Biochemistry of the menopause
Abstract
The life of a human female is characterized from teenage years by monthly menstruation which ceases (the menopause) typically between the age of 40 and 60 years. The potential for reproduction declines and ceases as the ovaries become depleted of follicles. A transition period in mid-life, for 2 to 10 years, when menstruation is less regular is called the perimenopause. The menopause is associated with a significant decline in plasma concentrations of sex hormones, an increase in the concentrations of the gonadotrophins and changes in other hormones such as the inhibins. These changes are superimposed with effects of aging, social and metabolic factors, daily activity and well-being. Although the menopause is entirely natural, in some cases ovarian failure can occur earlier than usual; this is pathological and warrants careful biochemical investigations to distinguish it from conditions causing infertility. Elderly females are affected by a range of clinical disorders including endocrine, cardiovascular, skeletal, urogenital tract and immunological systems, body mass, vasomotor tone, mood and sleep pattern. Reference intervals for many diagnostic biochemical tests for the menopause need to be used when interpreting results in clinical investigations for patient management. The standardization and harmonization of assays are being addressed. Many women now choose to develop their career before bearing children, and the health service has had to change services around this. This review does not cover screening for and tests during pregnancy. The review is timely since the population is aging and there will be more demand on healthcare services.
Introduction
The female menopause is a period of life when the ovaries are depleted of oocytes, and the cyclical activity of gonadotrophins, peptides and steroids is lost. Age at the menopause reflects the complex interactions of health and socioeconomic factors including for example ethnicity, diet, education, oral contraceptive use, weight, employment, exposure to endocrine disrupting chemicals, alcohol use, smoking and physical activity.1,2 The proportion of elderly women and men in the population is increasing,3 and many of them now maintain good health but at some stage a proportion will need medical help and laboratory investigations. An earlier age for the menopause due to premature ovarian failure (POF) can be due to stress, eating disorders or obesity, excessive exercise, dieting and from medication for fibroids or endometriosis. POF can be due to chromosomal disorders such as Fragile X or Turner syndrome, and genetic disorders are coming to light.4 Environmental pollution is a new threat to health including POF.5
Some of the biochemical changes at the menopause are attributed to oestrogen and or progesterone depletion. Both steroids act through cytoplasmic receptors and two receptors for each steroid, alpha and beta (ER-α and ER-β; PR-α and PR-β) are now recognized, sometimes they antagonize each other.6,7 Isoforms of each receptor exist that have different tissue expression patterns and function which affects gene expression in normal and tumour tissue and hence ligand treatment.8–10 ER-α is mainly expressed in reproductive tissues, breast, kidney, bone, white adipose tissue and liver. ER-β is present in ovary, central nervous system (CNS), lung, colon, kidney and immune system.11 PR-α and PR-β are virtually identical in structure except PR-β has additional 164 amino acids at the N terminus. PR-α and PR-β are expressed equally in human tissues. The classic view of steroids as dimeric transcription factors that bind a hormone response element is now thought to be oversimplified. The phosphorylation, SUMOylation of the receptors as well as the interaction of co-regulatory proteins, particularly with PR, affects transcriptional response.9,12,13 Oestrogens are important in maintenance of lipid and glucose homeostasis. They regulate food intake and energy expenditure by action on the CNS. Oestrogens also act to promote synthesis of proteins that maintain peripheral energy homeostasis. Progesterone leads to gene transcription of proteins that regulate functions of the uterus, ovary, mammary gland and brain.
General biochemistry
Many clinical laboratories will use reference intervals for biochemical tests without values in menopausal females. There is little information in the literature; in some cases, laboratories will have derived values from the laboratory database. Reference intervals in the elderly were reported in 1989 using Technicon instrumentation which is no longer in use.14,15 A comprehensive review in this journal has recently covered the variation of 85 analytes during menstrual cycles.16 Recent papers that address changes of liver, bone and kidney test results in elderly subjects using current methodologies on automated clinical chemistry analysers seem to be few in number,17–26 and only two papers have compared a wide range of results for premenopausal and postmenopausal females.25,26 The number of subjects studied for a reference interval varies considerably and tends to decrease with age as recruitment of normal healthy subjects becomes more difficult to define and older subjects are frail, prescribed drugs and can be house or institution bound. Cardiovascular problems and diabetes are usually exclusion criteria when collecting samples of the biological fluids of normal subjects. For many analytes, there are minimal differences between the genders and with age.
The detailed results and differences for liver, bone and kidney tests are summarized in Table 1 and enzymes in Table 2. Comparisons of the results for menopausal women reveal higher results than males of the same age for high-density lipoprotein cholesterol (HDL-cholesterol), but lower results for creatinine, glucose, urate and urea,17,19,20 alkaline phosphatase (ALP), aspartate aminotransferase, creatinine kinase and gamma-glutamyltransferase.20,21,25 No differences are seen between elderly females and males for apolipoprotein (APO A1) and Apo B, bilirubin, cholesterol and LDL cholesterol concentrations. The results for serum phosphate in females are similar to males.17,19,20,22 In comparing results for those studies, there are inevitable differences due to analytical factors, method standardization, conversion of results to SI units, ethnicity or living standards.Table 1. Summary of reference interval data from six publications17,19,20,22,25,26 for liver, bone and kidney tests in elderly males and females (columns 1 to 4) and pre- vs. postmenopausal women.
