TMEM16A chloride channels in the female reproductive tract and their role in normal and dysfunctional pregnancy and labour

Female fertility relies heavily on the health of the oviduct and uterus. Each of these organs consist of a muscular tube lined by epithelial cells. In this review, we discuss the role of the TMEM16A chloride ion channel, expressed in the epithelial and smooth muscle cells in these organs, with a focus on function and dysfunction. In the oviduct, infection and endometriosis are major causes of infertility and TMEM16A plays a major role. Oviduct epithelial TMEM16A is also essential for egg and sperm health and for the process of fertilization, which takes place in the oviduct. The role of TMEM16A in uterine function has focussed on the smooth muscle component. TMEM16A plays a major role in determining the sustained plateau component of the action potential in human myometrium. The plateau sits at the chloride equilibrium potential of −25 to −30 mV and, at this potential, calcium channels are active, permitting calcium influx and contraction. TMEM16A expression doubles in human labour and this is associated with a major increase in plateau and hence contraction duration. This prolongation of calcium influx permits contraction amplitude and duration to increase and appears to be important for successful labour.

The function of the uterus in mammals is to support the early development of the next generation (pregnancy) and to deliver the new individual into the world in a timely manner (birth). The wall of the oviduct (Fallopian tube) and uterus consists of a smooth muscle tube that is lined internally with epithelial cells. The oviduct is the site of fertilization, and tissue dysfunction and infections in this organ can lead to infertility. Oviductal damage predisposes to ectopic pregnancy, which can be difficult to diagnose and leads to loss of the fetus and very often oviduct removal, with subsequent serious consequences for fertility. Oviductal damage can be slow to diagnose and this can have serious consequences for maternal health and future fertility. The uterus is the site for embryo implantation, fetal growth and development, and uterine smooth muscle (the myometrium) must remain relatively quiescent throughout pregnancy to facilitate this. When the fetus is sufficiently mature and fetal oxygen and nutrient growth requirements outpace the ability of the maternal system to supply, uterine contractility must rapidly increase and deliver the fetus for independent life, in response to as yet imperfectly described hormonal and mechanical (stretch) signals. Labour is a relatively brief but complex process. During the active phase of labour, contractions are strong but must be interspersed with regular periods of relaxation (minutes duration), which permits oxygenated blood delivery to the placenta with consequential oxygen supply to the fetus. The frequent strong phasic uterine contractions and direct pressure from the fetal presenting part contribute to effacement of the cervix, as well as a probable increase in local inflammatory mediators. At full cervical dilation (second stage labour), the strong contractions are separated by more brief relaxations and birth occurs. Birth is usually followed promptly by a tonic uterine contraction that expels the placenta and also clamps closed the uterine spiral arteries and veins that supplied the placenta, thereby minimizing blood loss and post-partum haemorrhage. These processes involve hormonal and inflammatory changes initiated by both the fetus and the mother. Unfortunately, dysfunctional myometrial contractility arises frequently in obstetric practice. When labour occurs too early a vulnerable premature neonate is born (∼10% of pregnancies) (Ohuma et al., 2023) and this is associated with a 33% neonatal mortality and 70% perinatal morbidity (CCOPMM, 2025). In addition, there is ongoing increased long-term offspring mortality (through to the 36 years of age studied) (Ahmed et al., 2024). On the other hand, around 36% of women currently undergo caesarean delivery (CD). Of those who require CD in labour, for 70%, this is a result of poor labour contractions (dystocia) (Davis et al., 2015). Persistent ineffectual labour contractions in the active phase of labour that are resistant to oxytocin augmentation (failure to progress in labour, FPL) necessitate CD in labour. CD in labour increases the odds ratio (OR) for a range of significant issues: preterm labour in subsequent pregnancies (OR = 3.4) (Rosen O'Sullivan et al., 2025), hysterectomy (OR = 3.4) (Jamshed et al., 2021), postpartum haemorrhage (OR = 3) (Abecassis et al., 2024) and postpartum depression (OR = 1.5) (Cárdenas et al., 2025). Clearly, our understanding of the mechanisms underpinning uterine contractions in pregnancy and in labour requires further investigation. Excitation–contraction coupling A rise in cytoplasmic calcium is essential for contraction in all muscle. The increase in cytoplasmic calcium in the myometrium occurs