Questions Development

40

Elevated second trimester maternalserum unconjugated estriol is independently associated with a higher rate of spontaneous preterm birth (Olsen et al., 2014). The measurements of estriol from maternal saliva samples appear to correlate with maternal serum levels. Some studies demonstrated that elevated salivary estriol was a better marker for later sPTB in asymptomatic pregnant women, and the use of salivary estriol testing has potential advantages (i.e., noninvasive nature and ease of collection). However, one major limitation of the use of saliva as a biomarker is confounding by factors such as patient activity, posture, food consumption, and diurnal variations in estriol levels.

41

affecting several cell functions pivotal for inducing a defective placentation (Meroni, et al., 2008).

Complement-mediated placental inflammation has also been reported to play a key role in an experimental model of fetal loss induced by passive injection of large amount of aPL after the implantation (Girardi, et al., 2008). However, the real importance of complement activation in human pathology is still a matter of research (Cavazzana, et al., 2007). In addition, the thrombogenic effect of aPL may cause placental infarctions with the consequent impairment of the blood exchange between maternal and fetal circulation (Meroni, et al., 2008). It has been reported that 5–51% of patients with recurrent spontaneous miscarriage have aCL and 0–20% have LA (Vinatier, et al., 2001). Although aPLs are a risk factor for pregnancy loss, they are not pathognomic.

Attempts have been made to find the specific autoantibody responsible for pregnancy loss. There is evidence that the pathogenic antibodies are those directed towards β2GP1 (Meroni, et al., 2008).

Unfortunately, there are no cohort studies examining the natural history of untreated patients with aPL. There is a widespread belief, based on early studies that these antibodies are so pathogenic that they should always be treated. The nearest to a large study on the natural history is Empson et al.'s (Empson, et al., 2002) meta-analysis of 95 patients comparing treatment with aspirin with placebo.

There was an 86% live birth rate in the placebo group. Hence, the PPV seems to be only 14%.

However, as false-negative rates were not quoted, it is impossible to arrive at a valid conclusion about the PPV. Marai et al.,(2004) compared the results of autoantibody testing on 38 women with recurrent miscarriages (at least three consecutive miscarriages) in the first trimester of pregnancy, compared with a control group of 28 parous women with normal fertility. There was no association with aPL. However, the small numbers may have masked any difference in the results. A recent meta-analysis study reported that LA, aCL and aβ2GP1 all were strong risk factors for pregancy loss

42

(Opatrny, et al., 2006). A larger study of 269 patients (Shoenfeld et al. 2006) showed an increased prevalence of aPL (11% compared with 2.5%), with a PPV of 75%.

2.2.7.3.1.1 Obstetric complications of aPL

In APS, defined as the persistent presence of aPL (LA, aCL, aβ2GPI antibodies) and the loss of three or more miscarriages prior to 10 weeks, or the loss of one or more fetuses above 10 weeks, there is a higher incidence of obstetric complications including IUGR, PET, stillbirth and pre-term labour.

Although there have been follow-up studies of APS, showing an increased incidence of late obstetric complications compared with a healthy control group, there have been no follow-up studies of the obstetric complications in aPL-associated recurrent miscarriage compared with recurrent pregnancy loss without aPL. The incidence of obstetric complications is higher in women with recurrent pregnancy loss than in the general population (Carp, 2007).

2.2.7.3.1.2 Treatment of obstetrical APS

The standard treatment of aPL in pregnancy consists of low-molecular weight heparin and low-dose aspirin (Empson, et al 2005; Miyakis, et al., 2006). However, there are no comparative studies with which to judge this regimen. Hence, no predictive value can be given about treatment using evidence-based medicine. There are only descriptive reports. Recently, doubt has been cast on the role of aspirin, as a meta-analysis comparing three trials of aspirin compared with placebo showed a non-statistically significant OR of 1.05 in favour of aspirin (Vinatier, et al., 2001). Low-molecular weight heparins may lower the fetal loss rate; however, there is no effect on the incidence of late obstetric complications. Hence, when APS presents with late obstetric complications rather than

43

pregnancy loss, no prediction can be made about prognosis with anti-coagulant treatment.

Intravenous immunoglobulin (IVIg), however, actually lowers the titre of autoantibodies. Although it is not an anticoagulant and does not prevent thrombosis, there is some evidence that IVIg may lower the incidence of late obstetric complications (Carp, et al., 2001). However, there is insufficient data to allow any predictive value to be given.

