We also highlight where active pharmaceutical agents (APIs) with tocolytic properties have been used for other clinical indications and how these could inform the design of future therapeutics or be repurposed to diversify their application such as for use in preterm birth. Finally we outline and discuss the future challenges.The World Health Organisation (WHO) estimates 15 million babies worldwide are born preterm each year, with 1 million infant mortalities and long-term morbidity in survivors. Whilst the past 40 years have provided some understanding in the causes of preterm birth, along with development of a range of therapeutic options, notably prophylactic use of progesterone or uterine contraction suppressants (tocolytics), the number of preterm births continues to rise. Existing therapeutics used to control uterine contractions are restricted in their clinical use due to pharmacological drawbacks such as poor potency, transfer of drugs to the fetus across the placenta and maternal side effects from activity in other maternal systems. This review focuses on addressing the urgent need for the development of alternative therapeutic systems with improved efficacy and safety for the treatment of preterm birth. We discuss the application of nanomedicine as a viable opportunity to engineer pre-existing tocolytic agents and progestogens into nanoformulations, to improve their efficacy and address current drawbacks to their use. We review different nanomedicines including liposomes, lipid-based carriers, polymers and nanosuspensions highlighting where possible, where these technologies have already been exploited  This review outlines the importance of improving the treatment of preterm birth, summarises the use of nanomedicines for the delivery of active pharmaceutical ingredients and discusses the opportunities/challenges for enhancing future patient outcomes. Additionally, although the largest concern linked to preterm birth is immediate mortality, preterm infants often experience long-term morbidity including neurodevelopmental delay, cerebral palsy as well as respiratory disorders, cardiovascular disorders, infections, visual and hearing problems and learning disabilities owing to the underdeveloped fetal organs and systems.9 Indeed, preterm birth is one of the largest contributors to the disease burden and concomitant economic burden globally due to the lifelong disability often associated with being born preterm.10Preterm birth is defined as the birth of a child before 37 weeks' gestation; a full-term pregnancy lasts 39–41 week. According to the 2018 report from the World Health Organisation (WHO), preterm birth globally affects up to 15 million babies each year, with a total of 1 million infant mortalities.11 This includes poor fetal growth  often as a result of a poor functioning placenta, fetal anatomical concerns, or fetal distress. Alternatively, planned preterm birth can occur if the mother has a health condition such as preeclampsia or other obstetrical complications including placental abruption. In these situations, obstetricians induce birth early given that there is a clear clinical advantage of early labour based on maternal and fetal considerations.11,12 Given that here, labour onset is pre-planned and somewhat controlled, pharmacological intervention is not necessary and hence iatrogenic preterm births are not the focus of this review.Clinically, preterm birth is classified into different subtypes: In ∼30–35% of cases, preterm labour is pre-planned ) and instigated by obstetricians; these planned cases occur primarily because the mother or the fetus is suffering from a potentially life-threatening condition and hence earlier delivery is safer.11,13 Spontaneous preterm birth results from either preterm premature rupture of membranes (PPROM) or idiopathic (no known cause) spontaneous labour onset involving uterine contractions and cervical dilation. Around 40–45% of all preterm births are idiopathic and are the main focus of the treatments described here.The majority (∼65–70%) of preterm births are classed as sudden or unexpected and are referred to as spontaneous in onset.e.g. twins). Other possible etiologies include intrauterine infection and inflammation. That previous spontaneous preterm delivery increases the risk of subsequent preterm birth, suggests that there may be a genetic factor involved. Many studies to date have identified multiple genetic variants linked to spontaneous preterm birth, however, in most cases, outcomes cannot be replicated across different population cohorts.14 Furthermore, the function of the genes identified are often not validated and therefore a biological role is not provided. In addition, many genetic studies to do not consider the gene–environment interactions including external and in utero environmental factors.Risk factors for spontaneous preterm birth include preterm birth in a previous pregnancy, having a short (≤25 mm) cervix , either naturally or because of cervical surgery, and multiple pregnancy .18,19 The increased incidence of preterm birth in twins is multifactorial and includes maternal complications (discussed above) as well as uterine overdistention (stretch) which can trigger uterine contractions.Multiple gestations, 20 Conversely recent studies suggest that advanced maternal age (≥35 years) and concomitant increase in risk of gestational diabetes and preeclampsia, may contribute to the increased risk of preterm birth.21 Pregnancies conceived by assisted reproductive technologies  are also at higher risk of preterm birth.22Interestingly maternal age, both ≤19 years and ≥35 years, is often described as high risk for preterm birth. For those of a young maternal age (≤19 years), spontaneous preterm birth may be due to biological immaturity increasing their risk of PPROM.The treatment of preterm birth can be divided into two categories; either prophylactic treatment for preterm birth prevention for women at high risk of preterm birth, such as those with a history of previous preterm birth and/or current evidence of a short cervix, or treatment at the onset of spontaneous preterm labour to suppress uterine contractions, known as tocolysis. The types of active pharmaceutical ingredients (APIs) used, and the clinical findings will be briefly discussed in this section.P) and lower aqueous solubility than progesterone and is therefore administered as an intramuscular injection  have recommended vaginal progesterone for high risk women. For OHPC, in 2003, a multi-centre, double-blind, placebo-controlled trial evaluating progesterone in women with prior history of spontaneous preterm birth found clear benefit with significant reduction in the rate of recurrent preterm birth and significantly prolonged the duration of pregnancy25 and was granted approval by the U.S. Food and Drug Administration (FDA). However, a subsequent follow-up confirmatory trial could not replicate the initial findings and raised other safety concerns.26 In October 2022, the FDA held a hearing to discuss the proposal to withdraw the approval of OHPC for the treatment of preterm birth, the decision has not yet been announced.The APIs used for prophylactic treatment to reduce the risk of preterm birth occurring are progestogens . For these pregnancies, there is currently a lack of evidence-based interventions for the prevention of preterm birth. The most studied is prophylactic progesterone administration  in twin or triplet pregnancy. Neither vaginal progesterone in twin nor OHPC in twins or triplets has been shown to reduce preterm birth before 34 weeks.28,29 However, a higher dosage of daily vaginal progesterone (600 mg) for women with twins may improve preterm birth outcomes.30 Additionally, in a subgroup of women with twins and a short cervix, vaginal progesterone reduced the risk of delivering before 34 weeks.31 The same, however, has not been found for use of OHC in twin pregnancies and short cervix.32 Guidelines, such as those by the International Society of Ultrasound in Obstetrics and Gynecology (ISUOG), therefore recommend screening for preterm birth in twin pregnancy with cervical assessment on vaginal ultrasound but without clear guidance on management regime to follow. Due to insufficient evidence, routine use of intramuscular or vaginal progesterone for prevention of preterm birth in multiple pregnancies is not recommended.33 Unlike in singleton pregnancies, the use of tocolytics in multiple pregnancies has been less studied.34 Questions also arise over whether tocolytic treatments demonstrate the same efficacy in preventing or delaying preterm birth or improving infant outcomes for multiple gestation pregnancies. There is insufficient evidence for the prophylactic use of β2 adrenergic receptor agonists in twins,35 whilst nifedipine appears safe and effective in delaying labour in both singleton and twin pregnancies with spontaneous preterm labour onset.36 Indomethacin has not been investigated specifically in multiple pregnancy, but twin gestations were included in a network meta-analysis comparing tocolytic therapies for preterm delivery. Indomethacin showed the highest delay in delivery by 48 hours and least maternal side effects,37 albeit its use was restricted to before 32 weeks due to known fetal complications. Given the weaker evidence basis for the preterm birth treatments for multiple gestations compared to singleton pregnancies more research is needed to identify the best treatment options.For multiple gestations the aetiology of preterm birth is likely to be multifactorial and different to singleton pregnancies  to enhance fetal lung maturation prior to delivery; to reduce neonatal respiratory complications. Furthermore, the woman may also be administered magnesium sulphate to provide neuroprotection and reduce brain injury in the infant. These APIs can be divided into subcategories of tocolytics based on their mechanism of action (e.g. nifedipine), β2 adrenoceptor agonists (betamimetics) , oxytocin receptor antagonists  and non-steroidal anti-inflammatory drugs (NSAIDs) (e.g. indomethacin).37,42,43 For further discussion of mechanism of action of these drugs, see Arrowsmith et al. 2010 and Wray et al. 2022.44,45 These tocolytic APIs, with the exception of salbutamol, generally possess limited aqueous solubility , while all except atosiban can be administered orally , which is important for the production of prostaglandins within intrauterine tissues, and has been shown to reduce uterine contractility; thus, extending gestation. However, it also poses concerning side effects including premature closure of a major blood vessel, the ductus arteriosus, in the fetus as well as intraventricular bleeding.57,58Currently in clinic, calcium channel blockers are the most common first line treatment due to their enhanced tocolytic activity over