predominantly during the action potential (AP), which is a result of the opening of long-lasting (L-type), voltage-gated calcium channels (L-VGCCs) (Wray & Arrowsmith, 2021). Release of calcium from the endoplasmic reticulum stores can also contribute to contraction. Because the L-VGCCs are voltage dependent, the initiation of APs, and thus contraction, depends on the level of the resting membrane potential: too negative and no APs occur, whereas if the membrane depolarizes to threshold, APs and contractions will ensue. Depolarization to threshold can occur through pacemaker-like activity (Fig. 1). The L-VGCC inhibitor nifedipine is used clinically to temporarily stop preterm labour contractions. In the most studied myometrium, the outer longitudinal muscle layer of the rat uterus, multiple fast spike APs overshoot 0 mV (Fig. 1A). However, spikes are followed by a more prolonged period of depolarization from −20 to −35 mV (Fig. 1B) in the inner circular myometrial layer in rodents (Osa & Ogasawara, 1984), the myometrium of women (Nakajima, 1971; Parkington et al., 1999) and sheep (Parkington et al., 1988), and in the oviduct smooth muscle of baboons (Talo & Hodgson, 1978), guinea-pigs (Parkington, 1983) and mice (Dixon et al., 2009). This results in prolonged calcium influx through L-VGCCs and therefore increased contraction (Parkington et al., 1999) (Fig. 1). In human labour, the plateau duration is increased (Fig. 1D) resulting in more sustained contraction, facilitating timely labour progression and birth. The importance of the plateau for expeditious labour raises the question as to the mechanism(s) underpinning the plateau. A study by Casteels & Kuriyama (1965) demonstrated that the concentration of cytoplasmic chloride in rat myometrial cells during pregnancy is not passively distributed, it is elevated. This elevated cytoplasmic chloride was found to be a result of the activity of chloride/bicarbonate (Dai & Zhang, 2002) and sodium/potassium/2-chloride (Danielsson et al., 2014; Mitsui & Hashitani, 2016; Zhu et al., 2016) exchangers. The measurements of resting membrane potential by Casteels established a chloride equilibrium potential of −20 to −35 mV (Casteels & Kuriyama, 1965). Thus, the opening of chloride channels would result in chloride exit, membrane depolarization from the resting membrane potential, activation of L-VGCCs and contraction. This mechanism has been demonstrated in smooth muscle cells (SMCs) using inhibitors of the relevant exchangers (Dai & Zhang, 2002; Danielsson et al., 2014; Mitsui & Hashitani, 2016; Zhu et al., 2016). Such an increase in chloride conductance could explain the post-spike prolonged depolarization in the myometrium and oviduct in both rats and humans. Epithelial cells line the female reproductive tract and are critically important in maintaining the health of the egg, sperm and zygote in the oviduct, as well as in the uterus, nourishing the early fetus before the establishment of the placenta. SMCs and epithelial cells both express a calcium-activated chloride channel (CaCC) protein. In 2008, three laboratories identified a protein product of the ‘orphan’ transmembrane 16 (TMEM16) gene (Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008) that fulfilled a broad range of the characteristics of the CaCC channel described previously in a diverse array of tissues. The CaCC protein, called TMEM16A, attracted wide attention not only because of its role in smooth muscle and epithelial cell function, but also because of its association with diseases of the cardiovascular and respiratory systems, the intestines, neurons and in a variety of cancers. As a general principle, possible sources of calcium for activation of TMEM16A include L-VGCCs, release from ER stores and mitochondria, and calcium influx through calcium-permeable cation channels [e.g. Orai, some transient receptor potential (TRP) channels]. Apart from the significant contribution of L-VGCCs, the relative importance of the various other sources of calcium for the activation of TMEM16A remains to be elucidated for the myometrium. However, blockade of TMEM16A produces depolarization of the resting membrane potential rather than the expected hyperpolarization in pregnant human myometrium and this may reflect a complex interaction between the sources of calcium and other ion channels; for example, those sensitive to calcium or voltage (Parkington et al., 2025). Because activation of TMEM16A channels can result in chloride efflux and thus prolonged depolarization, their activation has important functional consequences, including prolonged activation of L-VGCCs and smooth muscle contraction. TMEM16A activity explains published observations of CaCCs in smooth muscle, including the myometrium. In this review, we provide an update of the role of TMEM16A (also known as anoctamin-1, ANO1) and the chloride channel accessory CLCA proteins in the function of the female reproductive tract during pregnancy and labour. The oviduct Ciliated epithelial cells The