2.2.7.3.2 Anti-thyroid antibodies

Anti-thyroid antibodies (ATAs) have been suggested to be independent markers of ‗at-risk‘

pregnancy. Euthyroid women with recurrent miscarriage have increased levels of autoantibodies either against thyroglobulin (aTG) or thyroid peroxidase (TPO) while the probability of abortion in women with ATA has been shown to be greater than in controls (Matalon, et al., 2001). There are animal models in which active immunization of mice with thyroglobulin has raised antibodies to thyroglobulin, and leading to increased fetal wastage and lower fetal and placenta weights (Tartkover-Matalon, et al., 2003). However, the pathophysiological role of these antibodies is still unclear. In recurrent pregnancy loss, the association between pregnancy loss and thyroid antibodies may be a result of: (i) a direct effect of ATAs on fetal tissue or (ii) the thyroid antibodies representing an underlying more generalized defect in autoimmunity. However, the prevalence of ATA has been reported to be 15–20% in normal pregnant women, compared with 20–25% in women with recurrent miscarriages (Ghazeeri, et al., 2001). In Marai et al.'s (2004) study, anti-TPO antibodies were the only autoantibodies found to have a significant association with recurrent miscarriage. Similar study by IIjima et al (1997) showed similar outcome. The aTPOs were found in 21% of the women with recurrent miscarriages (8/38) as compared with 0% in women with

44

infertility and no miscarriages (0/20). However, in Shoenfeld et al.'s (2006) larger study, there was no association with recurrent miscarriage as a whole, but anti-TG antibodies were associated with late pregnancy loss compared with controls, (OR 8.44; 95% CI 1.6, 43.8), PPV = 40%. Study by Mannisto et al (2009) showed that thyroid dysfunction and antibodies were not associated with pregnancy complications. Overt hypothyroidism was associated with subsequent maternal thyroid disease [hazard ratio (HR) (95% confidence interval), 17.7 (7.8–40.6)] and diabetes [6.0 (2.2–16.4)].

Subclinical hypothyroidism [3.3 (1.6–6.9)], TPO-Ab-positivity [4.2 (2.3–7.4)], and TG-Ab-positivity [3.3 (1.9–6.0)] were also associated with later thyroid disease. No association was found between thyroid dysfunction/antibodies and hypertension or overall mortality. Thyroid dysfunction and antibodies during pregnancy seem to predict later thyroid disease. Thyroid dysfunction and antibodies during pregnancy seem to predict later maternal morbidity to thyroid diseases. Therefore, the prognostic value of ATA remains uncertain.

2.2.7.3.3 Anti-nuclear antibodies

Cubillos et al. (1997) found that the incidence of ANAs was 31.8% in patients with a history of miscarriages (110 patients), but only 5.7% in 35 healthy patients with proven fertility and no history of pregnancy loss or autoimmune disease.

In previous studies, a high prevalence of low-titre ANA have been reported in the sera of patients with both explained and unexplained pregnancy losses (Xu, et al., 1990). However, the significance of these findings is still unclear. ANAs were found with a higher prevalence in patients with autoimmune disease in Shoenfeld et al. (2006) study. However, they were not found to occur with a higher prevalence in patients with infertility or recurrent pregnancy loss.

45 2.2.7.4 Calcium

Calcium is the fifth most abundant element in the human body, with about 1000 g present in adults.

It plays a key role in skeletal mineralization, as well as a wide range of biologic functions. It is an essential element that is only available to the body through dietary sources. Current dietary calcium recommendations range from 1000 to 1500 mg/d, depending on age. In some individuals, particularly the elderly (McCabe et al., 2004), calcium supplements may be needed to achieve the recommended dietary calcium intake (Munro, 2010). Calcium requirement is dependent on the state of calcium metabolism, which is regulated by three main mechanisms: intestinal absorption, renal reabsorption, and bone turnover. These in turn are regulated by a set of interacting hormones, including parathyroid hormone (PTH), 1,25-dihydroxyvitamin D [1,25(OH)2D], ionized calcium itself, and their corresponding receptors in the gut, kidney, and bone (Munro, 2010). During pregnancy, total calcium declines in parallel with serum albumin, whereas free calcium is unchanged (Vasudevan et al., 2010). The fetal circulation is relatively hypercalcemic as evidenced by higher total and free calcium in cord blood than in maternal serum. Calcium concerntrations decline after birth in healthy term neonates during the first few days but soon rise to concerntrations slightly greater than those observed in adults (Burtis et al., 2012).