Table 2. Summary of reference intervals from 5 publications17,19,20,22,25,26 for enzyme tests in elderly males and females (columns 1 to 4) and pre- vs. postmenopausal women (column 5).
| Rustad et al.17 | Chan et al.19 | Carlsson et al.20 | Ryden et al.22 | Adeli et al.25,26 | |
|---|---|---|---|---|---|
| Publication year | 2004 | 2008 | 2010 | 2012 | 2015 |
| Location | Norway | Hong Kong | Upsaala, Sweden | Canada | |
| Menopausal cohort | 1608 | 335 | 1016 | 727 | 12,000 |
| Normal females | 226 | 50.2% | 373 | 440–2500 | |
| Ages normal females (y) | 18–49 | 21–81 | 70 | 75 | 50–79 |
| Analytical platform | Various | Roche Modular | Abbott Architect | Ortho-Vitros | Ortho-Vitros |
| Comparison | Female (F) vs. male (M) | Female vs. male | Female vs. male | Female vs. male | OF vs. YF |
| Apolipoprotein A1, g/L | ND | ND | 1.2–2.54 (F); 1.06–2.29 (M) | 1.2–2.7 (F); 1.07–2.3 (M) | 1.0–2.1 (OF) 1.0–2.4 (YF) |
| Apolipoprotein B, g/L | ND | ND | 0.66–1.66 (F); 0.6–1.61 (M) | 0.7–1.76 (F); 0.59–1.67 (M) | 0.4–1.2 (OF) 0.5–1.5 (YF) |
| Bilirubin, μmol/L | ND | 4–30 (F); 6–37 (M) | 3.4–19 (F); 4.5–26 (M) | ND | 1.7–15.0 1.7–17.0 |
| Cholesterol, mmol/L | ND | ND | 3.8–7.9 (F); 3.2–7.5 (M) | 3.6–8.2 (F); 3.0–7.6 (M) | 5.2 (3.2 -7.3) (OF) 4.8 (3.8–7.3) (YF) |
| Creatinine, μmol/L | 50.2–87 (F); 62.8–101.8 M) | 45–85 (F); 59–112 (M) | 44–107 (F); 57–133 (M) | 51–107 (F); 61–143 (M) | ND |
| Glucose, mmol/L | 3.86–5.99 (F); 4.08–6.5 (M) | ND | ND | ND | 4.1–5.2 (OF) 4.4–6.1 (YF) |
| HDL cholesterol, mmol/L | 0.99–2.66 (F); 0.79–2.16 (M) | ND | 1.0–3.0 (F); 0.7–2.3 (M) | 0.98–3 (F); 0.72–2.48 (M) | ND |
| LDL-cholesterol mmol/L | ND | ND | 1.9–5.48 (F); 1.62–5.23 (M) | 1.6–5.7 (F); 1.2–5.7 (M) | 1.12–4.8 (OF) 1.82–5.1 (YF) |
| Phosphate, mmol/L | 0.84–1.5 (F); 0.73–1.63 (M) | 1.0–1.59 (F); 0.85–1.58 (M) | 0.83–1.54 (F); 0.74–1.36 (M) | 0.93–1.5 (F); 0.82–1.42 (M) | 0.30–0.49 (OF) 0.31–0.51 (YF) |
| Protein, g/dL | ND | N | ND | ND | 6.5–7.8 (OF) 6.4–8.6 (YF) |
| Urate, μmol/L | 148–365 (F); 225–481 (M) | ND | 178–491 (F); 222–577 (M) | 170–467 (F); 201–515 (M) | 417–1130 (OF) 595–1548 (YF) |
| Urea, mmol/L | ND | 2.7–7.4 (F); 3.7–8.7 (M) | ND | ND | 2.7 (1.7 -4.3) (OF) 2 (1.2–3.2) (YF) |
N.D: not determined; OF: old female; YF: young female.
| Rustad et al.17 | Chan et al.19 | Carlsson et al.20 | Ryden et al.22 | Adeli et al.25,26 | |
|---|---|---|---|---|---|
| Year | 2004 | 2010 | 2012 | 2012 | 2015 |
| Location | Norway | Uppsala | Uppsala | Northern China | Canada |
| Total cohort | 1016 | 727 | 1364 | 12000 | |
| Normal females | 50.2% | 373 | 440–2500 | ||
| Comparison | Female (F) vs. Male (M) | Female (F) vs. Male (M) | Female (F) vs. Male (M) | Old (O) vs. young (Y) female | Old (O) vs. young (Y) female |
| Analytical platform | Various | Roche Modular | Abbott Architect | Roche | Ortho-Vitros |
| Alanine amino transferase, U/L | 6.7–49 (F); 8.9–74 (M) | <0.15–1.01 μkat/L (F); <0.15–0.82 μkat/L (M) | 27 (14–42) (O); 24 (14–42) (Y) | ||
| Alkaline phosphatase, U/L | 24–98.8 (O): 25–151 (Y) | ||||
| Aspartate aminotransferase, U/L | 31–233 (F); 45–487 (M) | ||||
| Creatinine kinase, μmol/L | 0.35–3.3 (F); 0.39–5.72 (M) | ||||
| Gamma-glutamyltransferase μkat/L | 0.13–1.39 (F); 0.17–2.27 (M) | 0.24–2.20 (F); 0.28–2.38 (M) | |||
| Lactate dehydrogenase U/L | 375 (269–510) (F); 472 (253–593) (M) | ||||
| Lipase μkat/L | 0.61–6.68 (F); 0.52–6.34 (M) | ||||
The results in menopausal females compared with younger females show higher concentrations of cholesterol and lower ALP.25 The comparisons are not statistically tested. Changes in most biochemical investigations through the menopause are relatively minor but may be clinically relevant. There is no evidence of hypomagnesaemia in postmenopausal women, but there is significant hypermagnesuria (467 ± 20 [mean and SEM] vs. 355 ± 13 mmol/moL creatinine)27 which is attributed to the increased intestinal magnesium absorption. Other results relevant to cardiovascular and skeletal systems will be discussed later. Most data in menopausal women are the markers for endocrine, bone and cardiovascular functions and this review focuses on these areas. Treatment of menopausal symptoms will not be discussed but interpretation of laboratory results will affect patient management.