fimbriae of the free open end of the oviduct gather up ovulated egg(s), mainly achieved by the ciliated oviductal epithelial cells. Subsequently, these cells strongly support movement of the egg from the fimbriae in the direction of the uterus. Fertilization takes place in the ampulla (approximately half way along the oviduct) and the ciliated epithelial cells assist movement of the zygote along the ampulla and isthmus of the oviduct towards the uterus. Similar to the epithelial cells lining the airways, oviductal epithelial cells express TMEM16A. Secretory epithelial cells In the oviduct, these cells release peptides, growth factors, glucose, immune factors, bicarbonate and chloride that support the survival of both egg(s) and sperm, and are also critical for fertilization. Oviduct fluid chloride is required for sperm motility and capacitation (Fasoli et al., 2025; Wertheimer et al., 2008). A study in macaques showed that TMEM16A is strongly expressed in the secretory epithelial cells of the oviduct, being at maximal levels in the days immediately prior to ovulation, and this rise in expression fails to occur in animals with endometriosis (Luo & Slayden, 2025). The exit of chloride via TMEM16A is associated with water secretion, via aquaporins (Im et al., 2020; Ribeiro et al., 2021), which decreases the viscosity of the epithelial secretion, facilitating egg/sperm/zygote movement. Inflammation in human airways epithelial cells is associated with over-expression of TMEM16A. TMEM16A interacts with the inositol-trisphosphate receptor, facilitating release of calcium from the endoplasmic reticulum store (Akin et al., 2023). This increase in cytoplasmic calcium results in enhanced release of mucin from epithelial cells, which increases tubal content viscosity, reducing ciliary activity and leading to tubal blockade (Cabrita et al., 2021). Although ampullary TMEM16A mRNA expression in epithelial cells from tissue obtained from healthy women and from women with a tubal pregnancy did not differ, TMEM16A protein expression was doubled in epithelial cells from tubal pregnancy tissue (Ning et al., 2023). This slowed embryo transit along the oviduct, with fewer embryos reaching the uterus. Thus, it is probable that either over- or under-expression of TMEM16A by epithelial cells has a major impact on fertility/infertility. As a consequence, agents that modulate TMEM16A activity, selective activators and blockers, have the potential to improve fertility. Also, a possible role for microRNAs (miRNAs) in regulating TMEM16A protein synthesis has been reviewed (Bai et al., 2021). miRNAs regulate protein expression and TMEM16A expression is down-regulated by miRNA-9 (Sonneville et al., 2015), which is enhanced during inflammatory infection and hence could have consequences for oviduct function and fertility (Dixon et al., 2019). Sperm The first direct patch clamp recordings of ion channels in sperm (human) were made by Orta et al. (2012). They identified a CaCC current that was blocked by T16Ainh-A01 and less selective inhibitors of TMEM16A: niflumic acid (NFA) and 4,4-diisothiocyano-2,2-stilbene disulphonic acid (DIDS). TMEM16A blockers inhibit sperm motility, capacitation and the acrosome reaction (required for sperm penetration of the vitelline layer of the egg during fertilization) (Orta et al., 2012; Roa-Espitia et al., 2025). NFA, DIDS and T16Ainh-A01 prevented fertilization in a dish. More recently, agents such as NFA and DIDS that block TMEM16A activity or other agents that activate TMEM16A activity have been used therapeutically to a minor extent (Laghetti et al., 2025). However, new efforts have been instigated, and recently developed TMEM16A activators are under investigation (Al-Hosni et al., 2025). Smooth muscle Smooth muscle forms the major mass of the oviduct and it consists of a substantial inner component of circularly-arranged smooth muscle fibres and a thin outer layer of longitudinally arranged fibres. The muscle mass becomes thicker as the oviduct approaches the uterus: the uterotubal junction. The first recordings of oviductal circular smooth muscle activity demonstrated rhythmic oscillations in membrane potential in the oviduct of guinea-pig, mouse and baboon (Talo & Hodgson, 1978). We later demonstrated in guinea-pig isthmic circular smooth muscle that each contraction was associated with a ∼1.5 min oscillation in membrane potential of 40 mV, from a resting membrane potential of around −65 mV to a plateau at −25 to −30 mV. These membrane potential oscillations were underpinned by an increase in chloride conductance that was calcium sensitive, that is, a CaCC (Parkington, 1983). This electrical activity persisted in the presence of the L-VGCC blocker verapamil, similar to well-studied pacemaker potentials in guinea-pig stomach (Dickens et al., 1999). We found that the amplitude and frequency of the membrane potential oscillations and contractions in the isthmic region increased on day 4 following ovulation. All of these experiments were before