Prevention of complications in neck dissection

Cyrus J Kerawala
Manolis Heliotos
North West London Regional Maxillofacial Unit
Watford RoadHA1 3UJNorthwick Park Hospital, HarrowMiddlesexUK

Fulham RoadSW3 6JJLondonUK

Prevention of complications in neck dissection
10.1186/1758-3284-1-35BioMed Central Page 1 of 6 (page number not for citation purposes) Head & Neck Oncology Review Address: 1 Head and Neck Unit, The Royal Marsden NHS Foundation Trust, * Corresponding author
Background:The neck dissection has remained a pivotal aspect of head and neck cancer management for over a century. During this time its role has expanded from a purely therapeutic option into an elective setting, in part promoted by efforts to reduce its morbidity.Objectives: This review will consider the potential complications of neck dissection and on the basis of the available evidence describe both their management and prevention.Conclusion:Although the neck dissection continues to provide clinicians with a method of addressing cervical disease, its reliability and safety can only be assured if surgeons remain cognisant of the potential complications and aim to minimise such morbidity by appropriate management in the peri-operative period.

Introduction

Since its original description by Crile in 1906 and subsequent popularisation by Hays Martin in 1951 the radical neck dissection (RND) remained the standard treatment for palpable or potential cervical metastasis from head and neck cancer for many decades [1,2]. This operation provides a safe and reproducible method of comprehensively addressing cervical lymph nodes. Recurrence rates after RND vary according to the bulk of the disease present, ranging from less than 10% in the N0 neck to over 70% in patients with positive nodes at multiple levels [3]. The addition of either pre-or post-operative radiation therapy further reduces the incidence of failure in the neck by at least 50% for all N stages [4,5]. However, whilst the RND provided a reliable method of treating patients with head and neck cancer, it became increasingly apparent that it carried substantial morbidity and complications. Nahum described a syndrome of decreased range of abduction in the shoulder joint and pain following RND which has been termed 'shoulder syndrome' [6]. The aeti-ology of this syndrome is in part sacrifice of the spinal accessory nerve (SAN) with preservation of that structure during neck dissection ameliorating the syndrome [7]. The morbidity of the classical RND thus gave impetus to the development of modified procedures that attempted to reduce the adverse effects of the classical operation and yet preserve its effectiveness in oncological terms. The realisation that it was possible to perform a complete en-block lymphadenectomy with preservation of structures such as the SAN led to the development of less morbid procedures, the original concept of which is credited to Bocca [8]. The ability to harvest neck nodes in an operation that limits morbidity has led to a more proactive approach to cervical disease with many clinicians advocating elective neck dissections in those patients whose primary site characteristics would suggest a high rate of occult metastasis. Although this has been demonstrated to be of survival benefit, even in sites such as the tongue with rates of occult cervical disease approaching 50% in some series the majority of patients undergoing an elective, staging neck dissection will ultimately demonstrated to be disease-free [9,10]. As such it is imperative that complications of such surgery and their attendant morbidity are minimised.

Morbidity is best minimised by a meticulous approach which begins in the out-patient setting. This discussion will therefore present an evidence-based approach to the care of patients undergoing neck dissection beginning with pre-operative evaluation and peri-operative care prior to consideration of surgical technique itself. Although the ultimate complication of neck dissection is residual or recurrent tumour a logical approach such as this should permit maximum eradication of disease and improve survival whilst minimising hospital stay, complications and even mortality.

General considerations

Co-morbidities such as cardiac, respiratory and hepatic disease are common place in patients undergoing neck dissections in either an elective or therapeutic sense. Additional immunosuppression caused by conditions such as diabetes or relative malnutrition should be optimised since they predispose to complications including as wound infection.

Pre-operative evaluation

Reduced nutritional intake may be a direct result of symptoms from the index tumour such as dysphasia or odynophagia and in itself is an independent contributory factor to poor gastrointestinal function. In appropriate cases enhancement of nutritional status with either a percutaneous or radiologically-inserted gastrostomy should be considered since this will not only help to reverse any malnutrition pre-operatively but will also ensure prompt enteral feeding in the post-operative period. Patients undergoing isolated neck dissections rarely require transfusion, but if such surgical approaches are combined with ablation of the index tumour and in particular microvascular free tissue transfer allogenic blood is often indicated with it attendant problems including communicable disease transmission. In prognostic terms the immunosuppressant effects such blood transfusions have not been shown to have a detrimental impact on survival but autogenous transfusion or peri-operative normovolemic haemodilution remain viable alternatives [11,12].

The issue of how long it takes to benefit from a tobacco cessation is not well established but even avoidance for 12-24 hours may be advantageous to the cardiovascular system in terms of carbon monoxide and nicotine elimination. Only rarely do smoking withdrawal symptoms such as agitation and disorientation occur in such circumstances. Cessation of several days greatly improves ciliary flow but post-operative respiratory morbidity is only reduced if tobacco is avoided for a minimum of two weeks. Likewise excessive alcohol use amongst head and neck cancer patients is common with both minor and major post-operative withdrawal symptoms increasing morbidity. Delirium tremens confers appreciable mortality and its prevention should therefore be an achievable goal.

Anaesthesia

The importance of communication between the anaesthetic and surgical teams cannot be over emphasised. There are theoretical advantages to using low-molecular weight as opposed to standard low dose heparin in the prophylaxis of deep vein thrombosis and pulmonary emboli since it lessens bleeding complications, has a prolonged duration of action and is less likely to induce thrombocytopaenia [13]. Physical methods of prophylaxis including external pneumatic compression and gradient elastic stockings should be universal. Intra-operative hypovolemia is a potential cause of organ dysfunction, increased post-operative morbidity and death. Whilst over-administration of crystalloids can lead to pulmonary oedema, goal-directed volume expansion during the intra-operative period using oesophageal Doppler has been shown to improve not only stroke volume, cardiac output and post-operative recovery but also reduces the time until patients are declared medically fit for discharge [14,15]. The use of LidCo for fluid optimisation in the intensive care setting is well-established. Intra-operative LidCo rapid monitoring, particularly when combined with pre-operative cardiopulmonary exercise testing, may confer similar advantages [16,17].

Post-operative care

Peri-operative cardiac complications can often be minimised by proactive management, e.g. the use of intraoperative lignocaine on the carotid sinus to counter potential arrhythmias caused by digital manipulation. Early hypertension may follow RND possibly as a result of either carotid sinus denervation or a Cushing's reflex associated with intra-cranial hypertension [17]. If such hypertension is encountered its incidence is increased if the contra-lateral neck is subsequently dissected. Prolongation of the Q-T interval has been reported following rightsided neck dissection and under such some circumstances special attention should be given to potassium levels [18]. Aggressive respiratory support is mandatory in encouraging patients to clear secretions and is especially important in the tracheotomised individual in whom pain may further limit pulmonary function. Early mobilisation further decreases morbidity. If shoulder function is impaired proactive treatment with physiotherapy should be instituted [19].

Surgical technique

Asepsis

Although preparation of a surgical site prevents wound contamination by removing transient pathological bacteria and decreasing resident flora counts, good surgical technique with minimal tissue damage still has a role to play. Removal of large amounts of beard hair may increase the rate of infection unless carried out immediately prior to surgery. If concurrent en-bloc resection of a primary oral or oro-pharyngeal lesion is to be carried out the resultant through-and-through defect increases the rate of wound infection in the neck although topical treatments with either antiseptics or antibiotics have been demonstrated to be beneficial [20]. Reducing the length of peri-operative treatment with intra-venous antibiotics limits the development of drug-resistant bacterial infection, e.g. Methicillin-resistant Staphalococcus Aureus [21].

Incisions

A variety of approaches exist for the approach to neck dissections that usually simply rely on surgeon preference, e.g. wine glass incision, apron access. The vascular supply of the cervical skin is derived from the external carotid artery superiorly and the subclavian artery inferiorly. Trifurcations or incisions parallel to the carotid artery should be avoided particularly in salvage cases after radiotherapy. In the latter instances some surgeons prefer the security of a McFee incision which avoids some of the potential problems of three-point access.

Flap elevation and closure

Flaps should be elevated in the sub-platysmal plane in order to maximise their blood supply unless local disease dictates otherwise. Tissue should be incised in stages particularly if bilateral flaps are employed since this limits blood loss. Skin flaps should not be allowed to dry out and if necessary before closure 2-3 mm of the edges should be excised. If a tracheostomy is to be performed it should not be incorporated into the main surgical field since not only may sepsis become a major consideration but also the ability to preserve vacuum for drains at closure may be compromised. Careful approximation of tissues should be a balance between preventing dead space formation on the one hand and causing necrosis by overtightening on the other.

Progression of neck dissection

The sequence of the neck dissection is dictated by the type of procedure being undertaken, whether such surgery is being performed simultaneously with other ablative procedures, e.g. thyroidectomy or pharyngolaryngectomy, and ultimately by the preference of the surgeon. In selective neck surgery the first step is often to divide the fascia overlying the sternocleidomastoid muscle. A plane can then be developed around both the anterior and posterior borders of the muscle as required. Dissection at the base of the neck around the anterior aspect of the sternocleidomastoid muscle exposes the omohyoid muscle which is an important landmark. Most surgeons will dissect the upper deep cervical chain from inferior to superior, exposing the internal jugular vein (IJV), carotid system and vagus nerve in so doing. In the upper neck the digastric muscle should be exposed in its entirety and forms another useful landmark. Dissection along the anterior belly brings the surgeon into the midline and directs the dissection to the inferior border of the mandible whereas the hypoglossal nerve and its associated troublesome venous plexus can be identified above the tendon of the muscle. The posterior belly of digastric delineates a safe area lateral to the upper end of the internal jugular vein. If the posterior triangle needs to be addressed it is often best to identify the trapezius muscle in the first instance and then incise parallel to its fibres onto the pre-vertebral fascia. It is important to identify this fascia since it not only protects the underlying neural structures but also provides a plane along which dissection is readily facilitated.

Air embolus

This is a rare event which can occur following injury to the IJV. Large emboli can produce sudden falls in end-tidal carbon dioxide and arterial blood pressure. A pre-cordial Doppler probe may detect the characteristic murmur of venous embolus. Local pressure should be applied and the anaesthetist informed so the patient can be placed in the Trendelenburg position and rotated to the left. In severe cases attempts can be made to pass a catheter and aspirate air from the right side of the heart. Hyperbaric oxygen therapy, were available, is the ultimate and effective treatment.

Pneumothorax

This may occur when working low in the neck particularly if the lung apex is high as may occur in over inflation secondary to inadvertent one-lung intubation. Any tears in the pleura should be closed and their integrity tested by hyperinflating the lung, placing the patient in the Trendelenburg position and irrigating the area with clear fluid to observe bubbles. On table imaging may be necessary to determine the need for open chest drainage.