Endocrine function
During the life of a human female, there are many changes in ovarian function starting before birth. The reproductive system ages faster than other organs.28 At 20 weeks of gestation, the ovary contains about 7 million oocytes; from that point the numbers of follicles decrease. One million oocytes are present at birth falling off rapidly to around 200,000 by the end of puberty, and only about 350 follicles ovulate during the reproductive years. Further follicle loss is largely by apoptosis. A regular cycle frequency is around 30 days between the ages of 20 and 45 years. At the onset of menstruation (menarche), the pattern of menstrual cycles can be irregular with frequency up to 70 days. There may be short menstrual cycles, sometimes interspersed with normal cycles. By the last cycle, there may only be about 1000 follicles in the ovaries.29 Hormonal changes in the menopause were last reviewed in this journal in 199330 and elsewhere more recently in the context of hormone replacement.31 Changes in menstrual flow with age result from lower oestrogen and progesterone production; in some cases, there are high oestrogen to progesterone ratios,32 thus placing a risk for cancer of the uterus.
Hypothalamic–pituitary–ovarian hormones
During the reproductive years, pituitary gonadotrophins are under negative feedback control by steroids and inhibins. Every 30 days, on average, a number of follicles in the ovary (antral follicles) develop under the influence of follicle-stimulating hormone (FSH) secreted by the pituitary. FSH concentrations in the blood increase over 15 days and this stimulates several follicles to be activated to produce oestradiol and inhibin B. One follicle (so-called dominant follicle) ovulates in response to a mid-cycle luteinizing hormone (LH) surge. Highest concentrations of inhibin B are seen at mid-cycle. The granulosa cells are the source of progesterone which is produced in greater amounts as the dominant follicle becomes a corpus luteum. FSH production is inhibited by inhibin B.32 Ovulation separates the early follicular phase and the later luteal phase. Inhibin A is also a product of the granulosa cells. The dominant follicle secretes inhibin A in the follicular phase with highest concentrations in mid-cycle. The corpus luteum takes over inhibin A production.
In perimenopausal women, an increase in the cyclical early follicular concentrations of FSH is a consistent finding. Concentrations of oestrogens and inhibins are lower than seen in earlier years which correlates with the depletion of follicles in the ovaries and the resistance of the ovaries to gonadotrophins with a reduction in oestrogen and inhibin production. The menopause transition goes through several phases33 (Table 3) from normal ovarian activity, through a stage with prolonged follicular phase and no luteal activity. There is a stage with normal follicular activity and insufficient luteal phase, and finally the menopause with low oestrogens and progesterone. This division is based on data from multiple, annual blood tests in 13 women for between 4 and 9 years up to the menopause34 Blood samples were taken three times a week for a four-week period in each year. The changes at each stage are summarized in Table 3. Cycles that became longer may be delayed ovulatory cycles or anovulatory cycles. As the process moves on, FSH concentrations are higher and inhibins are lower than in the follicular phases. Intervals of 24 up to 38 days separate menses in early phase and the interval can be greater than 60 days in later phase. FSH concentrations above 25 IU/L are seen in the late transitional period. Once menstruation has ceased, FSH is above 60 IU/L with LH raised (10–45 IU/L), but oestradiol (<200 pmol/L), progesterone and inhibins (B < 25; A < 10 ng/L) concentrations are low.31,34 Hormone measurements other than FSH during the perimenopause are generally considered to be of little diagnostic value.31,34 The transition may take four or more years. The current guidelines from the National Institute for Clinical Excellence (NICE) for diagnostic tests in the menopause are shown in Table 4.35Table 3. Staging of menopause.
Table 4. NICE guidelines for diagnosis of the menopause.
| Terminology | Early menopausal transition | Late menopausal transition | Early postmenopause | Late postmenopause |
|---|---|---|---|---|
| Perimenopause | Perimenopause 1–3 years | Perimenopause | ||
| Menstrual cycles | Variable length, >7-day difference in cycle length | >60 days amenorrhoea | ||
| FSH | High | >25 IU/L | High variable | Very highly stable |
| AMH | Low | Low | Low | Very low |
| Inhibin B | Low | Low | Low | Very low |
FSH: follicle-stimulating hormone; AMH: antiMullerian hormone.
| 1.2 Diagnosis of perimenopause and menopause |
| 1.2.1 Diagnose the following without laboratory tests in otherwise healthy women aged over 45 years with menstrual symptoms |
| • Perimenopause based on vasomotor symptoms and irregular periods |
| • Menopause in women who have not had a period for at least 12 months and are not using hormonal contraception |
| • Menopause based on symptoms in women without a uterus |
| 1.2.2 Take into account that it can be difficult to diagnose menopause in women who are taking hormonal treatment for example for the treatment of heavy periods |
| 1.2.3 Do not use the following laboratory and imaging tests to diagnose perimenopause or menopause in women age over 45 years |
| • AntiMullerian hormone |
| • Inhibin A |
| • Inhibin B |
| • Oestradiol |
| 1.2.4 Do not use a serum follicle stimulating hormone (FSH) test to diagnose menopause in women using oestrogen and progestogen contraception or high dose progestogen |
| 1.2.5 Consider using a FSH test to diagnose menopause only |
| • In women aged 40 to 45 years with menopausal symptoms including a change in their menstrual cycle |
| • In women aged under 40 years in whom menopause is suspected |
FSH: follicle-stimulating hormone.