the discovery that the orphan TMEM16A and B proteins were responsible for the well-established CaCCs previously identified in many tissues. However, the electrical properties of the cells mirror those found in TMEM16A-expressing interstitial pacemaker cells in the muscle wall of the intestines (Huizinga et al., 1995; Sanders et al., 2014). Previous studies has provided convincing evidence that, although cilia are important for egg capture and translocation along the ampulla, transport of the zygote along the terminal ampulla and isthmus to the uterus involves smooth muscle contraction, with TMEM16A playing a major role (Dixon et al., 2009; Dixon et al., 2012). Functional implications Endometriosis can affect up to 10% of women and may have devastating long-term effects including, but not restricted to, infertility (Agrez, 2026; Vercellini et al., 2026). Endometriosis results from uterine lining-like epithelial cell migration to inappropriate locations, most commonly in the peritoneum and serosa of the ovary. Also, endometriosis of the lining of the oviduct has been described in up to 72% of patients with pelvic endometriosis. It causes scarring and/or blockade of the oviduct. It has been described only in humans and the old-world monkeys, restricting the detailed testing of direct function and agents that might alleviate the consequences. The pre-ovulatory rise in ampullary endometrial TMEM16A fails to occur in rhesus monkeys suffering endometriosis. In addition, peritoneal fluid from those with endometriosis induced the disease in controls (Luo & Slayden, 2025). It was concluded that an element present in the peritoneal fluid suppressed TMEM16A protein expression in endometriosis. These findings are consistent with the low fertility in endometriosis. In humans, 1–2% of pregnancies are ectopic, mostly tubal. In ampullary tissue samples from women undergoing surgery to remove the ectopic tissue, TMEM16A expression was significantly elevated, and ciliary beating and muscle contractions reduced (Ning et al., 2023). Ciliary beating and muscle contraction in these samples responded to the TMEM16A activator Eact and blocker CaCCinh-A01, supporting a role for TMEM16A in oviduct function/dysfunction. To investigate the functional consequences this group used mice and tested the effects of Eact and CaCCinh-A01 on embryo transport in the oviduct and uterus. Both of these agents independently halved the number of eggs that remained in the oviduct, never reaching the uterus, with only 12% (for Eact) and 8% (for CaCCinh-A01) of early embryos successfully implanting in the uterus (Ning et al., 2023). Thus, TMEM16A functionality appears to hang in a delicate balance between over- or under-expression, because either extreme has significant deleterious effects on fertility. The uterus Epithelial lining The lining of the uterus, the endometrium, consists of epithelial cells that provide essential critical support for the early blastocyst until implantation (around day 9 for human, day 4 for mouse) and until subsequent placental formation is fully established. Endometrial expression of TMEM16A is robust in early pregnancy in mice. Maternal subcutaneous injection of the TMEM16A inhibitor T16Ainh-AO1 in mice on days 4–7 of pregnancy halved the decidualization rate (determined on day 8 of pregnancy) (Qi et al., 2018). In a study by Dodds et al. (2015), TMEM16A expression was not observed in the myometrium of virgin mice, but the chloride channel accessory protein CLCA4 was expressed in the endometrial cells. CLCA4 has been implicated in regulating TMEM16B function, with no effect on TMEM16A (Sala-Rabanal et al., 2024). In pigs, an increase in CLCA4 expression was associated with a larger litter size (Hwang et al., 2017), supporting an improvement in endometrial function. Clearly, further study is required to enhance understanding of the mechanisms involved, including the factors controlling the expression of TMEM16A, TMEM16B and CLCA4 in the endometrium. Smooth muscle The wall of the uterus provides substantial housing for the developing fetus and appropriate modulation of contractile activity of the muscle component, namely the myometrium, is essential for both healthy development of the fetus and for the birth process. Throughout pregnancy, uterine contractions must remain modest (at most). Then, when the maternal/placental systems can no longer provide the nutritional and oxygen needs of the exponentially growing fetus, concerted contractile activity must develop expeditiously, sending the mother into labour. The contractions required for successful labour must be large in the active phase, pressing the fetal head forward to thin (efface) and dilate the cervix. However, because the uterine spiral arteries to the placenta are surrounded by the thick contracting myometrium, each contraction compresses the spiral placental arteries closed. This deprives the fetus of oxygen, and fetal brain oxygen decreases. Because cervical dilation and descent of the fetus takes