other tocolytic categories and reduced fetal effects.in vivo pharmacological performance of tocolytic agents, whilst addressing dosage restrictions and maternal and fetal safety concerns. The development of new APIs for the treatment of preterm birth is outside the scope of this review, instead the reader is directed to the review by Zierden et al.59With significant disadvantages still associated with the most potent tocolytics, there is a clear requirement for the development of new tocolytics or new formulations of pre-existing APIs that can be engineered to improve their pharmacokinetic and safety properties. The key desirable outcomes are to improve the 60–62 Most notably, nanomedicine technologies offer the potential to alter the pharmacokinetics and biodistribution of APIs. An example of this is in the clinical success of targeted liposomes for the delivery of anticancer therapies: paclitaxel loaded liposomes for lung cancer, using d-α-tocopheryl polyethylene glycol 1000 succinate triphenylphosphine conjugate to actively target the mitochondria in cancerous cells, have shown a strong inhibitory effect of 73% and increased apoptosis of cancerous cells.63 In the setting of preterm birth, better drug pharmacokinetics and biodistribution to reduce the dosage profile is clearly attractive for improving the clinical performance of tocolytic agents where side-effects in both the mother and the fetus are often limiting factors for their use. As noted for the example in cancer therapy, the targeting of nanomedicines can be used to further enhance the pharmacokinetics of an API, typically using intravenous (IV) as the route of administration. Nanomedicines can be designed to incorporate passive and/or active targeting. Passive targeting uses the properties of the nanoparticles themselves  in combination with the physiology of the disease site to increase the percentage of the API that reaches the target site. An example of this such of targeting is the enhanced permeation and retention effect which exploits the pathophysiological characteristics observed in some solid tumours.64,65 Active targeting involves the conjugation of a high-affinity ligand onto the surface of the nanoparticle. This ligand is then able to bind to a specific receptor that is found on the targeted cells.66 Targeting of nanomedicines is a considerable opportunity for improving the efficacy of treatments.Nanomedicine, the application of nanotechnology for therapeutics and diagnostics, has provided significant advantages for medicine in recent decades.P ≤ 5, H-bond donors ≤5, H- bond acceptors ≤10).67 This is because the oral bioavailability is controlled by the aqueous solubility and permeability of the drug through the gastrointestinal membrane; APIs with low solubility and low permeability are often associated with poor therapeutic efficacy after oral administration.68 However, the use of nanomedicines can be used to circumvent the limits caused by an API's physicochemical properties. For example, nanosuspensions can be used to enhance the dissolution kinetics of poorly solubility APIs,69 while lipid nanoparticles can show mucoadhesive behaviour in the gut or drive lymphatic uptake of their payload.70 Such approaches are of interest for a wide range of conditions including cancer.71 Conversely, moving from an oral dose to an IV nanomedicine can be beneficial, as the properties of the nanomedicine can influence the elimination mechanisms. Thus the properties of the nanomedicine will also influence the pharmacokinetics of the treatment rather than these being purely controlled by the physiochemical properties of the dissolved API. In the treatment of preterm birth, where therapies are given in a clinical setting (compatible with IV administration), it may be attractive to move from the oral dosing of an API to IV dosing of a nanomedicine with the aim of improving targeting to the uterus. This could potentially allow for a dose reduction and reduced side-effects. Furthermore, because nanomedicines can offer different behaviour in terms of penetration across biological barriers there is also the potential to delivery APIs topically or vaginally. Vaginal administration is an attractive route for the treatment of preterm birth as it avoids hepatic first-pass metabolism that is seen for oral dosing and is also successful in delivering a local effect at the uterus.72,73 The vagina is lined with mucosal tissue and as such the efficacy of vaginally administered APIs is strongly influenced by the interaction of the formulation with the mucus lining in the vagina. In terms of nanomedicine, both mucoadhesive or mucoinert nanoparticles can be designed, each with its own strengths and weaknesses. Mucoadhesion can be used to prolong the residence time of an API in the vagina, however, this can also limit the penetration of the API through the mucus to the underlying cells.74 Conversely mucoinert/mucus penetrating nanoparticles can improve the delivery of an API to the vaginal epithelium.75 The ability to introduce specific moieties to target specific organs and the ability to address the aqueous solubility issues surrounding the lipophilic character of many drugs, including tocolytics, makes this technology exciting and viable for novel therapies for preterm birth, as discussed next.76Another opportunity offered by nanomedicines is the potential to open new routes of administration. For ‘conventional’ (non-nano) formulations of an API the route of administration is limited by the physicochemical parameters of the API. For example, for oral dosing the API needs to typically possess physiochemical parameters within Lipinski's rule of 5  and its biodistribution. Typically, initial development of nanomedicine formulations focusses on producing stable nanoparticles with the required surface coating, the highest possible drug loadings  and the appropriate drug release kinetics. The ability to obtain the highest drug loadings possible for a formulation is important for the potential translation of a nanomedicine formulation to clinical use. While nanomedicines offer the potential to increase the therapeutic index of an API,79 allowing for dose reduction. Nanomedicine formulations also can consist of a considerable mass of excipients which reduce the drug loading in the formulation; ultimately low drug loadings can make it impractical to dose the therapeutically relevant amount of the API. Nanoparticle diameter is another key property and is possibly the most studied parameter with numerous studies investigating how this influences the biological behaviour of nanoparticles. The diameters of the nanoparticles produced are often driven by what is possible due to the processing method and nanomedicine type used; typically, it is possible to produce samples with diameters that vary up to five-fold. While the size of a nanomedicine is not the only factor that controls its biological properties, some generalisations have been provided. For IV dosing, a the distribution of particle sizes can not exceed 5 μm in diameter, this is because particles larger than this may lead to embolization.80 Nanoparticles in the diameter range of 20–200 nm do not generally leave the capillaries, while nanoparticles with diameters ≤10 nm can be filtered out of the circulation by the kidneys.81 For oral administration, there is no upper particle size limit and generally smaller sizes of nanoparticles can increase bioavailability of the API.82 The understanding of the effect of nanoparticle size on less common administration routes  is more limited; we will discuss the findings of specific studies in the later sections of the review.Whilst beneficial, the ability of nanomedicines to alter the pharmacokinetics and biodistribution of an API also introduces additional complexity to the formulation. A nanomedicine has many additional physical properties when compared to a dissolved API including the size, shape, surface coating and particle composition, which may all influence the biological behaviour of the nanomedicine.83 However, this behaviour might not be generalisable to other nanoparticles with different surface properties. In the following sections we review the different nanomedicine systems and discuss how they have been developed and investigated to date for the treatment of preterm birth, including the in vitro and in vivo models used to evaluate their efficacy. We also discuss nanomedicine formulations that have been developed for indications other than preterm birth but with APIs that have known tocolytic properties and could therefore be repurposed for use in treating preterm birth. To aid comparison of the different nanomedicine formulations each section contains a table that summarises the key feature of the formulations.In the treatment of preterm birth with tocolytics, the ultimate objective is to target the distribution of the API to the uterus to stop contractions and avoid effects in other organs and systems in the mother, as well as critically, to prevent the API from crossing the placenta and entering the fetal circulation. In terms of applications in the treatment of preterm birth, there are very few studies that have systematically varied particle size and investigated biodistribution. One study has shown that smaller gold nanoparticles (20 or 50 nm) displayed higher accumulation in the uterus than nanoparticles with diameters of either 100 or 200 nm.84 Due to this structure, a liposome possesses an aqueous core that is surrounded by a hydrophobic lipid bilayer(s). As such, liposomes can encapsulate both hydrophilic drugs (in the aqueous core) and hydrophobic drugs (within the lipid bilayers(s)) . Refuerzo et al. developed fluorescently labelled multilamellar liposomes (150–170 nm)  in the uterus, where they are abundantly expressed and are responsible for mediating the contraction stimulating effect of oxytocin during labour.76 Refuerzo et al. targeted the liposomes to the OTRs, by conjugating atosiban, a competitive OTR antagonist, to the surface of the liposome , as adapted from a method developed by Hua et al. for the development of antibody-conjugated liposomes.87 To add a uterine targeting functionality, a lipid 1,2-diastearoyl-sn-glycero-3-phospho-ethanolamine coupled to polyethylene glycol (PEG) with a maleimide chain end functionality was conjugated to an anti-OTR antibody. The liposomes were then synthesised by high pressure extrusion.86 Their key findings showed that OTR-targeted liposomes containing nifedipine, salbutamol or rolipram were able inhibit ex vivo human uterine contractions, whilst non-targeted liposomes did not reduce contractions at the equivalent concentrations tested .88 Both targeting approaches enhanced cellular internalisation compared to conventional liposomes; +78% with OTR antibody targeting and +82% with atosiban