Hot flushes, night sweats and vaginal dryness are common symptoms of the menopause from oestrogen withdrawal. A hot flush is a sensation of heat of unknown cause in the upper body, face and neck lasting for 3–5 min accompanied by higher heart rate and peripheral blood flow with a rise in skin temperature. With oestrogen loss, there is a reduction in bone density and an increase in cardiovascular risk, although the latter may be related more to concentrations of HDL-cholesterol (see later).4,36
Steroid analysis
Quantitative analysis of steroids by immunoassay (IA) has been the mainstay of endocrine tests for nearly five decades, but there has been criticism of the poor specificity of the assays. Initially, this was overcome by steps to isolate a steroid from the sample by selective extraction with or without chromatography which made the procedure impractical in routine hospital laboratories. Steroids in circulation are largely protein bound, specifically (steroid hormone-binding globulin – SHBG) and non-specifically (albumin) and are measured after extraction from plasma or serum so the combined bound and free steroids are measured. Steroids in some IAs are displaced chemically from binding proteins, but this can be overcome by exploiting the high antibody affinity. Kit manufacturers do not disclose the details of many steps in the procedure which is unfortunate when unusual results are encountered. Concentrations of steroids in plasma range from micromolar (dehydroepiandrosterone-sulphate – DHEAS), high nanomolar (cortisol), low nanomolar (testosterone, progesterone) to picomolar (oestrogens). The similarities in steroid structures make cross-reaction in IA inevitable. Oestrone concentrations are higher than oestradiol in the menopause and cross-reaction must be excluded in the assays. In addition to steroid interference through structural similarities, the IAs are subject quite often to the effects of human antimouse antibodies.37
In the 21st century, the use of liquid chromatography (LC) coupled with mass spectrometry (MS) has improved assay performances.38 LC coupled with tandem mass spectrometers (LC-MS/MS) methods also enable several steroids (panels or profiles) to be measured using isotope dilution in the same sample. Steroids are extracted from the sample by solvent or solid phase extraction techniques. Care must be taken to ensure that steroids of the same molecular weight are separated in the LC stage to avoid what is called isobaric interference in the mass spectrometric measurement if steroids co-elute. Cortisol39,40 and androgens41–45 are measured after electrospray in positive ion mode, but oestrogens alone46–54 and in combination with androgens55–58 and aldosterone59,60 are usually in negative ion mode. Accurate measurement of oestrogens at low concentrations (below 100 pmol/L) is still a challenge. Many LC-MS/MS methods for oestrogens incorporate the formation of derivatives in order to improve sensitivity, although some papers have overcome the problem, which makes the method acceptable in routine clinical laboratories.48,49,51 Lower concentrations of sex steroids have been found in LC-MS/MS than with IA, and new reference intervals have been developed.61 Interference from hormone replacement therapy (HRT) can be a problem.62 Steroid assays by extraction IA63 and automated IA on large analytical platforms have been validated against LC-MS/MS methods with closer agreement now, but the IAs can still give abnormal results due to interferences.64–67
Sex steroids
After the menopause, sex steroid concentrations decline (Table 5). Despite the lack of ovarian production, significant amounts of sex steroids are made in peripheral tissues from DHEAS which is still present in blood plasma at less than 4 μmoL concentration.68,69 This process is called intracrinology.70 Oestrogens are produced in adipose tissues, and hence increased plasma concentrations are seen with greater body weight. During the reproductive years, around 20% of women have polycystic ovaries. Women who had polycystic ovary syndrome (PCOS) go on to have higher testosterone concentrations after the menopause.71Table 5. Reference intervals for hormones with age and menopausal status.
| 40–49 y | 50–59 y | 60–69 y | Premenopause | Postmenopause | |
|---|---|---|---|---|---|
| Serum total T, nmol/L | 0.37–2.00 | 0.34–1.94 | 0.31–1.88 | 0.41–0.93 | 0.46–1.05 |
| Serum androstenedione, nmol/L | 0.89–4.77 | 0.66–3.79 | 0.52–3.04 | ||
| Serum-free T, pmol/L | 2.4–26.2 | 2.6–24.6 | 2.8–23.0 | 5.5–13.4 | 5.4–14.7 |
| Serum oestradiol, pmol/L | 8.9–24.2 | 9.8–27.1 | |||
| Serum SHBG, nmol/L | 33.6–60.5 | 34.3–61.7 | |||
| Mean inhibin B, ng/L | 50 | 25 | |||
| Mean inhibin A, ng/L | 25 L | 10 | |||
| AMH, pmol/L | <15 | ||||
| Saliva cortisol, nmol/L | 2–20 | ||||
| Saliva cortisone, nmol/L | 5–40 | ||||
| Mean FSH, IU/L | 10 | 100 | |||
T: testosterone; SHBG: sex hormone-binding globulin; AMH: antiMullerian hormone; FSH: follicle-stimulating hormone.