hours, regular, adequate periods of relaxation between contractions are essential for the restoration and maintenance of fetal brain oxygenation (Turner et al., 2020). However, if the rest periods between contractions are inadequate, CD in labour is required because of the risk of fetal brain hypoxia (most usually detected by the presence of consequential fetal heart rate abnormalities routinely recorded continuously throughout labour). On the other hand, if the driving contractions are weak and maternal intravenous oxytocin delivery fails to enhance contractions (failure to progress in labour), CD in labour is required. As mentioned in the Introduction, CD in labour is associated with a range of adverse long-term consequence for the mother and offspring. Thus, an improved understanding of myometrial contractility during labour, addressing both contraction and relaxation, is essential if we are to reduce the rate of CD in labour. The spiral arteries, delivering blood into the placental lakes, are very large in diameter and are devoid of most smooth muscle from weeks 10–12 of human pregnancy. That this structure is essential for optimal placental perfusion is evidenced by the fact that the persistence of spiral artery smooth muscle gives rise to pre-eclampsia consequent upon placental hypoxia and dysfunction resulting in placental angiogenic protein production imbalance, a key aetiological mediator of pre-eclampsia. Thus, healthy spiral arteries have minimal ability to contract to prevent substantial blood loss when the placenta is delivered (occuring 10–30 min after birth) and, as a consequence, strong sustained contractions of the myometrium during and immediately following delivery are essential. A consequence of weak myometrial contraction immediately following birth is postpartum haemorrhage (PPH) (Arenas & Lorca, 2024; Brooker et al., 2024; Gill et al., 2025). Despite the prophylactic use of currently available oxytocic medications PPH is a leading cause of maternal death and its incidence is increasing worldwide (Fitzgerald et al., 2024; Ford et al., 2007; Pettersen et al., 2023). Therapeutic enhancement of uterine contractions immediately following delivery is a possible mechanism whereby PPH may be minimized. A therapeutics-induced increase in the activity of TMEM16A could be a means of increasing uterine contractions when labour contractions are weak (dystocia) and/or when PPH threatens. The potential of TMEM16A as a drug target is enhanced by the multitude of isoforms that arise from its 26 exons (Ferrera et al., 2009; Mazzone et al., 2011). In human myometrium, western blotting showed three bands with molecular masses of 90, 97 and 114 kDa (Parkington et al., 2025), all of which are less than the expected 120 kDa of the full length TMEM16A (Mazzone et al., 2011). The presence of different isoforms raises the possibility of selective targeting of TMEM16A isoforms for particular tissues, such as the myometrium. Myometrial contractility during pregnancy and labour Myometrial APs and contractions occur spontaneously in excised myometrial strips obtained at all stages of pregnancy in all species studied (human, non-human primates, rodents, rabbits, guinea-pigs, sheep, cows). We recorded electrical activity in the myometrium of normally functioning conscious ewes throughout the latter half of pregnancy and through into labour. Electromyographic (EMG) electrodes (recording the electrical spikes that underpin contraction) were implanted (under anaesthesia) in the uterus on days 60–100 of the 143–148 day pregnancy (Harding et al., 1982; Parkington et al., 1988). EMG activity, although relatively small, occurred at a low frequency throughout pregnancy (Fig. 2A). Of interest in this context, women can be aware of low level Braxton Hicks contractions, even from early (∼12 weeks) in pregnancy (Raines & Cooper, 2025). In our ewes, EMG frequency and amplitude increased markedly during labour (Fig. 2B), as would be expected. Ongoing approaches, developed in sheep, are expanding our understanding in human labour (Wang et al., 2023). As shown in Fig. 1C and D, the plateau of depolarization as a result of the activity of TMEM16A channels is enhanced in labour and this is associated with a doubling of TMEM16A protein expression during normal labour (human tissues). Western blotting and immunohistochemistry identified a two-fold increase in myometrial TMEM16A protein expression in tissue from women in well progressing labour. This enhanced labour expression was not observed in tissue from those suffering failure to progress (Parkington et al., 2025). Blocking TMEM16A in myometrial strips from these women did not influence the small pacemaker potentials or spike Aps, although it markedly reduced the plateau component of the AP, especially during labour (Parkington et al., 2025). Arnaudeau et al. (1994) reported that the CaCC current in pregnant rat myometrium is enhanced by oxytocin. We found that oxytocin evoked a marked enhancement of the plateau component of the AP in