targeting.88Liposomes are artificial spherical vesicles made of one or more lipid bilayers.yers(s)) . Liposom–170 nm)  synthesimulation  and minimulation .85 The lliposome .76 Analynd fetus . When cond fetus . Finallynd fetus .76 Furths tested .86 A redwas used . In a si90 However, to date, other than the family of prostaglandin receptors which also change expression with gestation91 there have been very few alternatives put forward.From the targeted liposomal studies, it is evident that the conjugating of a targeting moiety such as one directed to the OTR and exploiting the upregulation of OTRs in pregnant uterine tissue is a significant angle to explore for the progression of tocolytic agent development. Nevertheless, although the OTR has shown significant targeting benefit, other receptors that are over expressed and/or are uterine- or gestationally-specific should also be considered as future therapeutic targets.e.g. for indomethacin the highest drug loading reported is only 3.7%  are made of lipid(s) that are solid at body temperature , therefore highlighting that progesterone-SLNs may be a viable route to provide sustained intravaginal progesterone administration, such as that which is required during assisted reproduction treatment e.g. for luteal support during embryo transfer and implantation.109 Importantly however, these studies also demonstrate the potential for SLNs to act as viable drug delivery vehicles for APIs for the treatment of preterm birth.SLNs have been successful at encapsulating a range of therapeutic agents for several acute and chronic conditions including ocular diseases and anticancer therapeutics.f 740 nm   which had a 1.9% drug loading. In vivo application of the NLC formulation produced a 5-fold higher concentration of indomethacin to ocular tissues than the SLN formulation, which was attributed to the higher drug loading.107 NLCs of indomethacin and a non-tocolytic API (celastrol) have also been produced for potential applications in the treatment of rheumatoid arthritis as a transdermal drug delivery system. The optimised formulation contained as Precirol ATO 5 (solid lipid) and Labrasol ALF (lipid) and had a mean diameter of 27 nm with a total drug loading of 3.6%. The particles showed no irritation on rat skin but successfully penetrated through the top ∼300 μm of skin, demonstrating the potential of these lipid carriers to enhance API penetration into epithelial tissue.105Nanostructured Lipid Carriers (NLCs) were developed as ‘second generation lipid nanoparticles’ to overcome drawbacks associated with some SLN formulations carrier , NLCs teet al. in 2007 produced NLCs by melt-emulsification using monostearin and stearic acid as the solid lipids, with oleic acid as the liquid lipid. Mean particle sizes of 340–385 nm were obtained ) and drug loadings were in the range of 3–11%.110 In 2017, Esposito et al. published work comparing both SLN and NLC formulations of progesterone using both ultrasound homogenisation and high-pressure homogenisation. The high-pressure homogenisation approach avoided the production of agglomerates and produced progesterone loaded SLNs of 181 nm or NLCs of 137 nm in diameter. The drug loading of the two formulations was ∼2%. This work showed that the composition of the lipids could be used to control the rate of progesterone release from the nanoparticles; the API was released more slowly when formulated into NLCs , has been produced for potential treatment of cardiovascular disease. Hassan et al. developed a lipid-based nanoemulsion with OHPC as the API with dimethylacetamide and medium chain triglycerides as the liquid core and Kolliphor HS 15 as the surfactant.114 Their system was a self-nanoemulsifying drug delivery system, an isotropic mixture of oil, surfactant and solvent that forms a stable nanoemulsion when dispersed in aqueous media.121 Their optimised system possessed a drug loading of 9% wt., had a mean diameter of 50 nm and was absorbed onto a powder of poly to allow processing into tablets along with other tabletting excipients. The vaginal administration of their nanoemulsion to a LPS infection-induced preterm birth model in mice showed a reduction in the number of mice delivering preterm: 60% compared to 100% for the mice treated with a blank formulation (no API). The mean time to delivery was increased from 14.5 hours for the blank formulation to 20.2 hours for the OHPC containing nanoemulsion.114 Much of the same team of researchers led by Guisto have demonstrated another nanoemulsion that used a liquid mixture of triglyceride and the co-solvent dimethylacetamide as the lipid carrier. The API used was a novel compound, a sphingosine kinase (SphK), inhibitor, 4-[[4-(4-chlorophenyl)-2-thiazolyl]amino]phenol, SKI II. SKI II had been shown to inhibit SphK, a key enzyme in the pathways that triggers contraction of myometrial tissue in response to lipopolysaccharide exposure.122 SKI II is a lipophilic compound with a solubility in aqueous media of less than 1 μg ml−1.115 The nanoemulsion was also a self-nanoemulsifying drug-delivery system. The authors studied different compositions of lipid, co-solvent and surfactant in order to determine an optimised nanoemulsion which had a mean diameter of 37 nm.115 Upon administration of the SKI II nanoemulsion via the vaginal route to LPS-infected mice, there was a significant reduction in rate of preterm birth. This work shows that SKI II could be a useful future option for treating preterm birth triggered by bacterial inflammation. However, SKI II is a compound still in early development and further work is needed to progress the API towards clinical trials.Nanoemulsions are the third type of lipid-based nanomedicine, they are nanoscale particles of liquid lipids. Similar to the other lipid-based nanomedicines previously discussed, the dispersed lipid phase is stabilised by stabiliser molecules. There are a few examples of nanoemulsions which have been investigated as carriers for APIs associated with the treatment of preterm birth. Patki 116,117 Klang et al. showed that positively charged nanoemulsions of Lipoid E-80 (mainly comprising of phosphatidylcholine) with stearyl amine providing the positive surface charge, produced prolonged duration of the API on the cornea.116 Yamaguchi et al. produced nanoemulsions of castor oil by high-pressure homogenisation and used chitosan to provide mucoadhesive properties. The resulting nanoparticles had a mean diameter of 117 nm and a drug loading of 0.048% with regards to total solids mass in the formulation. The use of chitosan as a coating enhanced the attachment of the formulation onto mucin and increased the residence time of the indomethacin in the tear fluid.117There are also published examples of the development of nanoemulsions of other APIs with tocolytic properties, although with a focus on use for other clinical indications. For example, indomethacin nanoemulsions have been shown for ocular applications.From the successful encapsulation of APIs with tocolytic properties such as indomethacin and nifedipine as well as progestogens in SLNs, NLCs and nanoemulsions, it is clear that there is also potential for these systems to be engineered specifically for applications in preterm birth , polyglycolic acid (PGA), their copolymer poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) are the most widely used polymers due to their well-documented use in biomedical applications.124 Whilst we were not able to find any examples of polymer nanoparticles that had been designed for application in preterm birth per se, APIs used to treat preterm birth have been formulated as polymer nanoparticles for use in other indications. For example, Alkholief et al. produced a PLGA nanoformulation of indomethacin; they used an emulsion solvent evaporation method to investigate the production parameters using two different surfactants , at a range of concentrations and different organic solvents. Depending on the conditions used, mean particle sizes of ∼280–610 nm were obtained, with higher surfactant concentrations yielding larger particle size. The indomethacin drug loadings were 5–6.8% of the formulations. Cytotoxicity tests showed that encapsulating the indomethacin in the polymer nanoparticles reduced the toxicity in HepG2 liver cells compared to the free indomethacin.125 The reduced toxicity of their formulation might have been caused by a reduction in the concentration of free indomethacin, or differences in the accumulation of the nanoparticles into the cells, although accumulation was not investigated. Muljajew et al. produced polymer nanoparticles containing indomethacin by a nanoprecipitation approach. They assessed six poly(ester amides)  polymer. These nanoparticles are coated with stabilisers that have a polar character and provide colloidal stability. amides)  as the c amides)  and mode amides)  were obt127 Therefore, it is highly feasible to utilise the OTR targeting (or others as they are identified) approaches as shown in liposomes and apply them to polymer nanoparticles for use in the treatment of preterm birth. To further investigate this however, it is imperative that the biodistribution and efficacy of the nanomedicines in vivo is examined.The potential to produce polymer nanoparticles containing indomethacin is attractive for the treatment of preterm birth, particularly as the examples show that it is possible to obtain higher drug loadings than in liposomes (see P) compounds. The hydrophobic surfaces of the particles are coated by the adsorption of stabilisers which provide colloidal stability.128 Nanosuspensions differ from the other nanomedicines in that they do not contain a carrier material, rather the nanoparticles are themselves composed entirely of the poorly water-soluble API. This means that nanosuspensions offer the highest drug loadings out of all nanomedicines. Nanosuspensions of a wide range of poorly water-soluble APIs can be produced via a number of routes, with milling,129–131 nanoprecipitation,132–134 high pressure homogenisation135 and emulsion-templated freeze drying136–139 being some of the most common methods.Nanosuspensions, also known as nanocrystals or solid drug nanoparticles, are sub-micron particles of water insoluble  as the stabiliser. Wet milling produced the smallest particle size with a mean diameter of ∼260 nm (in vivo comparison of their nanosuspension formulation to a gel formulation of progesterone (Crinone® 8%). They showed that when administered vaginally the gel formulation displayed significantly less progesterone uptake in the cervix compared to a nanosuspension formulation of progesterone. The maximum concentration (Cmax) of progesterone in the cervix using the nanosuspension was increased 5-fold compared to the branded gel  inhibitors.141 Histone deacetylation has been found to increase at