Free steroids can be measured after ultrafiltration or equilibrium dialysis before LC-MS/MS and give much lower concentrations than the total amounts (oestradiol < 1.8 pmol/L; testosterone < 14 pmol/L).72,73 Free steroid concentrations can be calculated after measuring total steroid and binding protein concentrations.74,75 Measures of free steroid are achieved from the steroid concentrations in saliva76–80 and urine,79,81,82 since both the salivary glands and kidney filter free hormones. Collection of saliva samples is non-invasive and permits repeated frequent sampling but has been little used for studies around the menopause. Cortisone is at higher concentration than cortisol in saliva because of high oxidative capacity of 11-hydroxysteroid dehydrogenase (HSD11B2) in salivary glands, and hence both steroids should be measured preferably by LC-MS/MS to avoid cross-reactivity in an IA.83 The menopause is associated with an increase in body weight and body fat particularly around the abdomen. Sex hormone-binding globulin concentrations decline with the menopause depending on the oestradiol concentrations which are determined by body weight.84 SHBG can now be measured by IA and by MS.85
Menopausal women who are obese can be investigated for cortisol excess (possible Cushing's syndrome). A high midnight salivary cortisol is a highly suggestive first test in these investigations.86 Total cortisol IAs show variable bias87 and different responses according to gender.88 The serum concentrations of cortisol and its binding globulin (CBG) are relatively constant with age. Extremely underweight and overweight states activate the hypothalamic–pituitary adrenal (HPA) axis.89 Hypercortisolaemia can lead to insulin resistance and decreased HDL-cholesterol concentrations,90 decreased bone mineral density and muscle wasting.86
During the perimenopause, there is still the possibility of a pregnancy, so women may choose to continue to take oral contraceptives. The progestogen-only pill (POP) is a useful option for contraception in older women. The Faculty of Sexual and Reproductive Healthcare recommend that women over the age of 50 years, who are amenorrhoeic and wish to stop oral contraception, can have their FSH concentrations checked. If FSH concentration is ≥30 IU/L, the FSH should be repeated after six weeks. If the second FSH concentration is ≥30 IU/L, contraception can be stopped after one year.91 Some women on POP have amenorrhoea and measurement of FSH will help the clinician to decide the perimenopausal status. If the FSH concentration is repeatedly above 20–30 IU/L (depending on method), then POP cessation is an option for discussion between patient and doctor, a barrier method may be recommended as a precaution. POP changes the cervical mucus and does not usually affect the ovulation pattern. Newer oral contraceptives such as Cerazette (desogestrel 75 μg daily) can suppress FSH and LH concentrations, thus making tests for perimenopause unreliable. Desogestrel can be substituted with an older POP before an FSH test.
Circulating concentrations of renin, angiotensin II and aldosterone in normotensive postmenopausal women are similar to those in normally cycling premenopausal women92 despite predicted lower concentration of hepatic angiotensinogen (the substrate for renin) due to the lower oestrogen production. There is a difference in response to orthostatic stress which is not activated in postmenopausal women. The axis may need further consideration when oral contraceptive or HRT is in use.93 New assays for renin activity measure the generation of angiotensin I by LC-MS/MS.94,95
AntiMullerian hormone, inhibins and kisspeptin
It is becoming more common that women are delaying when to have children compared with previous generations. The option for these women to ascertain their ovarian reserve is an appealing one. Direct ovarian markers such as antiMullerian hormone (AMH) are taken into consideration. There are issues with standardization and harmonization of assays in use.96–102 AMH is secreted into blood by small antral follicles. During the menstrual cycle, AMH concentrations are stable. AMH concentrations are related to inhibin B, FSH and antral follicle count; hence, serum AMH diminishes during the menopause (<20 pmol/L) and measurements have proved useful in assessing ovarian reserve in females who have delayed having children. Inhibin B is also produced by antral follicles but is less valuable in relation to follicle numbers.103,104 From around 40 years of age, inhibin B concentrations fall from a mean concentration of 50 ng/L as follicle numbers are depleted and as cell frequency lengthens. A direct, local role of kisspeptin signalling in the control of follicular dynamics and ovulation has been identified and defective ovarian actions of kisspeptins accelerates ovarian aging, leading to a state of POF.105 Plasma concentrations of kisspeptin are lower in adults (12 pmol/L) than children (40 pmol/L),106 but studies in relation to ovarian reserve and menopause have not been conducted. Kisspeptin as a therapeutic agent has potential use for infertility and sex-hormone-dependent malignancies.107
Thyroid function
The thyroid gland secretes thyroid hormones that are important for normal metabolism of body cells. The production of thyroid hormones is regulated in a negative feedback manner. The pituitary gland secretes thyroid-stimulating hormone (TSH) which acts on the thyroid gland to release thyroxine (T4) from thyroglobulin. Thyroid function and the hypothalamic–pituitary–gonadal axis are related both directly and indirectly. Thyroid hormones influence the reproductive system directly through the presence of receptors in the ovaries and pituitary. They increase the synthesis of thyroid-binding globulin (TBG), SHBG and androgens and lower the clearance of sex steroids and stimulate aromatase. At the menopause, however, thyroid uptake of iodine decreases, leading to reduced free thyroxine (FT4) and free tri-iodothyronine (FT3), and hence the concentrations of TSH increase through activation of the HPT axis.108
Many publications use reference intervals for TSH of <0.5 to 5 mU/L, but papers from 2000 to 2016 show upper limits increase to 5.8 in the range 60 to 79 years and 6.6 mU/L in subjects over 80 years of age.109–116 Normal TSH concentrations within the range of 0.35 to 5.5 mU/L were used in a study to determine the prevalence of hypothyroidism in a Latin-American population.117 TSH is positively associated with hypercholesterolaemia, hypertriglyceridaemia and high serum LDL cholesterol in postmenopausal women.114 Low TSH is found among postmenopausal women with osteoporosis and TSH can be brought into reference interval during treatment of hypothyroidism.115 A reference interval for FT4 of 9 to 22 pmol/L (0.7 ng/dL to 1.7 ng/dL) is applicable to subjects over 60 years of age.112 Some of the symptoms of thyroid disease are similar to signs of early menopause, so differentiating the two can be difficult.118 Signs of overt hypothyroidism include fatigue, weakness, constipation, cold intolerance, hair loss and dry skin. Hyperthyroidism can present with heat tolerance, tremor and anxiety.