human myometrium and this was entirely abolished by blockade of TMEM16A (Fig. 3) (Parkington et al., 2025). In addition, the enhanced hyperpolarization between the plateau APs induced by oxytocin now failed to occur because it is probably the result of a marked enhancement of calcium- and/or voltage-activated potassium channel activity initiated by the prolonged plateau. However, brief spike APs (involving L-VGCCs) and accompanying small, brief contractions persisted in the presence of the TMEM16A blocker. Labour contractions are associated with an increase in intracellular acidity in uterine SMCs as a result of the production of lactate that arises from labour contraction-induced occlusion of blood flow and the resulting hypoxia. This decrease in pH may result in an increase in the activity of TMEM16A in these cells because this mechanism occurs in vascular smooth muscle (Guo et al., 2021). This would tend to increase the duration of the plateau of depolarization and hence labour contractions. Other ion channels are also affected by low pH including some potassium, L-VGCCs and poorly-selective cation channels. Low pH can also reduce rho-kinase activity in smooth muscle and this would decrease the sensitivity of the contractile apparatus to calcium (Boedtkjer et al., 2011), thereby tending to reduce labour contractions. Overall, the effects of low pH on labour contractions depend on the complex interplay between a variety of ion channels and signalling processes (Wray et al., 2021). Smooth muscle pacemakers What are the mechanisms underpinning the spontaneous nature of the slow depolarization that give rise to APs and contractile activity in the myometrium? Marshall (1959) was the first to report that myometrial APs and contractions are preceded by slow spontaneous depolarizations. Spontaneous depolarizations, giving rise to APs and contraction, are clear in all layers of pregnant human and rat myometrium ex vivo (Fig. 1). This raises questions regarding the nature of the biophysical and biochemical mechanisms and the cell type underpinning these depolarizations. The mechanisms underpinning spontaneous contractility in smooth muscle tissues (e.g. gastrointestinal, urinary, lung, vascular and female and male reproductive – pacemaking – have received intense study over the past 50 years. The SMCs are responsible for contraction and express smooth muscle actin, heavy chain myosin, calponin and SM22α. However, a range of fibroblast-like stem cells have been identified in smooth muscle tissues. Of these, those that express the receptor tyrosine kinase c-Kit and/or vimentin have been called interstitial cells of Cajal (ICCs or ICC-like cells). Cells that express platelet-derived growth factor receptor α (PDGFRα) have also been identified, and cells that express c-Kit and CD34 were named telocytes. These mesenchymal stem cells all (i) possess a modest nuclear region; (ii) have long, branched processes that lie between the SMCs; (iii) generate spontaneous depolarizations; (iv) receive input from the neurons innervating the tissue; and (v) form gap junctions with the SMCs, permitting ICC depolarization to ‘invade’ the SMCs, thus taking their membrane potential to threshold for the opening of L-VGCCs and contraction: pacemaking (Hwang et al., 2019; Sanders et al., 2014). In addition, in the renal pelvis of some species, atypical SMCs provide a significant contribution to pacemaking (Iqbal et al., 2012). Mutations in c-Kit result in organ malfunction and/or death in animal models, and contribute to human diseases such as Hirschsprung's disease (Cil et al., 2021; Sanders et al., 2023). This demonstrates the essential nature of ICCs in rodent models and in human colon, at least. Early voltage clamp studies of single ICCs isolated from intestinal smooth muscle tissues demonstrated that the pacemaker depolarizations are underpinned by a chloride conductance, calcium dependent, blocked by NFA and resistant to nifedipine. A role for TMEM16A was demonstrated when mutations of the gene in mice resulted in the abolition of pacemaking in the gastrointestinal tract and early death (Rock et al., 2008). Myometrial pacemaking In vivo and ex vivo studies clearly demonstrate the occurrence of regular spontaneous contractility in the pregnant uterus (as discussed above). Contractions are modest throughout pregnancy and strong during delivery. So, which cells are responsible for the initiation of contractions, where are they located, and is TMEM16A involved in pacemaking? Cells with the characteristics of smooth muscle tissue ICC pacemakers [as described in the list (i) to (v) above] have been identified in the myometrium of humans, non-human primates, rodents and sheep. However, regarding the neuronal contribution to pacemaking [(iv) in the list above], the trophoblast invasion during placentation results in the disappearance of the autonomic nerves supplying the main body of the uterus (Nelson & Nelson, 2013). In rodents, cells expressing one or more of the ICC properties