Assessment of factors responsible for early menopause in Interior Sindh, Pakistan

Objective: To evaluate the factors leading to the early onset of menopause (<40 years) in the women in the


Introduction
The Word menopause is derived from the Greek word "meno" which means stop cease. This is an aging process due to a reduction in the ovarian hormones progesterone and estrogen. 1 The normal age of menopause is about 44-55 years, but some women suffer from menopause before 40 years of age. Menopause that occurs before 40 years of age, due to any reason, is called premature menopause. This can leads to earlier onset of chronic diseases of aging and therefore lengthen to later years as well. Menopause is a major factor for bone degeneration. [1][2][3][4] Furthermore; premature or early menopause, also known as a premature ovarian failure (POF), is an important disorder that affects a significant population of young women. 5 Characteristics of this condition are amenorrhea, elevated gonadotropins, and decreased gonadal steroids. The effect of this condition is on both physical and psychological components, it is so because of the long term effects of the gonadotrophin depletion in the body. Furthermore, the distressing result of this condition is infertility. 6 Proper functioning of the hypothalamus, pituitary gland, uterus, and ovaries are necessary for the normal menstrual cycle. The hypothalamus triggers the pituitary gland; the pituitary stimulates folliclestimulating hormone (FSH) and luteinizing hormone (LH). FSH and LH trigger the ovaries to produce hormones estrogen and progesterone. 7 Estrogens and androgens affect the skeleton during growth and skeletal homeostasis during adulthood. The decrease in estrogen concentration with menopause effects of female bone resorption. 8 The study conducted in Jordanian women with early menopause shows that the association factor for the early menopause history in first-cousin marriages. 1 Similar studies were carried out on early menopause and risk of fracture, osteoporosis, and mortality in Swedish women aged 48 years with early menopause, later on at the age of 77, they had a risk ratio of 1.83 for osteoporosis, for fragility fracture risk ratio was 1.68 and mortality risk was 1.59. 9 Premature menopause results in adverse effects, which include cardiovascular diseases, psychiatric diseases, mortality, neurological diseases, osteoporosis, Sheehan's syndrome, and early death. 10 Hormonal therapies have succeeded to moderate levels with some risks but many others are still under the line and faced by the patients. 11,12 This study was aimed to assess the factors which cause menopause at an early age and to check the prevalence of factors which are most common along with factors having raised FSH, LH & Prolactin levels and those having low FSH, LH & Prolactin levels in the women of Interior of Sindh as compared to the women of the same age without early menopause. Also, inform the healthcare personnel about the situation so that they may consider the prevailing factors while treating the patients.

Materials and Methods
A cross-sectional study, for which participants were selected from Interior Sindh. The duration of the study was about six months. A convenient non-probability technique has been used for sampling. The sample size was 218 subjects. Females with early menopause were included in the study as case, their age is 33.3 ± 3.23 whereas women with normal menstrual cycles were included as controls for the study of age 32.67 + 3.07 respectively. Females on medication and suffering from any disease particularly any hormonal disorder were excluded. Blood samples required for the study were collected from normal and affected individuals and written consent was obtained before the sampling. Samples were collected in 5ml gel tubes and serum was separated. The ELISA method was used to assess the concentration of Prolactin, LH, and FSH hormones 13,14 , in their blood by using Human diagnostic worldwide test kits with Elisa reader UVM 340. The principle is based on monoclonal antibodies and another in antibody-enzyme conjugate solution. The sample is reacted with antibodies at the same time with antibodies resulting in the sandwiching of sample molecules between the solid phase and enzyme-linked antibodies. For this 100 µl of the conjugate is dispensed into the wells. 50 µl of calibrator was added into the 5 wells. 50 µl of control & samples into the wells and then the plate is covered with an adhesive sheet and incubated at 37oc for about 60 minutes. The solution is then removed from the wells and was washed with a wash solution for any unbounded antibodies. 100 µl of the substrate was poured into the wells and was covered with aluminum foil and incubated for about 15 minutes at room temperature and stop solution (100 µl) is added into the wells for any further color development and finally, OD is observed at 450nm in a plate reader. All data obtained were put into SPSS version 17 for analysis of results. Ethical Consideration: The questionnaire was anonymously administered to the women/girls, with the permission of the ethical review committee of the Department of Physiology. Written consents were taken from the control and subject groups before managing the questionnaires. The present study will be used for the research purpose.