Thyroid function tests are subject to many problems. Results are subject to changes in concentration of binding proteins. HRT will increase concentrations of TBG. The use of biotin supplements by menopausal women for brittle nails and neuropathy, affects performance of automated IAs based on biotin-streptavidin chemistry. Mouse antibodies in patients' serum samples also lead to heterophilic interferences. Total T3 (TT3) concentration in blood is 100 times lower than total T4 (TT4) (around 1 nmol/L and 100 nmol/L, respectively) and T3 can be artefactually produced from T4 during sample preparation.119,120 The International Federation for Clinical Chemistry have recommended a protocol for free thyroxine (FT4) based on equilibrium dialysis LC-MS/MS.121 Most laboratories have high workloads for thyroid function tests, so LC-MS/MS methods may not be practical or financially viable. There are problems with direct analogue IA methods for FT4/FT3, and compared with results by tandem mass spectrometry more women can be wrongly classified therefore as having overt hypothyroidism.122,123 For the measurement of free thyroid hormones, physical separation methods, such as equilibrium dialysis or ultrafiltration, followed by tandem mass spectrometry, offer many advantages that improve patient diagnosis and care. Implementation of such assays should reduce the variability of results.121,124,125 A range of 11 thyroid hormones and metabolites can be measured in one method combining protein denaturation, SPE and LC-MS/MS126 that would only need 50 μL of serum or plasma (tested in adult frog plasma only). This method could be applied to human samples to assess thyroid homeostasis.
Cardiovascular system
Loss of ovarian function at the menopause and the resulting hypo-oestrogenic state are believed to increase the risk of cardiovascular disease. Postmenopausal women have higher apolipoproteins, cholesterol (total, and LDL cholesterol and triglyceride concentrations) than males and premenopausal women of the same age (Table 6).127–131 The pattern of change in HDL is variable and this may reflect the selection of subjects and differences in technology of measurement. Conventional methods of measuring lipoprotein subclasses involved determination of cholesterol content carried by particles separated with ultracentrifugation. Recently, lipoprotein particle size, distribution and concentration have been measured with nuclear magnetic resonance (NMR),132 high-performance liquid chromatography (HPLC)133 and MS.134,135 In a large study in Northern Europe, alterations in lipids were accompanied by changes in amino acid concentrations (glutamine, isoleucine).136Table 6. Reference intervals for cardiovascular tests in pre- and postmenopausal women.
| Premenopausal | Postmenopausal | References | |
|---|---|---|---|
| Plasma lipids | |||
| Total cholesterol | 153.4 ± 17.4 | 211.2 ± 29.6 | 127 |
| Triglycerides | 101.1 ± 7.4 | 125.5 ± 14.6 | 127 |
| HDL, mg/dL | 46.7 ± 7.3 | 27.7 ± 2.9 | 127 |
| HDL mmol/L | 1.88 ± 0.85 | 1.62 ± 0.59 | 128 |
| VLDL, mg/dL | 20.1 ± 1.5 | 25.1 ± 2.9 | 127 |
| LDL, mg/dL | 86.5 ± 22.2 | 158.4 ± 31.4 | 127 |
| LDL, mmol/L | 2.66 ± 0.93 | 2.99 ± 0.97 | 128 |
| Plasma lipids | |||
| Total cholesterol | 153.4 ± 17.4 | 211.2 ± 29.6 | 127 |
| Triglycerides | 101.1 ± 7.4 | 125.5 ± 14.6 | 127 |
| HDL, mg/dL | 46.7 ± 7.3 | 27.7 ± 2.9 | 127 |
| VLDL | 20.1 ± 1.5 | 25.1 ± 2.9 | 127 |
| LDL | 86.5 ± 22.2 | 158.4 ± 31.4 | 127 |
| CRP, mg/L | 2.1 ± 0.2 | 3.8 ± 0.8 | 128 |
| APO A1, g/L | 1.39 ± 0.4 | 1.42 ± 0.4 | 128 |
| APO B, g/L | 1.06 ± 0.4 | 0.93 ± 0.23 | 128 |
| ADMA, μmol/L | 0.4–0.8 | 0.82 ± 0.33 1.34 ± 0.58 (Obese) | 129 |
| Ischaemia-modified albumin, KU/L | 93.6 ± 14.4 | 130 | |
| Mean IL-6, ng/L | 3 | 128 | |
| Mean TNF-α, pg/mL | 7.9 | 128 | |
| NO, mmol/L | 2.4 | 128 | |
HDL: high-density lipoprotein; LDL: low-density lipoprotein; VLDL: very low density lipoprotein; CRP: C-reactive protein; APO A1: apolipoprotein A1; APO B: apolipoprotein B; ADMA: asymmetric dimethylarginine; IL-6: interleukin 6; TNF-α: tumor necrosis factor; NO: nitric oxide.
Atherosclerosis involves lipid deposits, oxidation and platelet aggregation to the arterial wall. Adherence of circulating leukocytes to the endothelium is an early step in the formation of atherosclerosis. Matrix metalloproteases (MMP) help to maintain protein components of the vasculature. Dysregulation or activation of these enzymes provides markers for endothelial injury and inflammation in many disorders of the heart and vasculature seen in elderly women. MMP2 activity by quantitative zymography (an electrophoretic technique) is higher in healthy postmenopausal women than in premenopausal women, although the number of subjects was small.137 MMP2 activity correlated with lipid profile, high sensitivity C-reactive protein (CRP) (immunoturbidometric assay) and soluble vascular cell adhesion molecules by enzyme-linked immunosorbent assay (ELISA).