term in both the myometrium (smooth muscle of the uterus) and the cervix. Trichostatin A is a potent HDAC inhibitor. Recently, Zierden et al. produced a nanosuspension formulation by milling trichostatin A with Pluronic® F127 to give a mean diameter of ∼200 nm as measured by DLS. They investigated the vaginal delivery of a combination of the trichostatin A and progesterone nanosuspensions (in 1.5 : 100 mass ratio of the two APIs) in an LPS-induced mouse model. The combination treatment was more effective at preventing preterm birth than either of the nanosuspensions independently. They also found combined application of the two APIs inhibited cell contractility in human myometrial cells in vitro.141Hoang  ∼260 nm . The drunded gel . The autrone gel .140 Furtet al. by using block copolymer of PEG and PCL (PEG–PCL) as the polymer stabiliser.142 They combined molecular simulations with experimental testing to investigate how different PEG chain lengths of block copolymers of PEG-PCL controlled the formation of polymer coated nanosuspensions using a nanoprecipitation type method. In this work, the indomethacin was processed as a coarse aqueous nanosuspension by sonication (mean diameter by DLS of ∼700 nm), then this suspension was mixed with an acetone solution of PEG–PCL in a T-piece. The authors hypothesised that the acetone solution of the polymer would partially dissolve the larger nanoparticles of indomethacin and also provide colloidal stabilisation. They observed a reduction in particle diameter, obtaining particles of ∼250 nm diameter, and drug loadings of up to 78% were also reported.A nanosuspension of indomethacin has been produced by Styliari 137,143,144 While examples of targeted nanosuspensions are less common than other nanomedicines, progress has been made in this area with the design of targeting ligands that can adsorb onto the surface of the nanoparticles.145Nanosuspensions offer the highest drug loadings out of all the nanomedicine options discussed in this review, with values of 80% reported  was approved by the FDA in 1995.146 This success de-risked the development of liposomal formulations; the majority of the earliest clinically approved nanomedicines were liposomes,147 and liposomes initially dominated early clinical nanomedicine success.148 However, more recently other nanomedicine types have now moved through the development pathway with 14% and 8% of clinical trials in investigating lipid nanoparticles and polymer nanoparticles respectively. This is a considerable growth in the development of such systems as lipid nanoparticles and polymer nanoparticles only represent ∼3% and 0% of currently approved nanomedicines.148 While liposomes are attractive nanocarriers they tend to be less stable than the other nanomedicine types, and are not typically suitable for oral dosing due to degradation under acidic conditions.149 As such, the opportunities offered by other nanomedicine types other than liposomes should be more effectively explored for preterm birth. As identified in this review, there are many examples of other lipid-based nanomedicines where tocolytic APIs could be formulated to potentially provide increased drug loading compared to liposomes. Such lipid-based nanomedicines can use the same approaches to incorporating targetting as liposomes, such as the use of lipid-PEG-ligand conjugates. Therefore, the successful targeting identified with liposomes can likely be adapted and used for lipid-based nanoparticles. Polymer nanoparticles are the nanomedicine type that has the fewest examples of the delivery of tocolytic APIs. However, this should not exclude polymer nanoparticles as a potential opportunity, they have good stability during preparation and storage in biological fluids. Additionally, polymer nanoparticles have been shown to offer many of the benefits of nanomedicine types 150 which should allow application in the treatment of preterm birth. Finally, nanosuspensions offer a route to deliver high concentrations of the API due to their very high drug loadings of up to ∼80% wt relative to excipients. Nanosuspensions are the second most progressed nanomedicine type (after liposomes) for the treatment of preterm birth with in vivo trials showing significantly more efficient in drug delivery for a nanosuspension of progesterone than unformulated progesterone. There is therefore a considerable potential for non-lipid systems to also be used as potential tocolytic therapeutics. Additionally, repurposing nanomedicine formulations from other indications may provide a considerable opportunity to diversify the range of formulations available for application in preterm birth.Preterm birth represents a major, pressing, global healthcare problem with an urgent need for more effective treatments. New formulations of APIs are required to prolong gestation, to improve dosage profiles, enhance targeting to the uterine tissue, reduce feto-maternal side effects and ultimately improve safety and efficacy. The use of nanomedicine for the delivery of tocolytics is still at an early stage; the first example of a nanomedicine delivering a tocolytic 90 However, there is not the data to generalise if this is true across all nanomedicine types. Furthermore, the density of the polymer coating on the nanoparticle may considerably alter the ability of nanoparticles to penetrate through tissue. For example, the use of a dense coating of PEG has been found to facilitate an increase in the mobility of polymer nanoparticles in tissue.151 Additionally, studies on targeted nanomedicines for other applications have shown that factors such as the density of the targetting ligand and the length of the polymer used between the nanoparticle and the targetting ligand can play a key role in influencing the targeting efficacy.152,153 Therefore, the careful selection of the polymer stabiliser is a key consideration for targeted nanomedicines as it has the potential to alter targeting efficacy or potential lead to increased transfer to the fetus. Currently, these knowledge gaps make it challenging to efficiently design nanomedicines for the treatment of preterm birth based on IV dosing. For vaginal dosing of nanomedicines with the aim to avoid mucoadhesion, there is data to support some elements of the nanomedicine design. Trends for enhancing muco-penetration have been shown across at least two nanoparticle types, with both polymer nanoparticles75,154 and nanosuspensions140 showing that particles with mean diameters in the range 110–260 nm and coated with polymers that provided a PEG coating have enhanced muco-penetration. In the future, the production of nanomedicines with systematic structural variation would allow more detailed studies in the relationships between particle properties and biological behaviour. Identifying the key design rules for nanomedicines for the treatment of preterm birth would considerably enhance progress in this field.Nanomedicines can be produced with many different particle properties including: size, surface charge, surface chemistry and carrier composition, all of which can considerably affect biological behaviour. The studies that show the most positive results in terms of reduction in preterm birth in mice have been using liposomes ∼200 nm in diameter for IV dosing or 200 nm nanosuspensions for vaginal dosing of progesterone. For IV dosing, targeting is critical to achieve a therapeutic effect, however with regards to the other particle properties there have not been studies to ascertain the optimal parameters. In terms of nanomedicine size, studies on the biodistribution of nanoparticles in pregnant animals have shown that there is a considerable difference in the maternal-to-foetal transfer based on the type and size of nanoparticles. Such analysis indicates that smaller nanoparticles (less than ∼10 nm) may be more likely to enter the foetus.155 Assuming that a similar IV dose of indomethacin would be used in a new nanomedicine formulation with a 1% drug loading, then potentially 2.5–10.0 g of a nanomedicine formulation would need to be infused. This amount of excipients in the formulation is much larger compared to other clinically used liposome systems such as liposomal doxorubicin (which has a API loading of 16.7%).156 Further optimisation of the current liposome formulations or use of higher drug loading lipid-based nanomedicines such as NLCs may address this issue.The drug loading of an API within a nanomedicine is another key factor that needs to be considered in order to allow clinical translation of novel formulations. With the exception of the nanosuspensions, many of the nanomedicines report API loadings of ∼1–4%. If we focus on indomethacin, the most widely investigated nanomedicine formulations of a tocolytic API, it is typically given orally in doses of 25–100 mg as a tocolytic and has a bioavailability of virtually 100%.etc.). Women in preterm labour currently receive IV medications such as magnesium sulfate, antibiotics and tocolytics. As such, the administration of nanomedicine by IV should be suitable for a clinical environment. The clinical application of nanomedicines is likely to be beneficial in the acute setting to allow reduction in dosing regimen to prevent side effects. Additionally, improvements in clinical management of preterm birth from the cessation of uterine activity would allow transfer to specialist neonatal facilities, administration of antenatal corticosteroids or potentially the prolongation of pregnancy all of which have strong associations with improved neonatal outcomes.From a clinical perspective, future developments in nanomedicines for the prevention of preterm birth are likely to be beneficial in the acute setting of clinical management of women in threatened spontaneous labour. Preterm birth is an attractive target due to the very high risks of morbidity and mortality to the neonate. As such, preterm birth is associated with a very significant financial cost both in the acute hospital setting of neonatal intensive care unit and to individuals and society in the longer term from caring for infants harmed by preterm birth (neurodevelopmental delay Ultimately, the treatment of preterm birth is a considerable opportunity for nanomedicine to address, improved therapies would offer significant health benefits globally. Addressing the challenges identified in this review should allow for the accelerated development of nanomedicine technologies that have a greater potential for clinical translation.SR is a Director of Tandem Nano Ltd and co-inventor of patents relating to drug delivery using nanotechnologies. SR has received research funding from GSK/ViiV, AZ and Gilead and a consultancy from Gilead. TM is a co-inventor of patents relating to drug delivery using nanotechnologies and has carried out consultancy work for Vifor Pharma.