Chyle leak

The thoracic duct arises from the cisternal chyli at the level of the second lumbar vertebra and rises into the neck between the aorta and the azygos vein. In the thorax it crosses to the left and after passing behind the aortic arch and left subclavian artery it lies on the anterior scalene muscles and phrenic nerve. The duct terminates most commonly in the left IJV although less commonly it may enter the left subclavian, left external jugular, left brachiocepalic (innominate) vein or right IJV. Up to 50% of patients exhibit more than one termination of the duct. The right lymphatic duct drain terminates at the junction of the right subclavian vein and IJV. The key to treatment of a chyle fistula is prevention which demands knowledge of the relevant anatomy. Whereas intra-operative identification can be aided by placing the patient in the Trendelenburg position or adopting a forced Valsalva manoeuvre, post-operative leaks are usually identified when feeding is commenced. Multiple approaches to the treatment of an established leak have emerged including nutritional, surgical and pharmalogical therapy. Although there are strong feelings amongst clinicians about the use of bowel rest, parental nutrition or low fat enteral formulae for the treatment of established chyle leaks, definitive evidence supporting one therapy over another does not exist [22].

Neural structures

Reparative processes of transected cervical nerves may lead to neuromas in the early or late post-operative period. A number of techniques have been advocated to eliminate such neuroma formation including ligation of the cut edge, cautery, alcohol injection and burying the cut end of nerves into muscle. None of these techniques have any proven value in preventing neuroma.

In limiting neural injury during neck dissection the surgeon arguably focuses most attention on the SAN which can be safely preserved without jeopardising the integrity of tumour exenteration as long as it not grossly involved by disease [23]. Care should be taken when elevating the skin flaps in the posterior triangle as occasionally the SAN lies surprisingly superficially. Consistent and rapid identification of the SAN in the upper neck during dissection that can be facilitated by a number of methods, many of which rely on the identification of Erb's points [24]. The attachment of the sternocleidomastoid muscle to the mastoid process is usually tendonous laterally, the inferior extent of this portion of the muscle often corresponding to the emergent point of the SAN from the medial muscle. A small vessel invariably lies immediately over the SAN in a relationship analogous to the artery that it often encountered whilst exposing the main trunk of the facial nerve. Whilst some authors have suggested cable grafting the SAN if formal resection is undertaken, intra-operative electroneurography and histochemical staining have demonstrated that maintenance of the C2, C3 and C4 branches to the trapezius muscle preserves useful function and can permit en-bloc resection of the proximal part of the SAN with the specimen if oncologically indicated [25]. If significantly reduced shoulder pain and disability is apparent in the early post-operative phase then progressive resistance exercise training should be instituted in an attempt to minimise its impact and improve quality of life [26].

Whereas the ansa cervicalis is frequently sacrificed the vagus, lingual, hypoglossal and marginal mandibular branch of the facial nerves should be identified and preserved. Identification of the latter should be attempted early on in the neck dissection following flap elevation. If the nerve cannot be identified by visual inspection through the layers of the deep cervical fascia its integrity should be preserved by dividing the facial vessels approximately 1 cm below the lower border of the mandible and then retracting the divided ends upwards, so lifting the marginal mandibular branch of the nerve away from the surgical field. The hypoglossal nerve can be identified crossing the external carotid artery and then emerging from underneath the posterior belly of digastric on the hyoglossus muscle -it is at risk of inadvertent damage at both sites. Transient neuropraxia to the phrenic nerve is often manifested sub-clinically in the post-operative period with changes on plain radiography but if a severe pulmonary problem exists, especially with concurrent pectoris major flat harvest, respiration may be compromised. Bilateral phrenic nerve palsies may necessitate periods of prolonged mechanical ventilation. Although the carotid plexus of the sympathetic trunk is at risk of incurring injury the presence of Bernard-Horner's syndrome in the post-operative period is less than 1% [27]. Paralysis of the cranial nerves occurs only rarely (less than 2% of neck dissections) and as such they are difficult to predict but are not associated with non-neurological complications [27].

The brachial plexus lies between scalenus anterior and medius muscles as it crosses the posterior triangle. It is not usually encountered other than in the extended radical neck dissection but knowledge of its location is important in preventing further readily avoidable complications. Intentional transection of the vagus nerve can result in intra-operative cardiac problems of which the anaesthetist needs to be forewarned.

Where possible integrity of the cranial nerves should be maintained unless this compromises tumour resection. Although it is well established that resection of the facial nerve in parotid malignancy provides similar survival benefit to preservation and post-operative radiotherapy, no similar data is available for other cranial nerves with relevance to neck dissection. Nerve resection therefore remains an individual decision and may well be influenced by other matters, e.g. the relatively low threshold for sacrificing a hypoglossal nerve if during the same operation a formal ipsilateral hemi-glossectomy is to be performed.

Drains

Drainage is used following neck dissection to prevent the collection of fluid and to aid healing. The placing of drain-age should be carried out separately from the incision to reduce the risk of infection. Although there is some controversy whether active or passive drains should be used when neck dissections are carried in conjunction with free tissue transfer, the evidence suggests that active drainage should be employed in both free flap and non-free flap cases [28].

Extended neck dissections

In addition to neural structures, vascular elements may also have to be sacrificed in certain clinically-determined circumstances. Major vessel involvement should be assessed pre-operatively with appropriate imagining, be it contrast-enhanced computed tomography, magnetic resonance imagining, magnetic resonance angiography, ultrasound, Doppler or conventional angiography. Selective sacrifice of the common or internal carotid arteries during extensive cervical operative procedures or their compulsory ligation after exposure for haemorrhage post-operatively can produce some of the most serious complications in head and neck surgery. The highest morbidity occurs in those patients in whom ligation has been performed during a hypotensive episode such as one due to proceeding haemorrhage rather than those in whom selective ligation or excision is undertaken [29]. Balloontest occlusion with hypotensive challenge offers a simple and reliable method of pre-operative risk assessment when internal carotid artery resection is planned for regional control of disease in advanced head and neck cancer [30]. However, this management option is still associated with a potential for neurological complication that must be weighed against the natural history of the disease and the risk and benefits of other treatment modalities.

Special considerations

Bilateral neck dissection

Increased morbidity and mortality has been demonstrated in patients undergoing simultaneous bilateral neck dissections [31]. Higher rates of infections and fistulae occur and complications such as facial oedema and swelling are commonplace, particularly if both IJVs are simultaneous transacted. Raised intra-cranial pressure (ICP) occurs following bilateral IJV ligation with secondary systemic hypertension (Cushing's reflex). This rise in ICP commonly requires aggressive treatment with hyperventilation, fluid restriction, steroids and mannotol. The ICP frequently returns to normal within 24 hours. There can be a significant rise in ICP in a staged second neck dissections even if the subsequent operation is undertaken many years after the initial surgery. Even in cases of bilateral neck dissections where one IJV is preserved post-operative imagining demonstrates thrombosis in up to 30% of cases [32,33] Meticulous technique with prevention of drying of the preserved IJV and minimisation of excess trauma probably reduces the incidence of this occurrence. If both IJVs are to be transected then preservation of conduits in the external venous system should be attempted wherever possible, eg external jugular veins. As in all cases of neck dissection where significant swelling may compromise the airway in the post-operative period the possibility of a prophylactic tracheostomy should be entertained.

The previously treated neck

Previous treatment of the neck, be it radiation, chemoradiation or surgery can have a significant impact both in terms of practicalities and post-operative complications. Previous radiation encourages fibrosis between tissue planes such that subsequent dissection can be a laborious process. There is no substitute to a painstaking approach and proactive haemostasis in such circumstances. These problems are often compounded when previous neck surgery has taken place and is of particular issue if vascular access needs to be preserved to form the basis of recipient vessels for free tissue transfer. In such circumstances some of the more commonly used vessels, e.g. facial or superior thyroid arteries, may well have been sacrificed. There is rarely a role for pre-operative arterial imaging but instead the surgeon should be wary of preserving vessels in and around the surgical field. The transverse cervical artery and vein are useful recipient vessels since they are rarely irradiated, are of near constant calibre throughout their course and are not commonly affected by atheroclerosis. In selected circumstances veins grafts may be advisable as an alternative to exposing the contra-lateral neck. In extreme circumstances one may resort to using the cephalic vein turned over the clavicle or the internal mammary system.

Carotid blow-out

This is associated with over 60% morbidity and 50% mortality. Neurological sequaelae of emergency ligation include hemiplegia, hemi-anaesthesia, aphasia and dysarthria. The incidence is increased following radiation and salary fistulae. Damage to the adventitial layer during surgery may be another contributory factor. If risk of exposure is anticipated vessels should be covered, e.g. dermal graft, fascia lata or levator scapulae muscle flap. This is particularly important in the post-irradiation subject. If impending blow out is suspected (sentinel bleed) endovascular techniques with stent-grafts may be indicated rather than open ligation although short-term complications still occur [34].

Conclusion

Over the past century the neck dissection has become the accepted face of head and neck oncology, be it performed in isolation or as integral element of a more major resection and reconstruction. Since it is carried out with such regularity in many units it has the potential to be considered as routine. However in common with many procedures the appreciable potential morbidity and indeed morbidity should not be underestimated by junior and seniors surgeons alike. Whilst a meticulous, almost protocol-driven approach should be employed throughout, any one patient's needs can only be truly addressed by an individual approach. Despite the best planning complications can still occur but their impact can be minimised by a vigilant and proactive emphasis in the entire peri-operative period.
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Competing interestsThe authors declare that they have no competing interests.Authors' contributions
Excision of cancer of the head and neck with special reference to the plan of dissection base of one hundred and thirty-two operations. G Crile, JAMA. 47Crile G: Excision of cancer of the head and neck with special reference to the plan of dissection base of one hundred and thirty-two operations. JAMA 1906, 47:1780-1786.

Neck dissection. H Martin, B Delvalle, H Enrlich, E G Cahan, Cancer. 4Martin H, DelValle B, Enrlich H, Cahan EG: Neck dissection. Cancer 1951, 4:441-499.

Preoperative radiation and radical neck dissection. E W Strong, Surg Clin N Amer. 49Strong EW: Preoperative radiation and radical neck dissec- tion. Surg Clin N Amer 1969, 49:271-276.

Combined surgery and postoperative irradiation in the treatment of cervical lymph nodes. D R Goffinet, W E Fee, R L Goode, Arch Otolaryngol. 110Goffinet DR, Fee WE, Goode RL: Combined surgery and postop- erative irradiation in the treatment of cervical lymph nodes. Arch Otolaryngol 1984, 110:736-738.

