at the reference level of the predictor, and OR < 1 indicates that good ovarian reserve and relatively good ovarian reserve are more likely at the level of the predictor than at the reference level of the predictor. Data were analyzed using SPSS version 25.0 software (IBM Corp., Armonk, NY, USA). A P value less than 0.05 was considered significant. (IQR 9-19) and the median AMH level was 3.28 ng/ml (IQR 1.73-5.92 ng/ml). Patients were stratified into three age groups (20-30, 31-36; ≥37 years): the median AMH levels were 4.11 ng/ml (IQR 2.36-6.97 ng/ml), 2.96 ng/ml (IQR 1.67-5.54 ng/ml), and 1.28 ng/ml (IQR 0.42-2.7 ng/ml), and the median AFC were 15 (IQR 11-21), 12 (IQR 8-17), and 6 (IQR 4-10), respectively. There was a remarkable positive relation between AFC and AMH (r = 0.7, p < 0.01), which was similar to the findings of a previous report. 20 The constituent ratio of patients by age and subdivided by AFC, AMH, and 17 candidate risk factors are displayed in the Supplementary material (Tables S1   and S2).

| RE SULTS
All selected factors in this study passed the test of parallel lines (p > 0.05). The results of the AFC univariate analyses are shown in

| DISCUSS ION
It is known that humans are facing a high risk of fertility decline.
Factors affecting female fertility include age, female reproductive function status, genetic factors, and ovarian function. However, there is a relative lack of evaluation of specific factors affecting fertility in women of different ages, especially young women. 5,12 In this study, risk factors for ovarian reserve were analyzed in three age groups of women, matched for optimal age, suboptimal age, and advanced age.
Reproductive history and physiologic cycle are generally considered closely related to female fertility. 8 Both univariate and multivariate analyses in this study showed that menarche age had a very significant negative impact on AMH in women aged 20-30 years, which was similar to the findings of previous studies. 6 important to female fertility protection, especially for women under 36 years of age. No association was found between smoking and AFC or AMH in our study, which was consistent with previous studies. 6,7 No association was found between coffee consumption and AFC or AMH in our study, which was consistent with a previous study. 9 Pelvic and abdominal cavity surgeries have been widely per- No significant correlations between any of the predictive risk factors and AFC or AMH levels were found in women aged 37 years and older. We speculated that women of this age group tend to have lower ovarian reserves already so other lifestyle insults or even adnexal surgery will not lower the ovarian reserves further. In addition, the small sample size may be part of the reason why external factors cannot affect the fertility of women from 37 years. Future studies should include a larger sample size of women aged 37 years and older.
Although female fertility has a certain ability to adapt to a variety of new lifestyles, environmental pollution, social behaviors, and medical factors, several age-specific risk factors including adnexal surgery, intense exercise, poor sleep quality and drinking, should be taken seriously. Our study revealed that female fertility is more susceptible to external risk factors with age. For women over 37 years, the effects of external risk factors on fertility may be due to reduced physical function, such as lower ovarian reserve, and are not considered as consequential. These results provide more detailed data for protecting female fertility.
However, this study had a few limitations. It was a single-center research and the study population was small, especially for the sub-group of women aged 37 years or older, which might cause bias.
Larger, multi-center studies are needed to confirm the results of the current study. In addition, although significant associations were found between adnexal surgery and AFC and AMH levels, this work cannot demonstrate that it is an independent risk factor for AFC or AMH decline because it is difficult to rule out the effects of the diseases themselves and of different surgical skills on AFC and AMH levels.

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interests.

AUTH O R CO NTR I B UTI O N S
WH contributed to the conception and design of the study. DM, XL, YG, and FW were responsible for data collection and checking.
YW, YY, and YL performed the data analysis, data interpretation, and manuscript drafting. All authors read and approved the final manuscript.