Preterm birth (<37 weeks of gestation) is a major cause of neonatal death and morbidity. Up to 40% of the variation in timing of birth results from genetic factors, mostly due to the maternal genome.We conducted a genome-wide meta-analysis of gestational duration and spontaneous preterm birth in 68,732 and 98,370 European mothers, respectively.HAND2 whose expression was previously shown to decrease during gestation, associated with gestational duration, and GC (Vitamin D-binding protein), associated with preterm birth. Downstream in silico-analysis suggested regulatory roles as underlying mechanisms for the associated loci. LD score regression found birth weight measures as the most strongly correlated traits, highlighting the unique nature of spontaneous preterm birth phenotype. Tissue expression and colocalization analysis revealed reproductive tissues and immune cell types as the most relevant sites of action.The meta-analysis detected 15 loci associated with gestational duration, and four loci associated with preterm birth. Seven of the associated loci were novel. The loci mapped to several biologically plausible genes, for example We report novel genetic risk loci that associate with preterm birth or gestational duration, and reproduce findings from previous genome-wide association studies. Altogether, our findings provide new insight into the genetic background of preterm birth. Better characterization of the causal genetic mechanisms will be important to public health as it could suggest new strategies to treat and prevent preterm birth. Annually, more than 15 million pregnancies are affected by preterm births all over the world. There are no effective ways to prevent preterm birth, and premature babies suffer from neonatal mortality and lifelong morbidities. Genetic factors of mother and fetus explain a large proportion, approximately 30–40%, of the of the variation in gestational age at delivery. To date, there have been just a few unbiased genome-wide investigations set out to locate these genes. Better characterization of the causal genetic mechanisms could suggest new strategies to treat and prevent preterm birth. In the current study, we aimed to identify maternal genetic factors that contribute to the timing of birth by meta-analyzing genome-wide association studies from European populations. We detected 17 independent loci that were associated with gestational duration and/or the risk of preterm birth. Ten of the loci replicated associations from previous studies, and seven were novel. The replicated associations provide strong evidence for the importance of these loci in the timing of birth, although the exact genes and causal variants and pathways still require further functional analysis. The seven novel associations provide further intriguing candidates that may account for the risk of preterm birth. Bioinformatics analysis proposed the associated loci have regulatory functions predominantly in immune cell types and reproductive tissues. The analysis further highlighted the unique nature or preterm birth as a phenotype, since the only traits with strong correlations were birth weight measures that are closely linked to the studied phenotypes. Our findings complement the knowledge of the genetic factors of preterm birth. Proper timing of birth is crucial for the survival and long-term health of newborn infants. Preterm birth, defined as birth that occurs prior to 37 completed weeks of gestation, is the most common cause of neonatal death and a prevalent cause of death among children under 5 years . MoreoveWNT4, EBF1, AGTR2, and KCNAB1 were associated with timing of birth in mothers [While intrauterine growth restriction and preeclampsia are the major causes of medically indicated preterm birth, approximately 70% preterm births occur after spontaneous onset of labor . There a mothers , 9, and  mothers .In the present study, our aim was to strengthen knowledge of the genetic background of SPTB by identifying and replicating associations of genetic loci in relation to timing of spontaneous singleton birth. To that end, we conducted a case-control meta-analysis of SPTB and a quantitative meta-analysis of gestational duration in 98,370 and 68,732 European mothers, respectively.n = 98,370) and gestational duration  was conducted with maternal GWAS data from the FinnGen study, 23andMe, Inc., and the cohort from Northern and Central Finland p<5×10–8 -based genomic inflation factor  indicatep-values indicated involvement of gonad development and steroid hormone biosynthesis in gestational duration, whereas kinetochore-microtubule and neuron differentiation were the top pathways in SPTB  detected DNAH2 and RAP2C as additional loci associated with gestational duration. The association for the RAP2C locus was previously known [DNAH2 was novel. Gene set analysis based on a list of genes corresponding to all significant loci in the current study identified enrichment of multiple pathways, with GO terms referring to regulation of morphogenesis and development of various organs and tissues among the top pathways , ADCY5, and KCNAB1 could play a role of regulating pregnancy duration in reproductive tissues, and further suggested that many previously known  or novel (GNAQ) loci have regulatory roles in immune cell types  in the meta-analysis, and one novel locus (DNAH2) in the joint analysis of the meta-analysis and replication data, for which the genes were assigned based on proximity in the lack of previous association with SPTB or evidence for colocalization. The case-control meta-analysis of SPTB replicated the associations for EBF1 and EEFSEC, and detected two novel associated loci , for which the genes were assigned based on proximity.Loci near nal data , 9, and ll types . In addiZBTB38 with a replicable association was associated with hyperplasia of prostate (BHP) in the FinnGen  were also associated with taller height, higher body mass measures, and increased risk of prostate cancer  (COL27A1 expression.Variants in portions . Gestatis (LCLs) . The allZBTB38, the polymorphisms in the WNT3A locus were associated with height and body mass indices, and the encoded protein was implicated in cell fate and patterning during embryogenesis [WNT3A expression in the placenta  and gestational duration  identified several associated loci. We detected loci that had no previous associations with gestational duration or SPTB, and our findings further reinforce the associations of genes from previous GWASs of mothers who gave birth preterm. The associated loci with strong replication in the current analysis included ZBTB38, HAND2, TET3, and KCNAB1. These loci, along with COL27A1, also showed association in the recent meta-analysis of the timing of parturition [The current genome-wide meta-analysis of SPTB n = 8,370 andZBTB38, zinc finger and BTB domain containing 38, with benign hyperplasia of prostate (BHP) is a compelling finding given that both gestational duration and BHP are affected by changes in estrogen and androgen levels [ZBTB38 was further associated with cell counts of various immune cells, and our results suggest that increased ZBTB38 expression in these cell types may play a role in regulating length of pregnancy. Alleles associated with longer gestational duration showed association with increased height, body mass, and risk of prostate cancer. It remains to be determined whether ZBTB38 confers its effect on birth timing through pregnancy-specific mechanisms or by contributing to more general immune pathways that influence gestation. Our findings for ZBTB38 associations are in keeping with reported correlations among maternal height, gestational duration, and fetal growth, and further comply with detected associations between maternal birth weight–elevating alleles and longer gestational duration and between maternal gestation-prolonging alleles and the risk of prostate carcinoma [Association of n levels , 27. ZBTarcinoma , 29.HAND2 showed strong replication. HAND2 encodes heart and neural crest derivatives expressed 2, a transcription factor best known for its roles in cardiac morphogenesis and limb development. Decreasing expression of HAND2 in the decidua during pregnancy may contribute to regulation of gestational duration [HAND2 in the human uterine tissue, and its gradually decreasing expression in the decidua during pregnancy [An association near duration . The expregnancy , makes iTET3) and potassium voltage-gated channel subfamily A regulatory beta subunit 1 (KCNAB1) were previously associated with birth weight of offspring, and alleles associated with longer gestational duration correlated with birth-weight measures, complying with known correlations of the length of gestation and fetal growth [KCNAB1 expression contributes to the timing of birth. Of note, TET3 was suggested to play a role in embryo implantation [TET3 and KCNAB1 represent interesting targets for further study to determine their specific roles related to the regulation of gestational duration.Variants in Tet methylcytosine dioxygenase 3 . COL27A1 is most abundantly expressed in the endometrium, and the gene encodes collagen type XXVII alpha 1 chain, which is a fibrillar, developmentally regulated protein. Further, the meta-analysis lead variant of the COL27A1 locus is near miR-455, which has roles in cartilage development, adipogenesis, and preeclampsia, and may protect endometrial cells against oxidative stress [Interestingly, e stress , 33.WNT3A and GNAQ, these genes were also supported as causal genes in the colocalization analysis. The genes further had some known functions consistent with a role in the timing of birth. Our results and previous assessments with single cell data suggest that WNT3A could play a role in regulation of pregnancy in both maternal and fetal tissues of the maternal-fetal interface during pregnancy [GNAQ  plays a role in survival of immune cells. Interestingly, it occurs as a part of a transcriptomic signature related to human labor in the choriodecidua [GNAQ is widely expressed in cells of the maternal-fetal interface [The novel meta-analysis loci associated with gestational duration, comprised further intriguing candidates. For the loci near regnancy –21. GNAQodecidua , 24, 34.nterface , 21.KCNN2, associated with gestational duration, had some roles that could link to regulation of pregnancy length. KCNN2 encodes a potassium calcium-activated channel subfamily N member 2, and potassium channel proteins have been linked to uterine function during gestation [KCNN2 to pregnancy-related regulation, but KCNN3, another molecule in the KCNN family of potassium channel genes, plays a role in uterine function [KCNN2 and other novel risk loci should be replicated in independent populations, and the role of the risk loci and corresponding candidate genes remains to be verified.The novel locus near estation . We founfunction . The assGC, encoding GC vitamin D binding protein, as of special interest because of its known involvement in vitamin D transport and storage. Protein encoded by GC is the primary carrier of vitamin D that binds to the vitamin and its plasma metabolites and transports them to their target tissues. Previous studies have suggested links among plasma vitamin D levels and preterm birth or other pregnancy- related outcomes including pre-eclampsia, polycystic ovary syndrome, and endometriosis [GC showed association with a reduced risk of certain immune-mediated diseases [GC in the context of human pregnancy and SPTB