Failure in the neck following multimodality treatment for advanced head and neck cancer. B Vikram, E W Strong, J P Shah, R Spiro, Head & Neck Surgery. 6Vikram B, Strong EW, Shah JP, Spiro R: Failure in the neck follow- ing multimodality treatment for advanced head and neck cancer. Head & Neck Surgery 1984, 6:724-729.

A syndrome resulting from radical neck dissection. A M Nahum, W Mullally, L Marmor, Arch Otolaryngol. 74Nahum AM, Mullally W, Marmor L: A syndrome resulting from radical neck dissection. Arch Otolaryngol 1961, 74:82-86.

Shoulder Pain and Function After Neck Dissection with or without Preservation of the Spinal Accessory Nerve. S O Short, J N Kaplan, G E Laramore, C W Cummings, Am J Surg. 148Short SO, Kaplan JN, Laramore GE, Cummings CW: Shoulder Pain and Function After Neck Dissection with or without Preser- vation of the Spinal Accessory Nerve. Am J Surg 1984, 148:478-482.

Functional neck dissection: an evaluation and review of 843 cases. E Bocca, Pignataro 0, C Oldini, C Cappa, Laryngoscope. 94Bocca E, Pignataro 0, Oldini C, Cappa C: Functional neck dissec- tion: an evaluation and review of 843 cases. Laryngoscope 1984, 94:942-945.

Elective neck dissection versus observation in the treatment of early oral tongue carcinoma. A P Yuen, W I Wei, Y M Wong, K C Tang, Head Neck. 197Yuen AP, Wei WI, Wong YM, Tang KC: Elective neck dissection versus observation in the treatment of early oral tongue car- cinoma. Head Neck 1997, 19(7):583-8.

T2 carcinoma of the tongue: the histopathologist's perspective. J A Woolgar, Br J Oral Maxillofac Surg. 373Woolgar JA: T2 carcinoma of the tongue: the histopatholo- gist's perspective. Br J Oral Maxillofac Surg 1999, 37(3):187-93.

Prognostic impact of blood transfusion in patients undergoing primary surgery and free-flap reconstruction for oral squamous cell carcinoma. M Fenner, E Vairaktaris, E Nkenke, V Weisbach, F W Neukam, M Radespiel-Tröger, Cancer. 1157Fenner M, Vairaktaris E, Nkenke E, Weisbach V, Neukam FW, Radespiel-Tröger M: Prognostic impact of blood transfusion in patients undergoing primary surgery and free-flap recon- struction for oral squamous cell carcinoma. Cancer 2009, 115(7):1481-8.

Acute perioperative normovolaemic haemodilution in major maxillofacial surgery. I R Parkin, G A Chiu, P A Schwarz, S C Hodder, Br J Oral Maxillofac Surg. 465Parkin IR, Chiu GA, Schwarz PA, Hodder SC: Acute perioperative normovolaemic haemodilution in major maxillofacial sur- gery. Br J Oral Maxillofac Surg 2008, 46(5):387-90.

Prophylaxis for venous thromboembolism in head and neck surgery: the practice of otolaryngologists. K W Ah-See, J Kerr, D W Sim, J Laryngol Otol. 1119Ah-See KW, Kerr J, Sim DW: Prophylaxis for venous throm- boembolism in head and neck surgery: the practice of otolaryngologists. J Laryngol Otol 1997, 111(9):845-9.

Goal-directed intraoperative fluid administration reduces length of hospital stay after major surgery. T J Gan, A Soppitt, M Maroof, H El-Moalem, K Robertson, E Moretti, P Dwane, P Glass, Anaesthesiology. 97Gan TJ, Soppitt A, Maroof M, El-Moalem H, Robertson K, Moretti E, Dwane P, Glass P: Goal-directed intraoperative fluid adminis- tration reduces length of hospital stay after major surgery. Anaesthesiology 2002, 97:820-6.

Haemodynamic optimisation of the surgical patient revisited. M Jonas, J Fennell, C S Brudney, Anaesthesia International. 21Jonas M, Fennell J, Brudney CS: Haemodynamic optimisation of the surgical patient revisited. Anaesthesia International 2008, 2:1.

Is cardiopulmonary exercise testing a useful test before esophagectomy?. M J Forshaw, D C Strauss, A R Davies, D Wilson, B Lams, A Pearce, A J Botha, R C Mason, Ann Thorac Surg. 851Forshaw MJ, Strauss DC, Davies AR, Wilson D, Lams B, Pearce A, Botha AJ, Mason RC: Is cardiopulmonary exercise testing a use- ful test before esophagectomy? Ann Thorac Surg 2008, 85(1):294-9.

Postoperative hypertension after radical neck dissection. S Celikkanat, M Akyol, C Koc, S Olcer, S Ensari, S Turgut, C Ozdem, Otolaryngol Head Neck Surg. 117Celikkanat S, Akyol M, Koc C, Olcer S, Ensari S, Turgut S, Ozdem C: Postoperative hypertension after radical neck dissection. Otolaryngol Head Neck Surg 1997, 117:91-92.

Prolongation of the Q-T interval and sudden cardiac arrest following right radical neck dissection. J Otteni, T Pottecher, G Bronner, H Flesh, J Diebolt, Anaesthesiology. 59Otteni J, Pottecher T, Bronner G, Flesh H, Diebolt J: Prolongation of the Q-T interval and sudden cardiac arrest following right radical neck dissection. Anaesthesiology 1983, 59:458-361.

Neck dissection and the clinical appearance of post-operative shoulder disability: the post-operative role of physiotherapy. D T Lauchlan, J A Mccaul, T Mccarron, Eur J Cancer Care (Engl). 176Lauchlan DT, McCaul JA, McCarron T: Neck dissection and the clinical appearance of post-operative shoulder disability: the post-operative role of physiotherapy. Eur J Cancer Care (Engl) 2008, 17(6):542-8.

The efficacy of topical antibiotic prophylaxis for contaminated head and neck surgery. J Grandis, R Vickers, J Rihs, V Yu, R Wagner, K Kachman, J Johnson, Laryngoscope. 92Grandis J, Vickers R, Rihs J, Yu V, Wagner R, Kachman K, Johnson J: The efficacy of topical antibiotic prophylaxis for contami- nated head and neck surgery. Laryngoscope 1994, 92:719-724.

Antimicrobial prophylaxis in otorhinolaryngology/head and neck surgery. F D Walker, L D Cooke, Clin Otolaryngol. 323Walker FD, Cooke LD: Antimicrobial prophylaxis in otorhi- nolaryngology/head and neck surgery. Clin Otolaryngol 2007, 32(3):204-7.

Chyle leaks: consensus on management?. A Smoke, M H Delegge, Nutr Clin Pract. 235Smoke A, Delegge MH: Chyle leaks: consensus on manage- ment? Nutr Clin Pract 2008, 23(5):529-32.

The eleventh nerve in radical neck surgery. J H Brandenburg, C Y Lee, Laryngoscope. 9111Brandenburg JH, Lee CY: The eleventh nerve in radical neck surgery. Laryngoscope 1981, 91(11):1851-9.

Upper neck spinal accessory nerve identification during neck dissection. T Tatla, J Kanagalingam, A Majithia, P M Clarke, J Laryngol Otol. 11911Tatla T, Kanagalingam J, Majithia A, Clarke PM: Upper neck spinal accessory nerve identification during neck dissection. J Laryn- gol Otol 2005, 119(11):906-8.

Trapezius muscle innervation from the spinal accessory nerve and branches of the cervical plexus. Y M Pu, E Y Tang, X D Yang, Int J Oral Maxillofac Surg. 376Pu YM, Tang EY, Yang XD: Trapezius muscle innervation from the spinal accessory nerve and branches of the cervical plexus. Int J Oral Maxillofac Surg 2008, 37(6):567-72.

Effect of exercise on upper extremity pain and dysfunction in head and neck cancer survivors: a randomized controlled trial. M L Mcneely, M B Parliament, H Seikaly, N Jha, D J Magee, M J Haykowsky, K S Courneya, Cancer. 1131McNeely ML, Parliament MB, Seikaly H, Jha N, Magee DJ, Haykowsky MJ, Courneya KS: Effect of exercise on upper extremity pain and dysfunction in head and neck cancer survivors: a rand- omized controlled trial. Cancer 2008, 113(1):214-22.

Neurological complications following functional neck dissection. M P Prim, De Diego, J I Verdaguer, J M Sastre, N Rabanal, I , Eur Arch Otorhinolaryngol. 2635Prim MP, De Diego JI, Verdaguer JM, Sastre N, Rabanal I: Neurolog- ical complications following functional neck dissection. Eur Arch Otorhinolaryngol 2006, 263(5):473-6.

Passive versus active drainage following neck dissection: a non-randomised prospective study. M D Batstone, D Lowe, R J Shaw, J S Brown, E D Vaughan, S N Rogers, Eur Arch Otorhinolaryngol. 2661Batstone MD, Lowe D, Shaw RJ, Brown JS, Vaughan ED, Rogers SN: Passive versus active drainage following neck dissection: a non-randomised prospective study. Eur Arch Otorhinolaryngol 2009, 266(1):121-4. Epub 2008 Jun 12

The internal carotid artery in the surgery of extensive oral cancer. C Martis, I Karabouta, N Lazaridis, Int J Oral Maxillofac Surg. 152Martis C, Karabouta I, Lazaridis N: The internal carotid artery in the surgery of extensive oral cancer. Int J Oral Maxillofac Surg 1986, 15(2):113-8.

Hypotensive endovascular test occlusion of the carotid artery in head and neck cancer. A O Dare, K J Gibbons, M D Gillihan, L R Guterman, T R Loree, W L HicksJr, Neurosurg Focus. 1435Dare AO, Gibbons KJ, Gillihan MD, Guterman LR, Loree TR, Hicks WL Jr: Hypotensive endovascular test occlusion of the carotid artery in head and neck cancer. Neurosurg Focus 2003, 14(3):e5.

Simultaneous bilateral neck dissection. M Ranzack, K Sako, R Baffi, J Patel, J Surg Oncol. 15Ranzack M, Sako K, Baffi R, Patel J: Simultaneous bilateral neck dissection. J Surg Oncol 1980, 15:387-392.

Intracranial pressure changes during bilateral radical neck dissections. K L Weiss, M K Wax, Haydon, H H Kaufman, M K Hurst, Head Neck. 156Weiss KL, Wax MK, Haydon RC 3rd, Kaufman HH, Hurst MK: Intracranial pressure changes during bilateral radical neck dissections. Head Neck 1993, 15(6):546-52.