Results
In this study, 109 cases and 109 healthy controls were studied. From cases, 104 were housewives, and 5 were office workers. The mean age of the cases involved in this study was 33.3 ± 3.23 while controls 32.67 + 3.07. The mean FSH, LH, and PROLACTIN were higher in study cases than controls. The mean BMI value of controls was (24.14 + 3.78) and that of study cases was (23.78 + 4.56). In the case of Anorexia Nervosa (17%), the mean FSH level was lower in cases than in controls, similarly, the mean LH level of cases was lower than in controls. Whereas, the mean Prolactin level was higher in subjects than in controls. The results were significant as shown in table-1. In subjects of Brain Tumor (7%), the mean FSH level was higher than in the control; similarly, the mean LH levels were also higher in study subjects than in controls. The mean Prolactin level was also elevated in subjects as compared to controls. Women undergoing Ovarian Cancer (6%) have a mean FSH level, mean LH level, and mean Prolactin level increased than controls.
In the case of Hormonal disorders (23%), the mean FSH level was greater in study subjects than controls, similarly the mean LH level and mean Prolactin level were also notable than controls. Post Hysterectomy (15%) cases had a high level of FSH, LH, and Prolactin as compared to the controls. In cases of Oophorectomy (5%) the mean FSH level, mean LH level and mean Prolactin level was higher than controls which herald early menopause. In the case of Pituitary Dysfunction (4%) the study subjects had a higher mean FSH level than in controls, similarly, the mean LH level of subjects was also higher than in controls and the mean Prolactin level was also at an elevated level in subjects than in controls. Similarly, in the case of Sheehan Syndrome, the cases had a greater mean FSH level mean LH level, and mean Prolactin level than controls. Women undergoing Polycystic Ovarian Syndrome (8%) had raised levels of mean FSH, LH, and Prolactin than normal women.  As illustrated in table no.2 BMI was slightly fluctuating in both case and control groups however not reaching the significance level. BMI is not a factor causing the onset of early menopause.

Discussion
Early menopause is linked with long term multiple health consequences including diabetes 15 cardiac problems, psychiatric disturbances, neurological disorders, osteoporosis along with increased mortality. 16,17 However, the adverse effects could be minimizing by giving estrogen therapy but it is not much beneficial in treating all these health issues. 16 This study is in conformation with reflective study 18 where the trends of hysterectomy were studied in the rural tertiary level teaching hospitals in Northern India. It was concluded that early menopause is either impulsive or driven. Women suffering from menopause, because of bilateral hysterectomy, oophorectomy, or cancer treatment. 19,20 This is all associated with early menopause. In contrast to this study, women with brain cancer had increased prolactin. The chances of infertility increase as a result of oncological treatment. It is postulated that chemotherapy and radiations used in cancer treatments lead to earlier menopause presented with much severe symptoms. 21 In this study women who have undergone surgeries like hysterectomy and oophorectomy are nearly at increased risk of early menopause. Furthermore, a study conducted in USA 22 also established that women undergoing surgeries have more risk for having early menopause. 23,24 also conducted a similar study in women with and without hysterectomy. She also concluded that surgeries are related to earlier onset of menopause. Also the study on the Jordanian Women, 2010 shows that the women who suffered from early menopause were having a history of premature menopause in their parents, siblings or child 1 furthermore it is reported that inheritance/genetic accounts for about 25-30% in the onset of premature menopause cases. 25 Apart from the above-mentioned reasons one of the abnormalities of early menopause is the osteoporosis, estrogen depletion is linked with increased loss in bone mass and density, which results in approximately 9 million cases of bone fractures per year all around the globe. 26,27 conducted a study that reveals that early menopause was associated with low (BMI) which is also in confirmation with this study that both low and high BMI are causes of early menopause. Anorexia is also a sign of early menopause. The loss of weight is a ubiquitous feature of amenorrhea. It is reported that being underweight may speedup menopause. 28 Likewise a study also demonstrated the Anorexia Nervosa is one of the causes of amenorrhea leading to early onset of menopause. 29 There are certainly other contributing factors that result in early menopause, females with low parity or null parity are highly prevalent to premature/early menopause, likewise, females who experience early menarche are also at greater risk. 30,31 Study conducted in the UK reported that twin females at higher risk of early menopause. 32 Apart from genetics and physiological factors many social and lifestyle factors are also linked with early and immature menopauselike cigarette smoking 33,34 childhood malnutrition, emotional stress, and cognitive function during childhood greatly influence timings of menopause. 35

Study Limitations
The study was conducted on a small population due to fewer resources, big sample size could portray a clearer picture of the story.

Conclusion
In the Interior of Sindh, women have symptoms of early menopause. Results of the study revealed that there are many underlying causes of early menopause but the common factors observed were certain pathological conditions, poor diet, surgeries, and cancer.