remains to be determined.The case-control meta-analysis of SPTB detected an association in etriosis , 25. Vitdiseases , 38. TheAltogether, our results highlight the unique nature of SPTB. At the genome-wide level, birth weight measures were the only traits that showed significant correlations with both gestational duration and SPTB in a comprehensive set of complex phenotypes. However, the Bonferroni correction deployed for the 773 tests is likely overly conservative since the traits include closely related phenotypes. Gene set enrichment for gestational duration and pathway analysis for gestational duration and SPTB highlighted involvement of gonad development and steroid hormone biosynthetic processes and GO terms referring to regulation of morphogenesis among multiple top pathways. Gene set enrichment analysis of SPTB implied kinetochore microtubule as the top pathway. Proper function of the kinetochore-microtubule pathway is essential for preserving genomic integrity and prevention of birth defects . Tissue Multiple variants in the candidate loci were individually associated with birth weight indices. Associated genes had primary roles in steroid hormone–regulating processes and tissue and organ morphogenesis. Reproductive tissues of the mother were among the principal locations where the associated genes were expressed. Our results suggest that many of the associated variants contribute to pregnancy outcomes by regulating expression of their target genes, mainly but not exclusively in reproductive tissues and immune cell types. Hence, our results indicate that those tissue and cell types are the most relevant when considering the regulatory events related to pregnancy and preterm birth, and should be the primary targets in future molecular biological studies of SPTB and gestational duration.The current analysis was restricted to individuals of predominantly European descent. Future studies should include ancestrally diverse populations to better understand the genetic architecture of the timing of birth and to ensure the broad applicability of results from genetic studies .In conclusion, the current meta-analysis detected multiple loci that were associated with gestational duration or SPTB and produced intriguing candidates for further studies. Our results highlight the intricate nature of spontaneous birth as a trait and emphasize the importance of reproductive and immune tissues and cell types. The new genetic discoveries prime further research including large-scale complex investigations and individual regulatory pathway analyses utilizing labor-inducing tissues and cells. Studies may eventually reveal signaling pathways that activate spontaneous preterm birth and contribute towards effective prevention of SPTB.n = 68,732) with data from mothers of European ancestry. The data originated from FinnGen, Northern/Central Finland, and 23andMe project.We conducted a meta-analysis of SPTB  and a quantitative meta-analysis of gestational duration (www.eandireview.com).FinnGen participants provided informed consent under the Finnish Biobank Act. Older cohorts with study-specific consents were transferred to the Finnish biobanks after approval by Fimea, the National Supervisory Authority for Welfare and Health. Recruitment protocols followed the biobank protocols approved by Fimea. The Coordinating Ethics Committee of the Hospital District of Helsinki and Uusimaa (HUS) approved the FinnGen study protocol (Nr HUS/990/2017). The FinnGen study is approved by the Finnish Institute for Health and Welfare , the Digital and Population Data Service Agency , the Social Insurance Institution , and Statistics Finland (permit numbers TK-53-1041-17 and TK-53-90-20). The Biobank Access Decisions for FinnGen samples and data utilized in FinnGen Data Freeze 6 include: THL Biobank BB2017_55, BB2017_111, BB2018_19, BB_2018_34, BB_2018_67, BB2018_71, BB2019_7, BB2019_8, BB2019_26, BB2020_1, Finnish Red Cross Blood Service Biobank 7.12.2017, Helsinki Biobank HUS/359/2017, Auria Biobank AB17-5154, Biobank Borealis of Northern Finland_2017_1013, Biobank of Eastern Finland 1186/2018, Finnish Clinical Biobank Tampere MH0004, Central Finland Biobank 1-2017, and Terveystalo Biobank STB 2018001. Women from Northern/Central Finland provided written informed consent, and studies were approved by ethics committee of Oulu University Hospital . Women from the 23andMe provided written informed consent and completed online surveys according to a human-subjects protocol approved by Ethical and Independent Review Services  combines genome information with health care data from national registries. The project aims to collect data from 500,000 Finnish participants. Preterm and term birth were defined as births before and after 37 weeks of gestation. FinnGen preterm endpoint in Preparatory Phase Data Freeze 6 comprised individuals with World Health Organization International Classification of Diseases, Eight, Ninth, and Tenth Revision  codes O60, 644, and 63497, respectively. We excluded births with ICD-9 code 644 (“early or threatened labor”) if they occurred after 37 weeks of gestation according to birth register data, and individuals with multiple gestation or birth, preeclampsia/eclampsia, and polyhydroamnios. Controls were people with spontaneous term birth. The GWAS of SPTB comprised 4,925 cases and 49,105 controls, and the GWAS of gestational duration comprised 24,391 mothers for whom gestational duration was available. We additionally performed GWAS with a “strict” definition of SPTB, in which we only included cases with births indicated as spontaneous and preterm in the endpoint data.Northern and Central Finland were sampled in Oulu and Tampere University Hospital districts. SPTB was defined as birth prior to 36 wk + 1 d of gestation. Term birth was defined as birth at 38–41 wk (38 wk + 0 d to 41 wk + 6 d) of gestation. We excluded births with multiple gestation, preeclampsia, polyhydroamnios, intrauterine growth restriction, placental abruption, anomalies of the fetus, clinical chorioamnionitis or acute septic infection in the mother, alcohol or narcotic use, and accidents. Term births were from families without previous preterm births. The analysis comprised 286 cases and 488 controls.The study subjects from 23andMe research program were obtained by request. The summary data comprised unrelated mothers of European ancestry with self-reported gestational duration for their first singleton live birth. Individuals reporting a medical indication for preterm delivery were excluded. Preterm birth was defined as birth before 37 weeks of gestation, and control samples were people with term deliveries (>37 weeks). The meta-analysis of gestational duration included data from 43,567 individuals, and the meta-analysis of SPTB comprised 43,566 samples .Summary statistics of the Nordic data sets including FIN cohort , MoBa , and DNBC , for which the summary statistics were obtained via collaboration. The characteristics of the data sets were described earlier [Data used in the replication and joint analysis originated from European women in the  earlier , 42. Bri earlier . The mot earlier  in Helsi earlier , 42.p<1×10-6) were excluded. Imputation was conducted against a Finnish population–specific SISuv3 reference with Beagle4.1 [https://github.com/FINNGEN/finngen-documentation).Various methods were used to extract DNA from the FinnGen samples. Genotyping was done with Illumina and Affymetrix arrays . Sample quality control (QC) entailed excluding individuals of uncertain sex, non-Finnish ancestry, high missingness (>5%), and excess heterozygosity (±4SD). For genotype QC, variants with missingness >2%, minor allele count (MAC)<3, and deviation from Hardy–Weinberg equilibrium (HWE) , Puregene Blood Core Kit , or prepIT-L2P kit . Genotyping was performed with the Infinium HumanCoreExome BeadChip  by the Technology Centre, Institute for Molecular Medicine Finland (FIMM), University of Helsinki. Variants with minor allele frequency (MAF)<1%, HWE SHAPEIT2 , and impSHAPEIT2 , againstSHAPEIT2 . Variantp<1×10-20, call rate <95%, or allele frequency discrepancy with 1KGP Europeans, were excluded. Imputation was done with Minimac242, using the 1KGP phase1 [DNA of the 23andMe samples was extracted from saliva samples, followed by genotyping with custom Illumina platforms by the National Genetics Institute (NGI). Samples with <97% European ancestry, and variants with HWE P phase1 .FinnGen GWAS was conducted with Scalable and Accurate Implementation of Generalized mixed model (SAIGE) . Gestatip-value-based meta-analysis of gestational duration. Sample size-based meta-analysis allows combining results when β-coefficients and standard errors from individual studies are in different units. Genomic coordinates of the meta-analysis cohorts were aligned into the GRCh38 coordinates. Genomic inflation factor was calculated with linkage disequilibrium score regression (LDSC) [We used METAL  to condun (LDSC) . We repoWe defined associated loci as genomic regions within a ±1 Mb window around the lead variant. The locus was defined as novel if there were no previous genome-wide significant associations for SPTB or gestational duration in the ±1 Mb window in the National Human Genome Research Institute–European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog or in the ±1 Mb window surrounding the loci reported in another recent meta-analysis of the timing of parturition, and no LD between the meta-analysis lead variants and previously reported index variants –11.We used LDSC to estimate SNP-based heritability and to test for genetic correlation between gestational duration or SPTB with a comprehensive set of phenotypes downloaded from the Integrative Epidemiology Unit (IEU) OpenGWAS Project , 13. We p<5×10-8. We further investigated gene expression of the novel loci with evidence for colocalization in reproductive tissues in publicly available single cell data from the reproductive cell types in a human cell atlas of fetal development (https://descartes.brotmanbaty.org/bbi/human-gene-expression-during-development/) [https://www.reproductivecellatlas.org/) [To gain insight into the associated loci, we checked previous associations in the FinnGen R7 data and performed phenome-wide association study (PheWAS) within 1 Mb window around the meta-analysis index variants by querying GWAS data in the IEU OpenGWAS Project, which includes approximately 40,000 studies . We perfopment/)  and in ras.org/) , 21. We n = 2,722) was available. Joint test of the replication data and meta-analysis variants with p<1×10-6 was done to see if additional loci reached genome-wide significance. We did not test replicated loci originally reported in Zhang et al. [We tested the lead variants within each locus for association in the replication data from the data set of Nordic birth studies. For most of the tested variants, replication data from two out of three cohorts Click here for additional data file.S2 FigThe meta-analysis of gestational duration was performed with 66,001 samples whereas the meta-analysis of SPTB included 94,781 samples .(PDF)Click here for additional data file.S3 FigEBF1 and EEFSEC are from the GWAS of gestational duration. The main GWAS of gestational duration was performed with data from 24,391 samples, and the GWAS with strict definition with 21,660 samples. The main GWAS of SPTB was based on 54,030 samples , whereas the strict GWAS of SPTB had 50,441 samples . The variants with a minor allele count (MAC) of five shown.The genes mapped to the meta-analysis risk loci for gestational duration and SPTB are shown in alphabetical order. Effect estimates for (PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 FigEBF1 and EEFSEC loci are shown because the meta-analysis lead variants were not present in the FinnGen-based GWAS.Two variants in (PDF)Click here for additional data file.S6 FigThe histograms show MAGMA gene set enrichment analysis for gestational duration (above) and SPTB (below). The heatmap features gene expression depicted as averaged expression value per tissue type, with hierarchal clustering for both genes and tissues. Across genes and tissues, the cells filled in red represent higher expression compared to the cells filled in blue.(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S9 Figp value is based on the strongest associating trait.In each category, (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Tablep<0.05 or top 10 gene sets shown.Gene sets with adjusted (PDF)Click here for additional data file.S7 TableWNT4, EEFSEC, ADCY5, EBF1, and AGTR2, originally discovered in NEJM 21;377:1156–1167 were not included, since the same replication data set was used in that study.Loci near (XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Tablep<1e-6 shown.Results with PP>0.6 and meta-analysis (XLSX)Click here for additional data file.S1 Acknowledgements(XLSX)Click here for additional data file.