Keller A: Internal jugular vein thrombosis following modified neck dissection: implications for head and neck flap reconstruction. D H Brown, S Mulholland, J H Yoo, P J Gullane, J C Irish, P Neligan, Head Neck. 202Brown DH, Mulholland S, Yoo JH, Gullane PJ, Irish JC, Neligan P, Kel- ler A: Internal jugular vein thrombosis following modified neck dissection: implications for head and neck flap recon- struction. Head Neck 1998, 20(2):169-74.

Stent-grafts in the treatment of emergent or urgent carotid artery disease: review of 25 cases. H Hoppe, S L Barnwell, G M Nesbit, B D Petersen, J Vasc Interv Radiol. 191Hoppe H, Barnwell SL, Nesbit GM, Petersen BD: Stent-grafts in the treatment of emergent or urgent carotid artery disease: review of 25 cases. J Vasc Interv Radiol 2008, 19(1):31-41.


Modulation of gene-specific epigenetic states and transcription by non-coding RNAs

Kevin V Morris
Modulation of gene-specific epigenetic states and transcription by non-coding RNAs
10.1007/s13148-011-0034-6Received: 18 March 2011 / Accepted: 7 April 2011 / Published online: 28 April 2011 # Springer-Verlag 2011REVIEWNon-coding RNA Epigenetic Transcription Pseudogene Tumor suppressor gene Antisense RNA
Emerging evidence points to a role for long noncoding RNAs in the modulation of epigenetic states and transcription in human cells. New insights, using various forms of small non-coding RNAs, suggest that a mechanism of action is operative in human cells, which utilizes noncoding RNAs to direct epigenetic marks to homology containing loci resulting ultimately in the epigenetic-based modulation of gene transcription. Importantly, insights into this mechanism of action have allowed for certain target sequences, which are either actively involved in RNA mediated epigenetic regulation or targets for non-coding RNA based epigenetic regulation, to be selected. As such, it is now feasible to utilize small antisense RNAs to either epigenetically silence a gene expression or remove epigenetic silencing of endogenous non-coding RNAs and essentially turn on a gene expression. Knowledge of this emerging RNAbased epigenetic regulatory network and our ability to cognitively control gene expression has deep implications in the development of an entirely new area of pharmacopeia.

Introduction

An enigma in modern-day molecular biology has been determining what exactly the role is for the massive amount of non-protein-coding transcripts found in the human cell. Noncoding transcripts have been estimated to account for ∼70-98% of the genome (reviewed in (Taft et al. 2007;Morris 2009a)). These non-coding RNAs (ncRNAs) emanate from non-coding DNA, regions in the genome that have been dubbed the "genomic dark matter"; as their functional relevance is questionable. If the non-coding regions of the human genome are not thought to be involved in protein coding then what, if anything, are these non-coding RNAs (ncRNAs) doing? And why would the cell transcribe vast tracks of DNA to never produce a protein?

RNA-mediated transcriptional gene silencing

Evidence addressing the central question of what the role is for ncRNAs in human cells has emerged from studies carried out with RNA interference (RNAi). RNAi is a process whereby small double-stranded interfering RNA (siRNAs) molecules functionally target and direct the degradation of a homology containing mRNA (reviewed in (Hannon 2002;Hannon and Rossi 2004)). This form of RNA-based regulation was termed; post-transcriptional gene silencing (PTGS). In PTGS, siRNAs are targeted to messenger RNA, recruiting the native cellular DICER machinery to degrade the transcript and provide gene suppression. While PTGS is a potent mechanism for the targeted degradation of targeted mRNAs, its gene suppressive effect is transient, lasting only until new transcripts are generated or the siRNAs are degraded. While several studies have been carried out using RNAi to suppress gene expression in a PTGS-based manner, one underappreciated study has created quite a wave of insight. In this study carried out in human cells, siRNAs were designed to target a gene promoter instead of the mRNA/coding region. The result of this siRNA targeting of a gene promoter was the suppression of gene expression at the transcriptional level, upstream of the well-documented PTGS pathway (Morris et al. 2004). Interestingly, this mode of RNA-based transcriptional suppression appeared to rely on an epigenetic component, as Trichostatin A and 5′ Aza-cytadine suppressed the obsiRNA-mediated transcriptional gene silencing (TGS). Notably, the observed siRNA-mediated TGS was similar to previous work carried out in plants and yeast (Matzke et al. 2004).

During the course of investigation as to how siRNAs were capable of directing TGS in human cells, one striking observation was generated. Weinberg and colleagues (2006) discovered, quite serendipitously, that only the antisense strand of the siRNA was required for TGS in human cells. Other studies have further validated this requirement (Turner et al. 2009), suggesting that the endogenous mechanism in human cells whereby ncRNAs regulate gene transcription may involve forms of RNA beyond doublestranded RNAs. Later work by several groups have collectively led to several insights suggesting a model for how small RNAs can function in controlling gene transcription in human cells (reviewed in (Morris 2009b)) ( Fig. 1). In this model, the RNA guides an epigenetic remodeling complex to the targeted promoter loci which contains homology to the RNA guide, ultimately resulting in the epigenetic remodeling of the target site to a transcriptionally silent state (Fig. 1). While much has been gleamed from these collective bodies of work, it had remained unknown whether or not human cells generated endogenous RNAs that might be regulating gene transcription in this manner.

RNAi-mediated suppression of the endogenous non-coding RNA suppressor results in activation of gene expression in human cells Despite earlier observations indicating that small ncRNAs, derived de novo could affect gene transcription, little was known as to whether or not there were active ncRNAs modulating gene transcription in human cells. Some studies provided evidence that micro-RNAs (miRNAs) could function as endogenous regulators of gene transcription (Klase et al. 2007;Omoto and Fujii 2005;Omoto et al. 2004;Tan et al. 2009;Kim et al. 2008). Hints to a deeper layer of RNA-based transcriptional regulation arose from one study which set out to target de novo derived siRNAs to the p21 and E-cadherin promoters, specifically targeting A:T-rich regions in these promoters. Notably, A:T-rich regions of the genome are generally found in the coding bodies of genes, while gene promoters tend to exhibit CpG islands and palindromic sequences with high GC content, often times used for transcription factor or protein binding (Alberts et al. 1994). In this study, it was determined that these p21 and E-cadherin promoter targeted siRNAs were able to activate gene expression, though activation of actual transcription was not demonstrated by nuclear run-on analysis or RNA polymerase occupancy at the respective promoters (Li et al. 2006;Place et al. 2008). In these studies, it was also determined that Argonaute 2 (Ago-2) was required for the observed activation, suggesting an RNAi-like effect was responsible for the observed activation (Li et al. 2006;Place et al. 2008). This observation is noteworthy as siRNAs shown to modulate TGS have generally been observed to require the action of Argonaute 1 (Ago-1), DNA methyltransferase (3a DNMT3a), and histone deacetylase 1 (HDAC-1) (reviewed in detail in (Morris 2009a;Morris 2009b))( Fig. 1). Nonetheless, the answer to the question as to how siRNAs targeted to A:T-rich regions in the p21 and E-cadherin promoters were utilizing Ago-2 to activate gene expression remained unknown.

During a foray into the mechanism of siRNA-mediated activation of p21 and E-cadherin, it was determined mechanistically that the A:T-rich siRNAs were actually targeting RNAi to an endogenous long antisense ncRNA (Morris et al. 2008). This observation suggested that possibly long antisense ncRNAs might be functioning as endogenous suppressors of p21 and E-cadherin, possibly regulating their epigenetic states and transcription. Notably, other tumor suppressor genes also appeared to be under this newly discovered regulatory mechanism of long antisense Fig. 1 Small antisense RNA-directed transcriptional gene silencing-a de novo derived small antisense RNAs, designed to target a promoter of choice, can interact with promoter-associated RNAs at the transcribed promoter (Hawkins et al. 2009;Han et al. 2007) and facilitate the recruitment of epigenetic remodeling complexes (Weinberg et al. 2006;Turner et al. 2009;Hawkins et al. 2009;Han et al. 2007;Kim et al. 2006;Suzuki et al. 2005;Suzuki et al. 2008). b The result of small antisense RNA targeting is the epigenetic remodeling and stable silencing of transcription from the targeted promoter non-coding RNA (Yu et al. 2008). A model has begun to emerge based on several different bodies of work suggesting that long antisense ncRNAs are active regulators of gene transcription in human cells (Fig. 2). Mechanistically, this model suggests that the siRNA targeting and degradation of the gene-specific long antisense ncRNAs affects the ability of these RNAs to interact with and guide epigenetic silencing complexes to their particular targeted loci in either gene bodies or promoters of the sense/mRNA loci for the gene. The result of this targeting is the activation of gene expression (Fig. 2) (Morris et al. 2008;Yu et al. 2008;Camblong et al. 2007;Cho et al. 2005;Ebralidze et al. 2008;Katayama et al. 2005;Knee and Murphy 1997;Li et al. 2009;Tufarelli et al. 2003;Wahlestedt 2006). Some antisense ncRNAs can function in trans to epigenetically regulate gene expression. In a recent study, an Oct-4 regulatory long-antisense ncRNA emanating from Oct-4 pseudogene 5 (Hawkins and Morris 2010) was found to be involved in epigenetically regulating Oct-4. Notably, this particular antisense ncRNA is not poly-adenylated, contains Oct-4 intronic sequences, and was found to specifically overlap segments of the Oct-4 promoter. The suppression of this Oct-4 antisense ncRNA resulted in a loss of H3K27 and H3K9 methylation at the Oct-4 promoter and activation of Oct-4 transcription (Hawkins and Morris 2010). The suppression of Ezh2 and G9a also had an impact on Oct-4 transcription; the loss of these factors resulted in increased transcription of Oct-4, suggesting that Oct-4 might be under epigenetic regulation via the action of this Oct-4 targeted long antisense ncRNA emanating from Oct-4 pseudogene 5 (Hawkins and Morris 2010).

Taken together, these data, along with those from studies carried out on p21 and p15 (Morris et al. 2008;Yu et al. 2008;Hawkins and Morris 2010), indicated that siRNA-directed gene activation is in essence "RNAi-mediated suppression of the endogenous ncRNA epigenetic suppressor", which results in a loss of endogenous epigenetic brakes being instilled in the particular gene by the action of the long antisense ncRNA (Fig. 2 and reviewed in (Morris 2009a;Morris 2009b)). Others have also found ncRNAs acting in the modulation of epigenetic states of imprinted genes (Tsai et al. 2010). These new findings strongly implicate long antisense ncRNAs as epigenetic regulators of gene expression in human cells. Such observations offer significant insights into a major question in biology: How do epigenetic changes get instilled at particular loci?