Calcium is required for the optimal activity of several extracellular digestive enzymes, including proteases, phospholipases and nucleases. Along the length of the gastrointestinal tract a calcium ion sensing receptor is expressed, and it is thought that the expression of this receptor plays a role in gastric acid secretion (Theobald, 2005). Calcium is essential for blood coagulation (clotting), a process that involves two pathways: the intrinsic pathway and the extrinsic pathway. Calcium plays a role in both pathways. The process of clot formation involves a cascade of proteolytic reactions, whereby inactive enzymes or clotting factors are activated. Each activated enzyme or clotting factor

46

then, in turn, activates another inactive factor, with the response amplifying each time, ultimately resulting in the production of thrombin from prothrombin and the production of a fibrin clot (Vasudevan et al., 2010; Burtis et al., 2012).

Calcium plays an important role in provoking neurotransmitter release from nerve cells and in muscle contraction. Both nerve and muscle cells are electrically excitable and their cell membranes contain calcium selective ion channels; these open when the membranes are depolarised (e.g. upon arrival of action potentials via nerves), causing an influx of calcium and an increase in cytosolic calcium concentration. In nerves, this results in the release of neurotransmitters (e.g. acetylcholine) (Sembulingam and Prema, 2010). A similar series of events causes heart muscle contraction. The role of calcium in the contraction of smooth muscle (e.g. the muscle lining the lungs and digestive tract) differs from that of skeletal and heart muscle contraction as the thin filaments of smooth muscle do not contain troponin. Rather, in smooth muscle, an increase in intracellular calcium ion concentration brings about binding of calcium to calmodulin (which is structurally similar to troponin) in the cytosol. The resultant calcium–calmodulin complex brings about the phosphorylation of myosin, allowing myosin crossbridges to form with actin and, thus, the muscle to contract. In smooth muscle only phosphorylated myosin can form cross-bridges with actin.

Relaxation is induced by dephosphorylation (Sembulingam and Prema, 2010).

47 Figure 2.2 Calcium homeostasis

Source: www. cnx.org

2.2.7.4.1 Calcium metabolism in pregnancy

The effect of pregnancy on the maternal skeleton has preoccupied the scientific medical community for decades. Fetal calcium deposition has been shown to peak at 350 mg/day in the third trimester,

48

(Sparks, 1984) and maternal calcium absorption increases to meet that demand, with greater increases reported among women with low intakes (Heaney and Skillman 1971;Ritchieet al., 1998;Vargas et al., 2004). Maternal bone turnover also increases, (Zeni et al., 2003; Hellmeyer et al., 2006) although women with low calcium intakes may respond differently to the additional demand (Ritchie et al., 1998).

Studies suggest that maternal calcium absorption increases significantly during the second and third trimesters (Cross et al., 1995).This increase in calcium absorption is directly related to maternal calcium intake. Ritchie et al. (1998) reported that women with a daily average calcium intake of 1,171 mg during pregnancy absorbed 57% during the second trimester and 72% during the third trimester. Several studies have reported that calcitriol [1,25(OH)2D] levels increase progressively each trimester, thereby influencing the increase in calcium absorption (Cross et al., 1995; Ritchie et al., 1998). Calcium absorption during pregnancy is mediated by changes in maternal calcitropic hormones. During the first trimester, parathyroid hormone (PTH) levels in Caucasian women consuming adequate amounts of calcium decrease to low-normal levels and then increase to the higher end of normal in the third trimester (Gallacher et al., 1994;Cooper et al., 2006) reflecting the increase in calcium transfer from mother to fetus. Although PTH levels typically do not increase above normal during pregnancy, levels of a prohormone, parathyroid hormone receptor protein (PTHrP), do increase in maternal circulation (Kovacs et al., 1997). PTHrP is recognized by PTH receptors and therefore has PTH-like effects. This prohormone is produced by mammary and fetal tissues to stimulate placental calcium transport to the fetus. (Kovacs et al., 1997). PTHrP may also protect the maternal skeleton from bone resorption (Kovacs et al., 1997) by increasing both calcium absorption in the small intestine and tubular resorption in the kidney. PTHrP may also support mineralization of trabecular and cortical bone in the fetus (Cooper etal., 2006).