Triplet pregnancies carry a high risk of pregnancy-related complications. The primary aim of this study was to describe maternal, pregnancy, and neonatal outcomes in expectantly managed triplet pregnancies in Sweden. The secondary aim was to compare outcomes in expectantly managed triplet pregnancies with triplet pregnancies where fetal reduction had been performed with the only indication to reduce the number of fetuses.+0 weeks between 2014 and 2019 were included.Nationwide cohort study based on linkage of data from three national Swedish registers. Triplet pregnancies with delivery at gestational age ≥ 22n = 106), 98% (312/318) of infants were liveborn with a mean gestational age at birth of 32+3 weeks and a mean birthweight of 1,726 g. Nine percent (n = 29) suffered from severe neonatal morbidity, and 4% (n = 12) died during the neonatal period. In the reduced cohort (n = 13 pregnancies), all infants were liveborn (n = 22). Mean gestational age at birth (36+0 weeks) and mean birthweight  were higher than in the expectantly managed cohort (P < 0.01 for both comparisons). There were no cases of severe neonatal morbidity (P = 0.24) or mortality (P = 1.00).In the main cohort of expectantly managed triplet pregnancies (+0 weeks of gestation in expectantly managed triplet pregnancies in Sweden was high. Nine out of 10 infants did not suffer from severe neonatal morbidity. Fetal reduction was performed in only a very small number of cases and was associated with higher gestational age at birth and higher birth weight.Overall neonatal survival from 22 During the last decades, multifetal birth rates have increased significantly in high-income countries, mainly due to assisted reproductive technologies (ART) –3. The S+0 weeks) from 28 to 10%  to retrieve detailed information on neonatal outcomes  code O30.1 (triplet pregnancy) at any time during pregnancy and/or the term ‘birth of three’ in the Swedish Pregnancy Register . Six of +0 weeks during the study period were identified from the Fetal Therapy Register (n = 21). Six pregnancies, where fetal reduction had been performed due to fetal indications such as twin-to-twin transfusion syndrome or fetal malformation, and two pregnancies with miscarriage before GA 22+0 weeks after fetal reduction were excluded from the cohort  [20]. Neonatal outcomes included neonatal mortality and the incidence of severe neonatal morbidity, analyzed as a composite variable which included intraventricular hemorrhage (IVH) grade 3 and 4, sepsis, necrotizing enterocolitis (NEC), bronchopulmonary dysplasia (BPD), and/or treated retinopathy of prematurity (ROP). Other outcomes were: Apgar at 5 min, neonatal resuscitation at birth, umbilical artery pH, and admission to the neonatal care unit. In SNQ, the definition of neonatal resuscitation included the need of mask ventilation, administration of continuous positive airway pressure (CPAP), intubation, cardiac compressions, correction of acidosis, or administration of epinephrine.As a core outcome set for multiple pregnancies according to the CoRe Outcomes in WomeN’s health (CROWN) initiative has not been established yet, we chose important outcomes that have been reported in previous studies on multifetal pregnancies .U-test was carried out for the comparison of continuous, non-parametric data, between the two cohorts. The statistical software IBM SPSS statistics 25 was used for all statistical analyses. A P-value <0.05 was considered to be statistically significant.Descriptive statistics were applied to present outcome variables in the cohorts. Outcomes were presented as numbers and percentages, mean ± standard deviation (SD), or median and interquartile range (IQR). Chi-square and Fisher’s exact test were applied to analyze differences in categorical data. Mann-Whitney Ethical permission for this study was given by the Swedish Ethical Review Authority with registration number: 2019-05160.In the expectantly managed cohort of 318 fetuses, six infants were stillborn . The redP < 0.01). Moreover, maternal BMI in early pregnancy was slightly higher in expectantly managed (26 ± 5 kg/m2) compared to reduced triplet pregnancies .Maternal age, parity, smoking in early pregnancy, level of education, country of birth, and pre-pregnancy comorbidity were not significantly different between women with expectantly managed and reduced triplet pregnancies . In contP = 1.00), a lower proportion of women was delivered by cesarean section , and the median estimated blood loss was lower . The proportion of pregnancy complications was lower than in the expectantly managed cohort, but without reaching statistical significance .There were no cases of maternal venous or pulmonary embolism and no cases of maternal death during pregnancy or the postnatal period in either group. Overall, consideration should be given to the very small number of pregnancies in the reduced cohort.Pregnancy and maternal outcomes are presented in n = 312) are presented in +3 weeks (min – max: 22+0 – 36+1 weeks). Six percent (17/312) of the infants were born before 28+0 weeks of gestation. The mean birthweight was 1,726 ± 474 g, and 34% (106/312) were born SGA. Nine percent (29/312) had an Apgar score <7 at 5 min, and 92% (287/312) of the infants were admitted to neonatal care, where the median length of stay was 21 days (IQR 14–37 days). Sixty-three percent (196/312) of the infants needed resuscitation at birth, and 9% (29/312) suffered from severe neonatal morbidity. Four percent (12/312) of the infants died during the first 28 days of life, whereof 10 during the early neonatal period (0–7 days). Seven of the 12 infants who died had been born extremely preterm (22+0–26+1 weeks), two other infants had lethal malformations, and the remaining three had been born between 28 and 32 weeks of gestation .Pregnancy and neonatal outcomes for liveborn infants  as was the mean birthweight , but there was no significant difference in the proportion of SGA births . Fewer infants were admitted to neonatal care , with a shorter median stay . Fewer infants needed resuscitation at birth , and none suffered from severe neonatal morbidity (P = 0.24). All infants were alive at 28 days of life (P = 1.0).In the reduced cohort – compared to the expectantly managed cohort – mean GA at birth was significantly higher . Perinatal survival rates were high, with 98% being liveborn, and 96% of liveborn infants being alive at 28 days of life. Nine out of 10 infants did not suffer from severe neonatal morbidity. Fetal reduction was performed in only a very small number of cases. In the reduced cohort, no infants were born extremely or very preterm, and mean birthweight and gestational age at birth were significantly higher compared to the expectantly managed cohort. There were no cases of severe neonatal morbidity or mortality and only one case of significant maternal pregnancy complication. However, consideration should be given to the very small number of pregnancies in the reduced cohort.In a country with excellent access to high-quality maternity, delivery, and neonatal care, all infants in a nationwide cohort of expectantly managed triplet pregnancies were born preterm, whereof 6% extremely and 23% very preterm (22The proportion of pregnancies conceived by ART was high in the expectantly managed cohort (31%). This is in sharp contrast to the overall proportion of pregnancies conceived by ART in deliveries in Sweden, which was 3.8% during the years 2014–2019 . DespiteIn the expectantly managed cohort, the proportion of cesarean sections was high 97%), which is in line with international guidelines, suggesting a planned cesarean section at 35 weeks of gestation in uncomplicated triplet pregnancies . A high %, which +0 rather than 22+0 weeks of gestation. Nevertheless, the rate of stillbirth and neonatal death in expectantly managed triplets in our study  is comparable to or even slightly lower than in other studies. Curado et al. found an incidence of stillbirth of 2% in trichorionic triamniotic (TCTA) and 5% in dichorionic triamniotic (DCTA) triplets   . The rate, TTTS) . While te, TTTS) , 30, 31.There were no significant differences in the proportion of severe neonatal morbidity or neonatal mortality between the expectantly managed and the reduced cohort. However, the non-significance is most probably due to the small number of neonates in the reduced cohort.+0 weeks of gestation when compared to data from Statistics Sweden during the years 2014–2019  is an important finding itself. Fourthly, data on some outcomes were missing to a higher extent in the reduced compared to the expectantly managed cohort, as for example Apgar score at 5 min of age , making comparisons between the cohorts more difficult.Firstly, the significant limitation in our study is the lack of information on chorionicity. Secondly, it was not possible to follow triplet pregnancies including miscarriages from early gestation through registers in Sweden, as a first trimester ultrasound is not offered to all pregnant women. In contrast, it was possible to identify basically all triplet pregnancies with a delivery ≥ 22+0 weeks of gestation was 0.018% during the years 2014–2019 in Sweden (According to Statistics Sweden, the proportion of triplet deliveries ≥ 22n Sweden . The pron Sweden . Prevent+0 weeks in expectantly managed triplets in our cohort, our data regarding triplets born GA ≥ 22+0 weeks are in line with previous studies (With the limitation that we do not have any data about miscarriage rates before GA 22 studies , 28, 33.+0 weeks of gestation in expectantly managed triplet pregnancies in Sweden was high, all infants were born preterm. Every tenth infant and every third mother suffered from severe neonatal morbidity or pregnancy complications, respectively. Fetal reduction was performed in only a very small number of cases and was associated with higher gestational age at birth and higher birth weight. In an international context, rates of multifetal births are very low in Sweden.While overall neonatal survival from 22Click here for additional data file.