While the insights gained from these bodies of work indicate that a vastly more complex regulatory network is operative in human cells they also suggest that some form of regulation can be instilled in a gene-specific manner, i.e., we can turn a gene on or off transcriptionally. Evidence with small RNA-targeted TGS has demonstrated that stable long-term silencing can be established after a relatively short duration of small RNA targeting to a gene promoter (Turner et al. 2009;Hawkins et al. 2009). This form of long-term silencing required Histone Deacetylase 1 (HDAC1), DNA methyltransferase 3A (DNMT3a), and epigenetic changes such as histone 3 lysine 9 di-methylation as well as histone 3 lysine 27 tri-methylation at the targeted site for the initiation of silencing. Conversely, the long-term maintenance of silencing required the action of DNA methyltransferase 1 (DNMT1) and DNMT3a as well as DNA methylation at the targeted promoter (Turner et al. 2009;Hawkins et al. 2009). The impact of a set of molecules that can govern this form of control cannot be overstated as long-lasting effects can, by extension, have an impact on the evolution of the cell. Interestingly, to date, the majority of genes exhibiting long antisense ncRNAs appear to be tumor suppressors (reviewed (Morris and Vogt 2010)). These observations might speak to a role for dysregulated long antisense ncRNA-based networks in the evolution of epigenetic silencing and cancer (Morris and Vogt 2010)?

Conclusion

The observations that antisense ncRNAs are involved in regulating gene expression in differentiated cells (reviewed in (Morris 2009b)) adds to the growing complexity of known long ncRNAs involved in cellular regulation. Examples include the Xist antisense ncRNA TSIX, which is involved in dosage compensation and X-inactivation in undifferentiated primordial cells (reviewed (Lee 2009)). Other examples of ncRNAs involved in epigenetic regulation can be found in the imprinted genes (reviewed in (Latos and Barlow 2009)). These long ncRNAs also appear to function by targeting the recruitment of different epigenetic regulatory complexes to their intended targets. Clearly, the emerging evidence suggests that in human cells many more genes than previously envisioned, beyond X-inactivation and imprinted genes might actually be regulated by long ncRNAs. In fact, ncRNAs might be actively switching on and off genes in an orchestral regulation that governs the fidelity of the cell and functions in cellular adaption. Knowledge of this mechanism can no doubt prove useful in future therapeutics aimed at controlling gene expression in a transcriptional manner. Future therapies may utilize small RNAs or various chemically modified oligonucleotides to epigenetically modulate a gene transcription or to target endogenous ncRNAs actively involved in regulating particular genes expression. While we may be clearly on the verge of a new therapeutic platform that allows for the targeted control of gene transcription via controlled epigenetic targeting, one issue will continue to remain: delivery.

Fig. 2
2Endogenous antisense non-coding RNA mediated transcriptional silencing and activation. Long antisense non-coding RNAs emanating from either a bidirectionally transcribed, b alternative alleles, or pseudogenes,(Morris et al. 2008;Hawkins and Morris 2010) can fold into secondary structures (c) and interact with various proteins as well as target these proteins to homology containing loci in the genome (d). The antisense RNA protein complexes can then target the corresponding homology containing loci in the genome where epigenetic remodeling of the local chromatin via directed histone and DNA methylation can occur (e). When the antisense non-coding RNAs are targeted by either single-stranded oligonucleotides or siRNAs, the secondary structure is changed or interactions with the epigenetic remodeling protein complexes are blocked (f). The result is a loss of antisense non-coding RNA-directed epigenetic regulation of gene expression and subsequent activation of gene transcription (g)
ClinEpigenet (2011) 2:433-437
Acknowledgments This project is funded by NIH R01 HL083473 and NIH R01 AI084406 to KVM.
Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae. Bbd Alberts, J Lewis, M Raff, K Roberts, J D Watson, Cell. 131Garland PublishingMolecular biology of the cellAlberts BBD, Lewis J, Raff M, Roberts K, Watson JD (1994) Molecular biology of the cell, 3rd edn. Garland Publishing, New York Camblong J, Iglesias N, Fickentscher C, Dieppois G, Stutz F (2007) Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae. Cell 131:706-717

Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF. D H Cho, C P Thienes, S E Mahoney, E Analau, G N Filippova, S J Tapscott, Mol Cell. 20Cho DH, Thienes CP, Mahoney SE, Analau E, Filippova GN, Tapscott SJ (2005) Antisense transcription and heterochromatin at the DM1 CTG repeats are constrained by CTCF. Mol Cell 20:483-489

. A K Ebralidze, F C Guibal, U Steidl, P Zhang, S Lee, B Bartholdy, M A Jorda, V Petkova, F Rosenbauer, G Huang, T Dayaram, J Klupp, K B O'brien, B Will, M Hoogenkamp, Borden KLEbralidze AK, Guibal FC, Steidl U, Zhang P, Lee S, Bartholdy B, Jorda MA, Petkova V, Rosenbauer F, Huang G, Dayaram T, Klupp J, O'Brien KB, Will B, Hoogenkamp M, Borden KL,

PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element. C Bonifer, D G Tenen, Genes Dev. 22Bonifer C, Tenen DG (2008) PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element. Genes Dev 22:2085-2092

Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. J Han, D Kim, K V Morris, Proc Natl Acad Sci U S A. 104Han J, Kim D, Morris KV (2007) Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. Proc Natl Acad Sci U S A 104:12422-12427

RNA interference. G J Hannon, Nature. 418Hannon GJ (2002) RNA interference. Nature 418:244-251

Unlocking the potential of the human genome with RNA interference. G J Hannon, J J Rossi, Nature. 431Hannon GJ, Rossi JJ (2004) Unlocking the potential of the human genome with RNA interference. Nature 431:371-378

Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5. P G Hawkins, K V Morris, Transcription. 1Hawkins PG, Morris KV (2010) Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5. Transcription 1:165-175

Promoter targeted small RNAs induce long-term. P G Hawkins, S Santoso, C Adams, V Anest, K V Morris, Nucleic Acids Res. 37Hawkins PG, Santoso S, Adams C, Anest V, Morris KV (2009) Promoter targeted small RNAs induce long-term. Nucleic Acids Res 37:2984-2995

Antisense transcription in the mammalian transcriptome. S Katayama, Y Tomaru, T Kasukawa, K Waki, M Nakanishi, M Nakamura, H Nishida, C C Yap, M Suzuki, J Kawai, H Suzuki, P Carninci, Y Hayashizaki, C Wells, M Frith, T Ravasi, K C Pang, J Hallinan, J Mattick, D A Hume, L Lipovich, S Batalov, P G Engstrom, Y Mizuno, M A Faghihi, A Sandelin, A M Chalk, S Mottagui-Tabar, Z Liang, B Lenhard, C Wahlestedt, Science. 309Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, Nakamura M, Nishida H, Yap CC, Suzuki M, Kawai J, Suzuki H, Carninci P, Hayashizaki Y, Wells C, Frith M, Ravasi T, Pang KC, Hallinan J, Mattick J, Hume DA, Lipovich L, Batalov S, Engstrom PG, Mizuno Y, Faghihi MA, Sandelin A, Chalk AM, Mottagui-Tabar S, Liang Z, Lenhard B, Wahlestedt C (2005) Antisense transcription in the mammalian transcriptome. Science 309:1564-1566

Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. D H Kim, L M Villeneuve, K V Morris, J J Rossi, Nat Struct Mol Biol. 13Kim DH, Villeneuve LM, Morris KV, Rossi JJ (2006) Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nat Struct Mol Biol 13:793-797

MicroRNA-directed transcriptional gene silencing in mammalian cells. D H Kim, P Saetrom, O SnoveJr, J J Rossi, Proc Natl Acad Sci U S A. 105Kim DH, Saetrom P, Snove O Jr, Rossi JJ (2008) MicroRNA-directed transcriptional gene silencing in mammalian cells. Proc Natl Acad Sci U S A 105:16230-16235

HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. Z Klase, P Kale, R Winograd, M V Gupta, M Heydarian, R Berro, T Mccaffrey, F Kashanchi, BMC Mol Biol. 863Klase Z, Kale P, Winograd R, Gupta MV, Heydarian M, Berro R, McCaffrey T, Kashanchi F (2007) HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR. BMC Mol Biol 8:63

Regulation of gene expression by natural antisense RNA transcripts. R Knee, P R Murphy, Neurochem Int. 31Knee R, Murphy PR (1997) Regulation of gene expression by natural antisense RNA transcripts. Neurochem Int 31:379-392

Regulation of imprinted expression by macro non-coding RNAs. P A Latos, D P Barlow, RNA Biol. 6Latos PA, Barlow DP (2009) Regulation of imprinted expression by macro non-coding RNAs. RNA Biol 6:100-106

Lessons from X-chromosome inactivation: Long ncRNA as guides and tethers to the epigenome. J T Lee, Genes Dev. 23Lee JT (2009) Lessons from X-chromosome inactivation: Long ncRNA as guides and tethers to the epigenome. Genes Dev 23:1831-1842

Small dsRNAs induce transcriptional activation in human cells. L C Li, S T Okino, H Zhao, D Pookot, R F Place, S Urakami, H Enokida, R Dahiya, Proc Natl Acad Sci U S A. 103Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, Dahiya R (2006) Small dsRNAs induce transcriptional activation in human cells. Proc Natl Acad Sci U S A 103:17337-17342

A noncoding antisense RNA in tie-1 locus regulates tie-1 function in vivo. K Li, Y Blum, A Verma, Z Liu, K Pramanik, N R Leigh, C Z Chun, G V Samant, B Zhao, M K Garnaas, M A Horswill, S A Stanhope, P E North, R Q Miao, G A Wilkinson, M Affolter, R Ramchandran, Blood. 115Li K, Blum Y, Verma A, Liu Z, Pramanik K, Leigh NR, Chun CZ, Samant GV, Zhao B, Garnaas MK, Horswill MA, Stanhope SA, North PE, Miao RQ, Wilkinson GA, Affolter M, Ramchandran R (2009) A noncoding antisense RNA in tie-1 locus regulates tie-1 function in vivo. Blood 115:133-139

Genetic analysis of RNA-mediated transcriptional gene silencing. M Matzke, W Aufsatz, T Kanno, L Daxinger, I Papp, M F Mette, A J Matzke, Biochim Biophys Acta. 1677Matzke M, Aufsatz W, Kanno T, Daxinger L, Papp I, Mette MF, Matzke AJ (2004) Genetic analysis of RNA-mediated transcrip- tional gene silencing. Biochim Biophys Acta 1677:129-141