discussed above have been demonstrated in the ovary, oviduct and uterus of virgin mice (Peri et al., 2015). These ICCs are widely and abundantly distributed, running between smooth muscle bundles, interspersed between SMCs within the bundles, within the endometrial linings and at the outer serosal edge of the virgin uterus. Their functional consequences are less clear. In virgin mice, although TMEM16A expression was identified in both the outer and inner myometrial layers by Bernstein et al. (2014), expression in either layer was not observed in a study by Dodds et al. (2015). In pregnant mice with SMC-specific TMEM16A deletion, myometrial contractility was normal (Qu et al., 2019), suggestive of IC- rather than SMC-mediated TMEM16A mechanisms, analogous to events in the gastrointestinal tract. Studies of pregnant rat (Arnaudeau et al., 1994; Jones et al., 2004) and guinea-pig (Coleman & Parkington, 1987) myometrium identified the presence of a CaCC current. This current was present in only ∼30% of the cells but its origin (i.e. myometrial or ICC cells) was not investigated. The CaCC current in single cells and spontaneous contractions in muscle strips were reduced by NFA and A9. High potassium induced contractions were resistant to these blockers. A subsequent study of isolated pregnant human and rat myometrium took into account the shape of the isolated cells: spindle-shaped SMCs (90%) or ICC-like cells that possessed spines and/or long branched processes (10%) (Duquette et al., 2005). The ICC-like cells did not generate spontaneous depolarizations, such as those frequently recorded in gastrointestinal SMCs. In addition, they did not display an inward current in response to depolarization, although they did possess an outward potassium current. This does not support the presence of a CaCC. The SMCs fired APs in response to depolarization and contracted. The ICC-like cells stained for vimentin, but not c-Kit. TMEM16A expression or the effects of more selective blockers was not determined because reliable CaCC channel blockers had not yet been developed (pre-2008). In primates, immunohistochemistry identified c-Kit, vimentin and PDGFRα in the ovary, oviduct and uterus of non-pregnant cynomolgus monkeys (Peri et al., 2015). ICC-like cells, including telocytes, are present in the uterine wall of pregnant women (Ciontea et al., 2005; Hutchings et al., 2009) (Fig. 4). Electrophysiological experiments following human myometrial culture found a CaCC current but an absence of a L-VGCC current in telocytes. Duquette et al. (2005) isolated single cells from rat uterus that stained for vimentin but did not stain for cKit. Using single-cell electrophysiology, they did not find any inward currents that could underlie spontaneous pacing activity and concluded that ICCs were not involved in the pacemaking process (Duquette et al., 2005). However, vimentin also stains fibroblasts. The isolated ICCs in a study by Ciontea et al. (2005) stained for cKit. These cells had spontaneous depolarizations, but this was following prolonged culture and observations conducted with cultured smooth muscle tissue require cautious interpretation (Chin-Smith et al., 2014; Duquette et al., 2005; House et al., 2008). We studied human myometrial strips and acutely isolated cells within hours of tissue acquisition and a nifedipine-resistant depolarizing current was not observed in ICC-/telocyte-like cells (Fig. 4), which is similar to the observations of Hutchings et al. (2009). Functional implications Setting out the implications of TMEM16A for uterine function is far from straightforward. First, the cell type expressing TMEM16A remains unresolved: SMCs or interstitial cells? Elegant studies have provided convincing evidence that myometrial contractile activity of the uterus is initiated in cells in very close proximity to the placental sites in rats (Lutton et al., 2017; Lutton et al., 2018). The nature of the cells involved is unclear, and further work is required to resolve this issue. Of relevance here is the observation that TMEM16A is expressed in SMCs rather than ICCs in the internal anal sphincter of mice (Lu et al., 2024). Experimentally, smooth muscle-specific TMEM16A knockout mice (TMEM16ASMKO) were studied and (i) myometrial contractility; (ii) sensitivity to oxytocin and prostaglandin (determined in isolated strips); and (iii) litter size (8 vs. 7) was not different between knockout and control pregnancies (Qu et al., 2019). In addition, immunohistochemical TMEM16A expression was entirely absent from the SMCs but was abundantly expressed in the endometrium. The idea that myometrial pacemaking follows the ‘rules’ set out by the classical notion of pacemaker activity in most other smooth muscle tissues has been questioned by Young (2018) in a review of the evidence in human myometrium. In a detailed, thoughtful review Young (2018) argues that pacemakers are widely distributed throughout the uterus. Our observations in pregnant and labouring ewes would support this view (Fig. 2).