The loss of oestrogen in the aging female contributes to accelerated dysfunction of mitochondria which results in a progressive decrease in lipid oxidation in mitochondria and increases the lipid storage in adipocytes with formation of fatty acyl intermediates in cardiomyocytes.138 Oxidative stress can cause hypertension, hypercholesterolaemia, diabetes and cardiovascular disease (CVD). Changes in vascular nitric oxide (NO) activity could lead to an increased risk of CVD in postmenopausal women.139 Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of the NO synthase pathway and may mediate effects on endothelial function.140 This study did not, unfortunately, include menopausal women. Concentrations of ADMA increase in postmenopausal women with exercise140 and body weight.129 Ischaemia-modified albumin (IMA) is albumin modified at the amino terminus by reactive oxygen species. IMA is increased in postmenopausal obesity associated with atherosclerosis risk.130 Advanced glycation end-products (AGE), the products of non-enzymatic glycation and oxidation of proteins, are raised (7.35 ± 1.23 IU/mL vs. 5.22 ± 0.5) in postmenopausal women with testosterone concentration in the range of 1.5–6 nmol/L compared with normal testosterone group.141 Stratification of patients for better or worse risk is a goal for biomarker discovery in this area. Many of the assays discussed here are not tests in routine hospital laboratory service.
Skeleton
Women who experience an early menopause are more susceptible to osteoporosis. The menopause is often accompanied with an increase in body weight and a reduction in bone mineral density. At the menopause, obesity can be the result of changes in lifestyle, exercise, insulin sensitivity, fat metabolism, fat partitioning to the periphery and to the abdomen along with changes in appetite or the gut microbiome.142 It is difficult to distinguish the effects of age per se and the extent of previous and current sex hormone and sunlight exposure.
The integrity of bone is the balance between bone formation and bone resorption. The balance is regulated by parathyroid hormone (PTH), vitamin D and steroid hormones. Bone turnover markers include enzymes and peptides from cellular and non-cellular compartments of bone. The collagen breakdown products include hydroxyproline and telopeptides (CTX). Bone formation is marked by pro-peptides of type 1 collagen (PINP) or osteoblast action (osteocalcin) and ALP. The biochemistry and assay variability (for technical and biological reasons) of bone turnover marker tests were reviewed by Hlaing and Compston143 and Woitge and Seibel.144 The clinical applications were published by Seibel145 and Szulc.146 The laboratory analyst needs experience for tests in this area with colorimetry, HPLC, LC-MS/MS, electrophoresis, immunoradiometric assay and ELISA. Collection of biological samples needs a number of analyte-specific precautions to prevent degradation within the sample, by exposure to high temperatures and daylight.
Around mid-life, less bone is deposited than is resorbed, although the menopause is associated with 50 to 100% increase in both bone formation and bone resorption markers. Results can vary with time of day, season, diet and exercise. Reference intervals for bone turnover markers across ethnic groups have been reported for example in UK and Europe,147–149 China150–152 and Japan.153,154 In Table 7, representative reference intervals for bone markers pre- and postmenopause are summarized.148,155–158 Serum activity of bone ALP increases with age20,21,25 as does the serum and urine excretion of CTX, NTX excretion in urine serum PINP and osteeocalcin.148,155 Results for CTX and PINP are method dependent;159 therefore, repeated measurements in one patient should be performed with the same assay particularly if monitoring treatment. Reference intervals for serum ALP in China were traced to the new IFCC reference procedure.160 The results were higher than other populations. Hypercortisolism is frequently investigated because of the effects on bone health and body weight as discussed earlier in this review. The stomach and adipocytes produce a number of signalling factors (ghrelin, leptin, adiponectin, resistin, visfatin and adipsin) that can affect appetite and body weight but measurements are not yet routine hospital tests and they remain confined to research studies.161,162Table 7. Reference intervals for bone marker tests in pre- and postmenopausal women
| Premenopausal | Postmenopausal | References | |
|---|---|---|---|
| Serum CTX, ng/mL | 0.19 (0.05–0.63) | 0.31 (0.10–1.00) | 148 |
| Urine NTX, nmoL BCE, per mmoL Cr | 38.1 (15.0–97) | 49.7 (21.2–116.4) | 148 |
| Serum PINP, ng/mL | 30.1 (4.2–74.5) | 41.3 (18.3–94.1) | 148 |
| Serum bone ALP, ng/mL | 9.8 (5.2–18.6) | 14.1 (7.2–27.6) | 148 |
| Serum osteocalcin, ng/mL | 17.9 (8.8–36.4) | 24.5 (12.7–47.4) | 155 |
| 25-OH vitamin D, ng/mL | 31.5 ± 7.9 | 26.5 ± 4.9 | 157 |
| 1,25-Dihydroxy vitamin D, pmol/L | 50–130 | 30–80 | 158 |
| DBP, mg/dL | 45.3 ± 6.2 | 41.7 ± 5.7 | 157 |
| PTH | |||
| Vitamin A, μg/dL | 1.53 (0.98–2.42) | 2.01 (1.07–3.41) | 158 |
| Vitamin B12, pg/mL | 358 (172–776) | 386 (161–1015) | 158 |
| Vitamin C, mg/L | 44.9 (4.5–92.6) | 38 (1.7–88) | 158 |
| Vitamin E, mg/dL | 19.9 (7.8–33.6) | 33.7 (16.8–82.4) | 158 |
PTH: parathyroid hormone; DBP: vitamin D binding protein; ALP: alkaline phosphatase; PINP: pro-peptides of type 1 collagen; NTX: N-terminal telopeptide; BCE: bone collagen equivalents; CTX: serum C-telopeptide.