Non-coding RNAs, epigenetic memory, and the passage of information to progeny. K V Morris, RNA Biol. 6Morris KV (2009a) Non-coding RNAs, epigenetic memory, and the passage of information to progeny. RNA Biol 6:242-247

Long antisense non-coding RNAs function to direct epigenetic complexes. K V Morris, Epigenetics. 4Morris KV (2009b) Long antisense non-coding RNAs function to direct epigenetic complexes. Epigenetics 4:296-301

Long antisense non-coding RNAs and their role in transcription and oncogenesis. K V Morris, P K Vogt, Cell Cycle. 9Morris KV, Vogt PK (2010) Long antisense non-coding RNAs and their role in transcription and oncogenesis. Cell Cycle 9:2542- 2545

Small interfering RNA-induced transcriptional gene silencing in human cells. K V Morris, S W Chan, S E Jacobsen, D J Looney, Science. 305Morris KV, Chan SW, Jacobsen SE, Looney DJ (2004) Small interfering RNA-induced transcriptional gene silencing in human cells. Science 305:1289-1292

Bidirectional transcription directs both transcriptional gene activation and suppression in human cells. K V Morris, S Santoso, A M Turner, C Pastori, P G Hawkins, PLoS Genet. 41000258Morris KV, Santoso S, Turner AM, Pastori C, Hawkins PG (2008) Bidirectional transcription directs both transcriptional gene activation and suppression in human cells. PLoS Genet 4: e1000258

Regulation of human immunodeficiency virus 1 transcription by nef microRNA. S Omoto, Y R Fujii, J Gen Virol. 86Omoto S, Fujii YR (2005) Regulation of human immunodeficiency virus 1 transcription by nef microRNA. J Gen Virol 86:751-755

HIV-1 nef suppression by virally encoded microRNA. S Omoto, M Ito, Y Tsutsumi, Y Ichikawa, H Okuyama, E A Brisibe, N K Saksena, Y R Fujii, Retrovirology. 144Omoto S, Ito M, Tsutsumi Y, Ichikawa Y, Okuyama H, Brisibe EA, Saksena NK, Fujii YR (2004) HIV-1 nef suppression by virally encoded microRNA. Retrovirology 1:44

MicroRNA-373 induces expression of genes with complementary promoter sequences. R F Place, L C Li, D Pookot, E J Noonan, R Dahiya, Proc Natl Acad Sci U S A. 105Place RF, Li LC, Pookot D, Noonan EJ, Dahiya R (2008) MicroRNA- 373 induces expression of genes with complementary promoter sequences. Proc Natl Acad Sci U S A 105:1608-1613

Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region. K Suzuki, T Shijuuku, T Fukamachi, J Zaunders, G Guillemin, D Cooper, A Kelleher, Journal of RNAi and Gene Silencing. 1Suzuki K, Shijuuku T, Fukamachi T, Zaunders J, Guillemin G, Cooper D, Kelleher A (2005) Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region. Journal of RNAi and Gene Silencing 1:66-78

Closed chromatin architecture is induced by an RNA duplex targeting the HIV-1 promoter region. K Suzuki, T Juelich, H Lim, T Ishida, T Watanebe, D A Cooper, S Rao, A D Kelleher, J Biol Chem. 283Suzuki K, Juelich T, Lim H, Ishida T, Watanebe T, Cooper DA, Rao S, Kelleher AD (2008) Closed chromatin architecture is induced by an RNA duplex targeting the HIV-1 promoter region. J Biol Chem 283:23353-23363

The relationship between nonprotein-coding DNA and eukaryotic complexity. R J Taft, M Pheasant, J S Mattick, BioEssays. 29Taft RJ, Pheasant M, Mattick JS (2007) The relationship between non- protein-coding DNA and eukaryotic complexity. BioEssays 29:288-299

Transcriptional inhibiton of Hoxd4 expression by miRNA-10a in human breast cancer cells. Y Tan, B Zhang, T Wu, G Skogerbo, X Zhu, X Guo, S He, R Chen, BMC Mol Biol. 1012Tan Y, Zhang B, Wu T, Skogerbo G, Zhu X, Guo X, He S, Chen R (2009) Transcriptional inhibiton of Hoxd4 expression by miRNA-10a in human breast cancer cells. BMC Mol Biol 10:12

Long noncoding RNA as modular scaffold of histone modification complexes. M C Tsai, O Manor, Y Wan, N Mosammaparast, J K Wang, F Lan, Y Shi, E Segal, H Y Chang, Science. 329Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, Shi Y, Segal E, Chang HY (2010) Long noncoding RNA as modular scaffold of histone modification complexes. Science 329:689- 693

Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. C Tufarelli, J A Stanley, D Garrick, J A Sharpe, H Ayyub, W G Wood, D R Higgs, Nat Genet. 34Tufarelli C, Stanley JA, Garrick D, Sharpe JA, Ayyub H, Wood WG, Higgs DR (2003) Transcription of antisense RNA leading to gene silencing and methylation as a novel cause of human genetic disease. Nat Genet 34:157-165

Mobilization-competent lentiviral vector-mediated sustained transcriptional modulation of HIV-1 expression. A M Turner, J De La Cruz, K V Morris, Mol Ther. 17Turner AM, De La Cruz J, Morris KV (2009) Mobilization-competent lentiviral vector-mediated sustained transcriptional modulation of HIV-1 expression. Mol Ther 17:360-368

Natural antisense and noncoding RNA transcripts as potential drug targets. C Wahlestedt, Drug Discov Today. 11Wahlestedt C (2006) Natural antisense and noncoding RNA tran- scripts as potential drug targets. Drug Discov Today 11:503-508

The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. M S Weinberg, L M Villeneuve, A Ehsani, M Amarzguioui, L Aagaard, Z X Chen, A D Riggs, J J Rossi, K V Morris, RNA. 12Weinberg MS, Villeneuve LM, Ehsani A, Amarzguioui M, Aagaard L, Chen ZX, Riggs AD, Rossi JJ, Morris KV (2006) The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells. RNA 12:256-262

Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. W Yu, D Gius, P Onyango, K Muldoon-Jacobs, J Karp, A P Feinberg, H Cui, Nature. 451Yu W, Gius D, Onyango P, Muldoon-Jacobs K, Karp J, Feinberg AP, Cui H (2008) Epigenetic silencing of tumour suppressor gene p15 by its antisense RNA. Nature 451:202-206


Antimullerian hormone is a predictor of live birth in patients with recurrent pregnancy loss

Gayathree Murugappan
Lora Shahine
Ruth B Lathi
Antimullerian hormone is a predictor of live birth in patients with recurrent pregnancy loss
10.1186/s40738-019-0054-zR E S E A R C H A R T I C L E Open Access
Background: Ovarian reserve testing is not routinely performed in the evaluation of recurrent pregnancy loss (RPL). The objective of this study was to determine if AMH levels are predictive of live birth rate in RPL patients pursuing expectant management (EM). Methods: Retrospective cohort study of RPL patients. Patients tried to conceive spontaneously for 12 calendar months or until they achieved a live birth, whichever occurred first. All patients with the intent to conceive were included regardless of final outcome.Results: One hundred fifty-five RPL patients treated from 2009 to 2017 were included. In a univariate logistic regression, AMH < 1 ng/mL was associated with decreased likelihood of live birth (OR 0.38; CI 0.16-0.87, p = 0.03) and increasing age (OR 0.91; CI 0.83-0.99, p = 0.04). Likelihood of live birth was not significantly associated with BMI (OR 1.21; CI 0.83-1.77, p = 0.31), three or four or more prior pregnancy losses (OR 0.93; CI 0.40-2.22, p = 0.87 and OR 0.52; CI 0.19-1.42, p = 0.20, respectively) and prior live births (OR 1.00; CI 0.48-2.08, p = 0.99). AMH < 1 ng/mL was shown to be a stronger predictor of live birth than age using a multivariate model adjusting for age, AMH, and time to conception.Conclusions: AMH < 1 ng/mL is associated with decreased likelihood of live birth among RPL patients pursuing EM, and may be a stronger predictor of live birth than age in this population.

Background

Recurrent pregnancy loss (RPL) is a challenging disorder for both patients and clinicians [1]. The most common cause of first trimester pregnancy loss is aneuploidy within the embryo and testing the patients having miscarriages for anatomic, parental karyotypes, immune, and hormonal issues results in no abnormal findings in the majority of cases [1][2][3][4][5]. The American Society for Reproductive Medicine (ASRM) and the European Society of Human Reproduction and Embryology (ESHRE) recommend expectant management (EM) as the standard of care for patients with unexplained RPL [1,2].

While ovarian reserve testing is not routinely recommended as part of the workup for RPL patients [1], this testing is recommended for women with infertility and has been shown to predict response to gonadotropin stimulation in fertility treatments like in vitro fertilization (IVF) [6,7]. The ability of biomarkers such as AMH to predict overall reproductive potential, however, is uncertain. A large prospective cohort study of fertile women trying to conceive spontaneously found that AMH did not correlate with fecundability [8]. In a subsequent analysis of pregnancy outcomes in this cohort, the authors report that AMH levels are inversely associated with the risk of miscarriage [9]. The role of AMH in predicting reproductive potential in RPL patients, defined as having two or more prior pregnancy losses, is unknown. A prospective cohort study of women attempting spontaneous conception after one or two prior pregnancy losses found that AMH was not associated with fecundability, while live birth was not separately examined [10]. In a population of women who have experienced multiple pregnancy losses, one can argue that their ultimate goal is to achieve live birth, as opposed to clinical pregnancy. The objective of our study is to examine the role, if any, of AMH in predicting live birth rate in RPL patients attempting spontaneous conception.

Methods

Patients and workup

This is a retrospective cohort study of RPL patients treated at one academic fertility center and one private practice setting between 2009 and 2017. This study was approved by the Institutional Review Board of Stanford Hospital and the Western Institutional Review Board. Patients with a history of at least 2 prior pregnancy losses, defined as loss of pregnancy from conception through 20 weeks gestational age, were included. This study was designed as an intent to treat analysis, so all RPL patients attempting spontaneous conception were included regardless of final clinical outcome. All patients had a complete RPL workup as recommended by the ASRM including blood work for parental karyotypes and to detect anti-phospholipid syndrome (APS) including the presence of anti-cardiolipin antibody, lupus anticoagulant and beta-2-glycoprotein as well as a uterine cavity evaluation. All patients also underwent screening for thyroid function; and, if they reported irregular menses, had screening with prolactin levels. Unexplained RPL patients as well as patients with APS and uterine cavity anomalies were included. Patients with APS were treated with low dose aspirin and prophylactic heparin or enoxaparin. Patients with uterine cavity anomalies including a uterine septum, submucosal fibroids or endometrial polyps underwent hysteroscopy with reection of the uterine septum, myomectomy, or polypectomy, respectively. Patient with translocations (either maternal or paternal) were excluded. Patients also had ovarian reserve testing with serum AMH prior to attempting pregnancy. Diminished ovarian reserve (DOR) was defined as AMH < 1.0 ng/mL. All patients were followed for at least 12 months from the initial clinic visit.