TMEM16A has a major role in female reproductive function. This encompasses transport of the ovum to the mid oviduct where fertilization takes place. It is essential for sperm function and for the process of fertilization. Although the role of TMEM16A in maintaining relative quiescence of the uterus throughout pregnancy is unclear, its enhanced expression in labour probably contributes to the strong contractions of labour and to strong postpartum contraction, preventing haemorrhage. The mechanisms underpinning smooth muscle pacemaking in a variety of smooth muscle tissues have been resolved as a result of decades of technically difficult experiments. TMEM16A is a major player. A similar effort is now required for our understanding of myometrial pacemaking because it is critical for successful birth. On the bright side, advancements in producing new activators and blockers of TMEM16A are under way (Al-Hosni et al., 2022; Al-Hosni et al., 2025; Sala-Rabanal et al., 2024), potentially providing additional therapeutic mechanisms that could extend pregnancy when preterm birth threatens, reduce the incidence of CD in labour when failure to progress occurs and present novel approaches to preventing postpartum haemorrhage. What is clear is that, once an AP spike is initiated, resulting in calcium influx, TMEM16A becomes involved. The consequent prolonged plateau of depolarization as a result of TMEM16A is critical for the strong contractions of the myometrium that are required for successful birth and prevention of postpartum haemorrhage. Biographies Helena Parkington is Professor in the Department of Physiology, Monash University. She uses ex vivo and in vivo electrophysiology, calcium imaging and transcriptomics to better understand the ionic mechanisms underlying smooth muscle contractile function and dysfunction in human uterine tissue during pregnancy and labour. These approaches give considerable insight into ionic mechanisms underlying smooth muscle contractile activity and clearly demonstrate the critical role of membrane potential in determining uterine contractile activity, including labour contractions. Penelope M. Sheehan is Director of Obstetrics at Eastern Health and Senior Lecturer at Monash University Department of Obstetrics and Gynaecology. She is also Head of the Preterm Labour Unit at Royal Women's Hospital. She is the Clinical Lead for Maternity and Newborn Improvement Program, responsible for the State's Improvement work in Maternity Services. She complements her clinical roles with an active program of research that specializes in parturition and preterm birth.

Additional information Competing interests The authors declare that they have no competing interests. Author contributions H.C.P. and H.A.C. contributed to the conception of the review and provided the first draft, and also had a major role in revising the manuscript. P.M.S. and S.P.B. provided critical comments on the clinical aspects of the review. M.T. provided critical comment on the scientific aspects of the review. All authors have approved of the final version of the manuscript submitted for publication. However, H.C.P. and H.A.C. are accountable for the overall submitted review. All authors meet the authorship criteria listed and thus qualify for authorship. Funding National Health and Medical Research Council of Australia grants 2000774 and 1127100. Australian Research Council (ARC) grant DP230101068.

This review includes original data for which we thank the research midwives for liaising with the pregnant women and facilitating the collection of tissue samples, as well as the women who donated tissue. Open access publishing facilitated by Monash University, as part of the Wiley - Monash University agreement via the Council of Australasian University Librarians