Vitamin D is important in the regulation of calcium and phosphorus. Vitamin D measurements vary with time of day, season and diet (particularly calcium intake) as well as the analytical method. 25-Hydroxy-vitamin D3 is the major metabolite and further hydroxylation occurs in the kidney and there are several di-hydroxyvitamin D metabolites in urine.163 There are many issues over interferences in the measurement of vitamin D3 products due to calibration, isomers, epimers and metabolites with IA and LC-MS/MS assays. Candidate reference methods have been published for 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3 and a metabolite (24R),25-dihydroxyvitamin D3.164–166 25-hydroxyvitamin D and 24,25 dihydroxyvitamin D can be measured simultaneously.167 Reference values for 1,25-dihydroxyvitamin D show an age-related fall.168 Free vitamin D3 can be calculated once the concentration of the vitamin D-binding protein (DBP) has been determined. The concentration of DBP is lower in the menopause, and assays for DBP are variable between assays and the results different by ethnicity.169 The results by LC-MS/MS are less affected by genotype.170 Sunlight exposure is an important factor in vitamin D synthesis so the housebound elderly will often have circulating vitamin D concentrations below the recommended 50 nmol/L.171 Vitamin D deficiency leads to hyperparathyroidism, and hence the measurements should be linked with PTH concentrations when establishing a reference interval.
IAs for PTH are continually changing in response to the advances in knowledge of PTH synthesis and breakdown. The identification of fragments and their impact on assay specificity has necessitated changes in antibodies such that automated methods now use Third Generation methodology. There are also issues around PTH stability in samples,172 biotin therapy173 and standardization.174 Reference intervals for PTH concentrations of 27.0 ± 3.7 in young women and 34.9 ± 3.3 pg/mL in older women (mean ± SEM) indicate that results are 25% higher in the menopause.175 The response of PTH to citrate and calcium infusions is much higher after than before the menopause.176 Assays for PTH by LC-MS/MS of trypsin-digested fragments are now available but may be subjected to interference from oxidized and phosphorylated variants.177
Urogenital tract
Menopause is associated with vaginal atrophy among many other problems. Thinning of the vaginal wall, dryness and changes of pH due to lack of oestrogen affect sexual function and make the vagina susceptible to infection. Vascular endothelial growth factor (VEGF) is a critical angiogenic factor whose down-regulation results in tissue ischaemia.178 Steroid hormones regulate VEGF concentrations, and both androgenic and oestrogenic hormones can directly modulate the expression of VEGF.179,180 High concentrations of oestradiol reduce VEGF concentration in rats.181 Recent studies suggest the importance of steroid-VEGF in maintaining the integrity of bladder structure.182,183 Urinary VEGF concentrations have not, however, been measured in human over-active bladder (OAB) studies. Nerve growth factor (NGF) is produced within detrusor muscle and urothelium.184 Studies have demonstrated that concentrations are elevated in patients with OAB and may be correlated with the severity of symptoms.185,186 The same research group has recently published a correlation between NGF and outcomes of anticholinergic and Botulinum toxin-A therapy.187,188 These studies have yet to be independently reproduced. Steroid hormones influence NGF in gynaecological/neural tissue,189,190 but have not been studied in bladder. These findings suggest the importance of steroid hormones and VEGF/NGF expression in maintaining the integrity of bladder function.
Immune system
Postmenopausal women are at a higher risk of autoimmune disorders.191 There are links between sex steroid changes at the menopause with immune senescence192 and osteoporosis.193 Menopausal women have higher concentrations of cytokines such as tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and CRP and lower numbers of white blood cells such as cluster of differentiation type 4 (CD4) T cells and B lymphocytes.194,195 In line with alterations in sex hormone concentrations, there is decreased interferon (INFγ)-gamma production and proliferation of T lymphocytes.196
Summary
Laboratories need to offer interpretation and advice for many investigations for menopausal clinical problems,2 and this requires relating the test results to appropriate reference intervals. In the menopause women, experience changes in the urogenital tract, bones, fat distribution and cognitive function that are different from the opposite sex. The time span just before periods cease is called the perimenopause with uncertain cyclical ovarian activity. This makes difficult interpretation of many biochemical tests at this stage. For convenience, many reference intervals are usually interpreted by age, even though the time/phase of the menopause and the duration of the transition from normal menstrual cycles to the end of menstruation vary considerably. This is reflected in the wider spread of results with age for many tests. This review considers changes to the reference intervals of concentrations of analytes relevant to the menopause. Laboratory scientists will need to be able to recognize and consider if obesity, lack of exercise, smoking, oral contraceptive or HRT use,3 and assay methodology have affected results of the analytes to avoid misinterpretation of results by the clinicians.
The menopause has significant effects on the functions of endocrine, cardiovascular, skeletal, immune and genitourinary systems. Gonadal hormones affect many of the processes largely by their effects on steroid-binding proteins and receptors, but the changes in lifestyle with aging are also contributory. Senescence without much exposure to sunlight affects many of the analytes considered in this review. Reference intervals appropriate for age and impact of the menopause are essential when assessing some biochemical tests in elderly female patients who are at risk of mortality and morbidity from failings in endocrine, skeletal, renal, cardiovascular, urogenital and immune systems. The IA methods for some analytes are being replaced with assays by LC-MS/MS which is more specific; hence the results and reference intervals can be different now to the earlier published values. There are many issues around standardization and harmonization of assays that need to be addressed. This review has discussed these areas. Additional analytes currently being studied in animal models may lead to further tests in hospital laboratories. The review is timely, since the population is aging and there will be more demand on healthcare services.
Acknowledgements
The review was commissioned by the Clinical Sciences Review Committee of the Association of Clinical Biochemistry and Laboratory Medicine. I am grateful to them and to Dr Adel Ismail for constructive comments, criticisms and suggestions for change that helped with the review.
Ethical approval
Not applicable.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.
Guarantor
JWH.
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Article first published online: November 2, 2017
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