Treatments offered

RPL patients attempting to conceive spontaneously were offered supportive care with serum bHCG levels and first trimester ultrasounds. In addition, patients were allowed low dose aspirin treatment and vaginal progesterone supplementation in the luteal phase or with positive bHCG at the discretion of their provider. Patients tried to conceive spontaneously for 12 calendar months or until they achieved a live birth, whichever occurred first. The main study outcomes were pregnancy rate (PR), live birth rate (LBR), and clinical miscarriage rate (CMR) per pregnancy.

Study definitions

Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. A clinical pregnancy was defined as a serum quantitative bHCG level > 5mIU/mL and the presence of a gestational sac on transvaginal ultrasound at 6-7 weeks of gestation. Pregnancies were then followed weekly by ultrasound until transfer of care at 10 weeks gestational age. A patient with a serum bHCG level > 5mIU/mL that never progressed to a gestational sac on transvaginal ultrasound was diagnosed with a biochemical pregnancy (BC). A patient with a serum hCG level > 5mIU/mL and an extra-uterine gestational sac was diagnosed with an ectopic pregnancy. A clinical miscarriage (CM) was defined as a loss of pregnancy after a gestational sac had been identified on ultrasound and prior to 20 weeks gestational age. A pregnancy loss was defined as loss of pregnancy from conception (defined as serum quantitative bHCG level > 5mIU/mL) through 20 weeks gestational age. A live birth (LB) was defined as birth of a neonate at or beyond 24 weeks gestation and was documented by patient report. When results were not available, patients were individually contacted for follow-up. Pregnancy rate was calculated per patient while live birth and clinical miscarriage rates were calculated per pregnancy. Time to pregnancy was calculated from date of new patient visit to date of positive bHCG result in both groups, regardless of when the cause of RPL was addressed. All pregnancies included were conceived after hysteroscopy or APS treatment.

Statistical analysis

The primary outcome for the study was live birth by 12 cycles of attempting spontaneous conception. Secondary outcomes included clinical pregnancy rate and pregnancy loss rate.

In order to compare patient demographics, the Shapiro-Wilk test was first used to determine normality of each distribution. Continuous data with a normal distribution was reported as a mean value with standard deviation. The unpaired two-tailed student t-test was used to analyze the difference between means. Continuous data with a non-normal distribution was reported as a median with inter-quartile range (IQR). The Mann-Whitney U Test was used to determine the difference between medians. Categorical data was presented as percentages and a Fisher's exact test was used to present the differences between the two groups. A logistic regression model was then used to assess the association between AMH (assigned a categorical value for AMH < 1 ng/mL vs AMH ≥1 ng/mL) and live birth while adjusting for potential confounders and adjusting for time to pregnancy as a proxy for exposure time. To test for interaction by age, a likelihood ratio test was used to compare the fit for the model without the interaction term with that of the model with the interaction term. A Cox regression model was used to assess the association between AMH (assigned a categorical value for AMH < 1 ng/mL vs AMH ≥1 ng/mL) and clinical pregnancy and pregnancy loss, since time to conception was known for each pregnancy. Kaplan-Meier curves with 95% confidence intervals were constructed to present time to successful conception and time to any conception during the 12-month study period in patients with AMH < 1 ng/mL compared to AMH ≥1 ng/mL. Difference between the groups was assessed using a log-rank test. Analyses were performed using R version 3.5.0. All testing was 2-sided. A p-value of < 0.05 was considered statistically significant.

Results

One hundred fifty-five RPL patients tried to conceive spontaneously for 12 months. 90% of patients had unexplained RPL, with 5% (n = 8) diagnosed with uterine anomalies and 5% (n = 7) diagnosed with APS. Seven patients achieved a conception twice during this 12-month interval; 4 of these patients had 2 consecutive pregnancy losses and 3 of these patients had a pregnancy loss followed by a live birth. Average age of the overall patient cohort was 35.4 ± 4.8 years, ranging from 25 to 44 years. Overall, patients had an average of 2.5 ± 1.1 prior miscarriages, 0.6 ± 0.7 prior live births and a median BMI of 23.5 kg/m2 with IQR of 6.5 kg/m2. Median AMH was 1.7 ng/mL with IQR of 0.63 ng/mL. The distributions of age and AMH are shown in Additional file 1: Figures S1, S2. 29% of patients (n = 47) had DOR based on AMH < 1 ng/mL. As shown in Table 1, patients with AMH < 1 ng/mL were significantly older than patients with AMH ≥1 ng/mL (p < 0.01, student's T test) but did not differ in terms of BMI (p = 0.45, Mann-Whitney U Test), number of prior live births (p = 0.10, student's T test) or number of prior pregnancy losses (p = 0.42, student's T test).

Clinical pregnancy rate per patient was 66% (n = 103). Among patients with DOR, clinical pregnancy rate was 60% (n = 28). LBR per pregnancy overall was 59% (n = 61) and CMR per pregnancy was 40% (n = 41). One pregnancy was terminated for XYY karyotype on amniocentesis. Among patients with DOR, LBR per pregnancy was 39% (n = 11) and CMR per pregnancy was 61% (n = 17). Biochemical pregnancy rate per attempt was 6% (n = 10) and ectopic pregnancy rate per attempt was 2% (n = 3). Average time to pregnancy was 5.0 ± 2.8 months. Outcomes were known for all pregnancies and are stratified by AMH level in Table 2. Kaplan-Meier curves comparing time to successful conception over the 12-month study period in patients with AMH < 1 and AMH ≥ 1 are shown in Fig. 1. Patients with AMH ≥1 ng/mL had a significantly higher likelihood of achieving a successful conception (p < 0.01, log-rank test) compared to patients with AMH < 1 ng/mL. The shape of the curve suggests that among patients who did not succeed within the first 6 months, those with AMH < 1 ng/mL were unlikely to succeed within the following 6 months. In contrast, patients with AMH ≥1 ng/mL increased their odds of success with each subsequent month of attempting conception. A logistic regression model was then used to assess the association between AMH and live birth while adjusting for potential confounders. In order to avoid double counting, patients with more than one conception during the study period were assigned an outcome of live birth only if at least one conception during the study period resulted in live birth. In the unadjusted univariate model, AMH < 1 ng/mL was associated with decreased likelihood of live birth (OR 0.38; CI 0.16-0.87, p = 0.03) and increasing age (OR 0.91; CI 0.83-0.99, p = 0.04). Likelihood of live birth was not significantly associated with BMI (OR 1.21; CI 0.83-1.77, p = 0.31), three or four or more prior pregnancy losses (OR 0.93; CI 0.40-2.22, p = 0.87 and OR 0.52; CI 0.19-1.42, p = 0.20, respectively) and prior live births (OR 1.00; CI 0.48-2.08, p = 0.99). AMH < 1 ng/mL was shown to be a stronger predictor of live birth than age using a multivariate model adjusting for age, AMH, and time to conception (p = 0.14 for age and p = 0.07 for AMH). The interaction between AMH and age was not significant. Using a cox regression model, AMH < 1 was A subgroup analysis was performed comparing clinical outcomes among patients less than 38 years of age (Additional file 1: Table 1). Among patients in this subgroup, LBR per pregnancy was lower but not statistically significant between patients with AMH < 1 ng/mL compared to patients with AMH ≥1 ng/mL (47% v 69%, respectively, p = 0.12). Among patients in this subgroup, CMR per pregnancy was higher but not statistically significant between patients with AMH < 1 ng/mL compared to patients with AMH ≥1 ng/mL (53% v 30%, respectively, p = 0.09).

Discussion

While markers of ovarian reserve such as AMH are routinely used to predict outcomes of IVF cycles in a general infertility population [6,7], the value of AMH as an overall predictor of reproductive potential is unclear. Studies in both a fertile population and among patients with one or two prior pregnancy losses show no association between AMH and pregnancy rate, while AMH < 1 ng/mL may predict increased likelihood of miscarriage [8][9][10]. Among patients with RPL, one can argue that the ability to each live birth is a more significant predictor of reproductive potential than pregnancy rate. The role of AMH in predicting prognosis among RPL patients is largely unknown. In this study, RPL patients attempted spontaneous conception and clinical outcomes including live birth rate were then stratified by AMH. We report that in this RPL patient population, AMH < 1 ng/mL is associated with decreased likelihood of live birth. AMH < 1 ng/mL and may be a stronger predictor of live birth than age in this patient population, however using a multivariate model adjusting for age, AMH, and time to conception we did not find any statistically significant relationships. We did not find that an AMH < 1 ng/ mL was associated with likelihood of pregnancy loss, likely because the study was underpowered to identify this association. AMH < 1 was also not associated with likelihood of clinical pregnancy, a finding that has previously been shown in an IVF population [11].  RPL patients often present a significant challenge in terms of treatment, as counseling these patients to pursue EM per clinical guidelines can feel like a passive approach. Providers may benefit from identifying a subset of RPL patients more likely to benefit from EM. Based on the results of this study, RPL patients with AMH ≥ 1 ng/mL have a favorable prognosis for achieving live birth while pursuing EM compared to patients with AMH < 1 ng/mL. Furthermore, we demonstrate that patients with AMH ≥1 ng/mL increased their odds of live birth with each subsequent month of attempting conception, while patients with AMH < 1 ng/mL who did not achieve live birth by 4 months were unlikely to succeed within the 12-month study period.

RPL patients have been noted to have lower AMH, higher serum FSH and higher percentage of DOR compared to age-matched fertile controls [12]. Furthermore, patients with unexplained RPL have a higher incidence of DOR compared to patients with an identified cause of RPL [13][14][15]. In this cohort of RPL patients pursuing EM, clinical miscarriage rate is higher than expected for age for RPL patients with AMH < 1 ng/mL, suggesting that ovarian reserve testing may also impact prognosis for these patients. Increased clinical miscarriage rate in patients with low AMH levels is multifactorial in etiology and may be due to decreased oocyte quality or other factors that disproportionately affect patients with diminished ovarian reserve. Since low AMH is also a negative prognostic factor for IVF outcomes in RPL patients less than 38 years of age [16], the benefit of applying preimplantation genetic testing for aneuploidy in embryos to an RPL cohort deserves further study.