S100A6 recognizes 26 of the cytokines from all families of this fold, with equilibrium dissociation constants from 0.3 nM to 12 µM. Overall, S100A6 interacts with ca. 73% of the four-helical cytokines studied to date, with a selectivity equivalent to that for the S100P protein, with the differences limited to the binding of interleukin-2 and oncostatin M. The molecular docking study evidences the presence in the S100A6 molecule of a cytokine-binding site, analogous to that found in S100P. The findings argue the presence in some of the promiscuous members of the S100 family of a site specific to a wide range of four-helical cytokines. This unique feature of the S100 proteins potentially allows them to modulate the activity of the numerous four-helical cytokines in the disorders accompanied by an excessive release of the cytokines.S100 is a family of over 20 structurally homologous, but functionally diverse regulatory -binding proteins of vertebrates. The involvement of S100 proteins in numerous vital (patho)physiological processes is mediated by their interaction with various (intra/extra)cellular protein partners, including cell surface receptors. Furthermore, recent studies have revealed the ability of specific S100 proteins to modulate cell signaling via direct interaction with cytokines. Previously, we revealed the binding of ca. 71% of the four-helical cytokines via the S100P protein, due to the presence in its molecule of a cytokine-binding site overlapping with the binding site for the S100P receptor. Here, we show that another S100 protein, S100A6 (that has a pairwise sequence identity with S100P of 35%), specifically binds numerous four-helical cytokines. We have studied the affinity of the recombinant forms of 35 human four-helical cytokines from all structural families of this fold to Ca The /Golgi pathway, or several unconventional passive and active mechanisms ,27,28. Wor CD146 ,35,36. Mor CD146 , S100A13or CD146 ,39, S100or CD146 ,41, and or CD146 . Severalor CD146 , S100A1/or CD146 ,44,45, aor CD146 . The S10or CD146 ; S100B ior CD146 , but favor CD146 ; S100A12or CD146 ; and theor CD146 ,44,45. Mor CD146 ,39.d, in the 1 nM–3 µM range  [The vast majority of the established S100–cytokine interactions correspond to cytokines belonging to the superfamily of four-helical cytokines (SCOP  ID: 3001of RAGE) . Using mof RAGE) ,51. Therof RAGE) ,51, S100of RAGE) ,53), speof RAGE) ,55,56).E. coli, and purified as described in [The human S100A6 protein was expressed in ribed in . The samribed in .2, Tween 20, and EDTA were purchased from Sigma Aldrich Co. .The HEPES and sodium chloride were from PanReac AppliChem . The CaCl2, and 0.05% Tween 20, pH 7.4. The S100A6 (63 nM–8 µM) solution in the running buffer was passed over the chip surface for 300 s. The dissociation of the ligand–analyte complex was triggered by the passage of the running buffer for 1200–2400 s. The SPR sensograms were described via a single-site binding scheme (S100A6-IL-2 interaction) or a heterogeneous ligand model (1). The latter suggests the presence of two populations of the ligand (L1 and L2) independently binding an analyte molecule (A):SPR studies of S100A6 binding to the cytokines immobilized on the sensor chip surface  were performed at 25 °C, mainly according to . The cytd and kd values were calculated for several S100A6 concentrations. The resulting values are the average of 3–5 estimates. The ligand was regenerated using 20 mM EDTA pH 8.0 solution for 100 s. The free energy change in the reaction was estimated using the equation: ΔGi = −RT ln(55.3/Kdi), i=1,2.Here, k and K refer to the kinetic and equilibrium association (‘a’) and dissociation (‘d’) constants, respectively. The Khttps://scop2.mrc-lmb.cam.ac.uk [The cytokines investigated in this study were classified according to the SCOP 2 database  software.The ClusPro docking server  was usedfrom PDB  entry 1Kfrom PDB ), while une 2023 ) (Table une 2023 . The mod2+-loaded (1 mM CaCl2) recombinant human S100A6 protein to the panel of 35 recombinant human four-helical cytokines  may reach the nanomolar level or below  are I44 (16 cytokines out of 30) of the ‘hinge’ loop region between helices α2 and α3, R55 and D59 of helix α3 (17 cytokines), and I83 (21 cytokines) and Y84 (18 cytokines) of helix α4 A. An anasubunits A, which 3 and α4 . Importa3 and α4  are pred3 and α4  is even 2+-bound S100A6 dimer via the residues of the N-terminus and helices α1 and α3 , long-chain cytokines , and cytokines of the interferon/IL-10 family  overlap by more than 50% with the residues similarly predicted for interaction with the S100P protein . On the As some of the predicted S100A6-binding residues of the specific four-helical cytokines  have previously been shown to participate in the recognition of their respective receptors , S100A6 Of note, the presented structural predictions based on the molecular docking suffer from their inability to take into consideration the structural flexibility inherent to the four-helical cytokines, as evidenced by the experimentally confirmed disorder in IL-15, THPO, G-CSF, LEP, and IL-10, and the theoretical predictions described in .d, are low enough for efficient S100A6 binding only for THPO, S100A6 monomerization could decrease the Kd values by 1.4–2.2 orders of magnitude, which was earlier shown for the S100P interaction with IL-11 and IFN-β [To get an insight into the possible relative in vivo concentrations of S100A6 and the S100A6-specific cytokines , we collnd IFN-β ,64,69. Ind IFN-β  (Table SThe binding of S100A6 to the cytokines may also promote their non-canonical secretion, as previously reported for S100A13 binding to IL-1α and FGF1 ,39 and fd, exceed 10−4 M. Thus, about 73% of the cytokines are specific to the Ca2+-loaded S100A6 dimer, with Kd values in the range from 0.3 nM to 12 µM (d estimates for the complexes of Ca2+-bound S100A6 with extracellular RAGE fragments from 28 nM to 13 µM [In this work, we have extended the list of the four-helical cytokines studied, with regard to their affinity to the promiscuous S100A6 protein ,51, fromto 12 µM , which ito 13 µM ,66,67. Tto 13 µM  cannot bThe promiscuous binding of S100A6 and S100P to a wide range of the four-helical cytokines belonging to all structural families of this fold can be partly explained via the molecular docking analysis, which revealed that both S100 proteins have a nearly identical cytokine-binding site formed by helices α1, α3 and α4, and the ‘hinge’ (