49

Other calcitropic hormones affecting maternal calcium metabolism are both the active [1,25 (OH)2D] and the inactive [25(OH)D] forms of vitamin D. Serum 25(OH)D levels do not change during pregnancy, but an increase in 1-a-hydroxylase and additional synthesis in the placenta allows for an increase in the conversion of 25(OH)D to 1,25(OH)2D. Maternal serum 1,25(OH)2D levels increase twofold during pregnancy, allowing the intestinal absorption of calcium also to double.-(Ritchie et al., 1998;Zeni et al., 2003) Both free and protein-bound forms of calcitriol increase during pregnancy,( Wilson et al., 1990; Kent et al., 1993) as do concentrations of vitamin-D binding protein.(Bouillon et al., 1981) Due to the corresponding changes in vitamin-D-binding protein and 1,25(OH)2D, the index of free 1,25(OH)2D does not increase until the third trimester,( Ritchie et al., 1998;Wilson et al., 1990) which may explain the large increase in calcium absorption seen during late pregnancy. Because maternal 25(OH)D does cross the placenta and because there is a positive association between maternal serum 25(OH)D, cord blood 25(OH)D, and infant 25(OH)D levels at delivery,(Kovacs et al., 1997) it is thought that vitamin D plays a role in fetal bone development. However, there is a paucity of research in this area, and it is not clear what effect maternal vitamin D status has on maternal and/or fetal bone outcomes. A more in-depth review of the role of vitamin D during pregnancy is provided by Dror and Allen (2010).

2.2.7.4.2 Calcium level and preeclampsia

In the 1980s, it was reported that there is an inverse relationship between calcium intake and pregnancy-induced hypertension (PIH) defined as systolic blood pressure of >140 mmHg and/or

50

diastolic blood pressure of >90 mmHg that has occurred on at least two occasions at least 4 hours to 1 week apart ( Villar et al., 2006). PIH has been estimated to complicate 5% of all pregnancies and 11% of first pregnancies (Villar et al., 2004) often resulting in preterm birth (Ananth and Basso, 2010).

Preeclampsia is a condition in which hypertension (as defined above) occurs during the latter half of gestation and is associated with an increase in urinary protein. Low calcium intakes during pregnancy may 1) stimulate PTH secretion, increasing intracellular calcium and smooth musclecontractibility, and/or 2) release renin from the kidney, leading to vasoconstriction and retention of sodium and fluid. These physiological changes can lead to the development of Pregnancy Induced Hypertension and preeclampsia (Theobald, 2005). A meta-analysis of the role of calcium supplementation during pregnancy in the prevention of gestational hypertensive disorders found a 45% reduction in the development of PIH in women receiving calcium versus placebo (relative risk [RR] 0.55; 95% confidence interval [CI] 0.36–0.85) (Imdad et al., 2011). A Cochrane review of 13 trials involving 15,730 pregnant women reported that the average risk of preeclampsia was reduced in those receiving calcium supplements (RR 0.45) and that the effect was greatest in women with low baseline calcium intakes (RR 0.36) (Hofmeyr et al., 2003; 2006; 2007). The review concluded that pregnant women consuming low amounts of calcium could reduce their risk of preeclampsia by 31% to 65% if they consumed an additional 1,000 mg of calcium each day (Hofmeyr et al., 2003). This review also reported that the risk of developing PIH could be reduced with calcium supplementation (RR 0.65; 95% CI 0.53–0.81), especially in women with low baseline dietary calcium intakes (RR 0.44; 95% CI 02.8–0.70) (Hofmeyr et al., 2010).

2.2.7.4.3 Calcium level and preterm birth (delivery)

51

Calcium supplementation has shown effectiveness in reducing the risk of preterm delivery in women with low calcium intakes. Among pregnant women who regularly consumed less than 600 mg of calcium per day and were supplemented with additional calcium (1,500 mg/day), decreases in the risk of preterm delivery, in maternal morbidity, and in the neonatal mortality index were observed (Villar et al., 2006). A Cochrane review reported that women who were chronically low consumers of calcium but who took 1,000 mg of calcium supplement per day also reduced their risk of preterm birth by 24% (Hofmeyr et al., 2006).