To our knowledge, this is the first study examining AMH as a predictor of clinical outcomes in RPL patients pursuing EM. One attempt at EM was defined as 12 calendar months trying to conceive. We did not, however, confirm that patients were trying for each month. The structure of the study as an intent to treat analysis allows for representation of all possible clinical outcomes, both positive and negative, as they occur in clinical practice, but has limitations as noted in prior studies [14]. Livebirth outcomes were compared using logistic regression while clinical pregnancy and pregnancy loss outcomes were compared using Cox regression models because gestational age was not known for the live births, therefore time to live birth was not available for analysis. However, we used Kaplan-Meier curves as an additional descriptive summary for time to successful conception because it is a common modality to present data. Sensitivity analyses showed no difference between Cox and logistic regression models for the outcome of live birth. In an adjusted logistic regression model, we demonstrate that AMH < 1 may be a stronger predictor of live birth than age. This conclusion is limited by the age range of our patient cohort, from 25 to 44 years. In a subgroup analysis of patients less than 38 years of age, there is a trend towards AMH being predictive of LBR and CMR but the statistical analysis is limited by the small size of the cohort. While there was no significant linear interaction between age and AMH, future studies would ideally examine this effect modification in a larger cohort. Finally, the retrospective nature of data collection is a limitation as patients with less or more favorable prognoses may have chosen EM, thus introducing bias into the study results. In addition, patients were allowed treatment with aspirin and/or vaginal progesterone which is a limitation of the study inherent in its retrospective design which we could not control. Larger prospective studies are needed to verify our findings.

Conclusion

In this intent to treat analysis, we report that AMH < 1 ng/mL is associated with decreased likelihood of live birth among RPL patients pursuing EM and may be a stronger predictor of live birth than age. Ovarian reserve testing is not currently recommended routinely in the workup of RPL. However, the findings of this study along with the higher incidence of DOR among RPL patients compared to the general infertile population may provide a more compelling argument towards screening RPL patients for DOR. RPL may be a predictor of DOR and providers will be able to counsel patients more thoroughly with ovarian reserve tests as a part of their standard RPL evaluation.

Additional file

Additional file 1: Figure S1. Distribution of age (N = 155). Figure S2. Distribution of AMH (N = 155). Table S1. Subgroup analysis of clinical outcomes among patients less than 38 years of age. (DOCX 17 kb)

Fig. 1
1Kaplan-Meier curves for time to successful conception stratified by AMH < 1 and AMH ≥ 1 (N=155, 61 events)

Table 1
1Baseline demographics for RPL patients pursuing expectant management, stratified by patients with AMH < 1 compared to AMH ≥1Parameter
AMH < 1 (n = 47 patients)
AMH ≥1 (n = 108 patients)
P-value

Maternal Age (years) (Mean ± SD)
37.5 ± 3.8
34.7 ± 4.0
< 0.01 1

BMI (kg/m2) 3 (Median (IQR))
23.9 (5.2)
22.9 (5.8)
0.45 2

Prior live births (Mean ± SD)
0.7 ± 0.8
0.5 ± 0.6
0.10 1

Prior miscarriages (Mean ± SD)
2.6 ± 1.2
2.5 ± 1.0
0.42 1

1

Calculated using Student's T-Test, 2-tailed, unpaired

2

Calculated using Mann-Whitney U Test, 2-tailed

3

Parameter not available for n = 7 patients

Table 2
2Clinical outcomes for RPL patients pursuing expectant management stratified by AMH levelOutcomes by AMH level:
(n = 155 patients)

Age (Mean ± SD)
Pregnancies per
patient, N (%)

LBR per pregnancy,
N (%)

CMR per pregnancy,
N (%)

Time to Pregnancy,
months (Mean ± SD)

AMH < 0.5 (n = 28)
37.9 ± 4.0
14 (50%)
5 (36%)
9 (64%)
4.8 ± 3.7

AMH 0.5-0.99 (n = 19)
36.4 ± 3.1
14 (74%)
6 (43%)
8 (57%)
4.8 ± 2.7

AMH 1-1.99 (n = 41)*
35.3 ± 6.2
30 (73%)
17 (57%)
12 (40%)
3.9 ± 2.2

AMH 2-2.99 (n = 24)
35.0 ± 3.9
16 (67%)
12 (75%)
4 (25%)
4.6 ± 2.3

AMH ≥ 3 (n = 43)
33.7 ± 3.4
29 (67%)
21 (72%)
8 (28%)
4.6 ± 3.3

*1 pregnancy was terminated for XYY karyotype on amniocentesis

AcknowledgmentsThe authors would like to acknowledge Jason Bentley for his generous assistance with statistical analysis and interpretation of results.FundingNone.Availability of data and materialsThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Authors' contributions GM, LS and RL participated in study design, data acquisition, data analysis and interpretation and manuscript preparation. All authors read and approved the final manuscript.Ethics approval and consent to participateThis study was approved with the Stanford IRB and the Western IRB.Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Publisher's NoteSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Early pregnancy guideline development group. Guidelines of the European Society of Human Reproduction and Embryology: Recurrent Pregnancy Loss. Eshre, ESHRE. Early pregnancy guideline development group. Guidelines of the European Society of Human Reproduction and Embryology: Recurrent Pregnancy Loss; 2017.

The Practice Committee of the American Society for Reproductive Medicine. Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Feril Steril. 98The Practice Committee of the American Society for Reproductive Medicine. Evaluation and treatment of recurrent pregnancy loss: a committee opinion. Feril Steril. 2012;98:1103-11.

Recurrent miscarriage. G M Stirrat, Lancet. 336Stirrat GM. Recurrent miscarriage. Lancet. 1990;336:673-5.

Evaluation and Management of Recurrent Pregnancy Loss. M Stephenson, W Kutteh, Clin Obstet Gynecol. 50Stephenson M, Kutteh W. Evaluation and Management of Recurrent Pregnancy Loss. Clin Obstet Gynecol. 2007;50:132-45.

Frequency of factors associated with habitual abortion in 197 couples. M Stephenson, Fertil Steril. 66Stephenson M. Frequency of factors associated with habitual abortion in 197 couples. Fertil Steril. 1996;66:24-9.

Serum antimullerian hormone predicts ovarian response and cycle outcome in IVF patients. W Cheng-Hsuan, Yu-Ching , C Hsin-Hung, W Jyuer-Ger, Y Yu-Jun, C Horng-Der, T , JARG. 26Cheng-Hsuan W, Yu-Ching C, Hsin-Hung W, Jyuer-Ger Y, Yu-Jun C, Horng- Der T. Serum antimullerian hormone predicts ovarian response and cycle outcome in IVF patients. JARG. 26:383-9.

Antimullerian hormone as a predictor of IVF outcome. D N Lekamge, M Barry, M Kolo, M Lane, R B Gilchrist, K P Tremellen, Repro BioMed Online. 145Lekamge DN, Barry M, Kolo M, Lane M, Gilchrist RB, Tremellen KP. Antimullerian hormone as a predictor of IVF outcome. Repro BioMed Online. 2007;14(5):602-10.

Association between biomarkers of ovarian reserve and infertility among older women of reproductive age. A Z Steiner, D Pritchard, F Z Stanczyk, J S Kesner, J S Meadows, A H Herring, JAMA. 318Steiner AZ, Pritchard D, Stanczyk FZ, Kesner JS, Meadows JS, Herring AH, et al. Association between biomarkers of ovarian reserve and infertility among older women of reproductive age. JAMA. 2017;318:1367-76.

Antimullerian hormone as a risk factor for miscarriage in naturally conceived pregnancies. Bml Schumacher, Amz Jukic, A Z Steiner, Fertil Steril. 109Schumacher BML, Jukic AMZ, Steiner AZ. Antimullerian hormone as a risk factor for miscarriage in naturally conceived pregnancies. Fertil Steril. 2018; 109:1065-71.

Is anti-Müllerian hormone associated with Fecundability? Findings from the EAGeR trial. S M Zarek, E M Mitchell, L A Sjaarda, S L Mumford, R M Silver, J B Stanford, JCEM. 100Zarek SM, Mitchell EM, Sjaarda LA, Mumford SL, Silver RM, Stanford JB, et al. Is anti-Müllerian hormone associated with Fecundability? Findings from the EAGeR trial. JCEM. 2015;100:4215-21.

Antimullerian hormone as predictor of implantation and clinical pregnancy after assisted conception: a systematic review and meta-analysis. R Tal, O Tal, B J Seifer, D B Seifer, Fertil Steril. 103Tal R, Tal O, Seifer BJ, Seifer DB. Antimullerian hormone as predictor of implantation and clinical pregnancy after assisted conception: a systematic review and meta-analysis. Fertil Steril. 2015;103:119-30.

Diminished ovarian reserve: is it a neglected cause in the assessment of recurrent miscarriage? A cohort study. M Atasever, Z Soyman, E Demirel, S Gencdal, S Kelekci, Fertil Steril. 105Atasever M, Soyman Z, Demirel E, Gencdal S, Kelekci S. Diminished ovarian reserve: is it a neglected cause in the assessment of recurrent miscarriage? A cohort study. Fertil Steril. 2016;105:1236-40.

Do women with unexplained recurrent pregnancy loss have higher day 3 serum FSH and estradiol values?. S W Trout, D B Seifer, Fertil Steril. 74Trout SW, Seifer DB. Do women with unexplained recurrent pregnancy loss have higher day 3 serum FSH and estradiol values? Fertil Steril. 2000;74:335-7.

High basal estradiol level and FSH/ LH ratio in unexplained recurrent pregnancy loss. B Gurbuz, S Yalti, S Ozden, C Ficicioglu, Arch Gynecol Obstet. 270Gurbuz B, Yalti S, Ozden S, Ficicioglu C. High basal estradiol level and FSH/ LH ratio in unexplained recurrent pregnancy loss. Arch Gynecol Obstet. 2004;270:37-9.

Diminished ovarian reserve may explain otherwise unexplained recurrent pregnancy loss. K A Wald, L K Shahine, Ann Pregnancy Birth. 111005Wald KA, Shahine LK, et al. Diminished ovarian reserve may explain otherwise unexplained recurrent pregnancy loss. Ann Pregnancy Birth. 2018; 1(1):1005.

Higher rates of aneuploidy in blastocysts and higher risk of no embryo transfer in recurrent pregnancy loss patients with diminished ovarian reserve undergoing in vitro fertilization. L K Shahine, L Marshall, J D Lamb, L R Hickok, Fertil Steril. 106Shahine LK, Marshall L, Lamb JD, Hickok LR. Higher rates of aneuploidy in blastocysts and higher risk of no embryo transfer in recurrent pregnancy loss patients with diminished ovarian reserve undergoing in vitro fertilization. Fertil Steril. 2016;106:1124-8.