Alexandre J Vivanti
email: alexandre.vivanti@aphp.fr

Barbara Maraux

Marie Bornes

Émile Daraï

Frédéric Richard

Roman Rouzier

Emile Daraï

Threatened preterm birth: Validation of a nomogram to predict the individual risk of very preterm delivery in a secondary care center

Keywords: In utero transfer, nomogram, perinatal network, secondary care center, threatened preterm delivery

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Situations involving very preterm birth require optimal care to limit morbidity whenever possible. In utero transfer to a tertiary level center reduces perinatal morbidity and mortality when the delivery occurs before 32 weeks gestation. [START_REF] Shlossman | An analysis of neonatal morbidity and mortality in maternal (in utero) and neonatal transports at 24-34 weeks' gestation[END_REF][START_REF] Towers | The effect of transport on the rate of severe intraventricular hemorrhage in very low birth weight infants[END_REF][START_REF] Rautava | The effect of birth in secondary-or tertiary-level hospitals in Finland on mortality in very preterm infants: a birthregister study[END_REF] Accurate assessment of the risk of preterm birth remains a challenge in improving neonatal outcome. In clinical practice, most women treated for preterm delivery risk actually deliver at term. [START_REF] Ferguson | A comparison of tocolysis with nifedipine or ritodrine: analysis of efficacy and maternal, fetal, and neonatal outcome[END_REF] Threatened premature delivery is defined as the combination of painful uterine contractions and cervical modifications. Nonetheless, approximately 15% of women are hospitalized for uterine contractions only or cervical modifications only, and 5% show neither symtoms. [START_REF] Parant | Management of threatened preterm delivery in France: a national practice survey (the EVAPRIMA study)[END_REF] Even if both symptoms are present, the likelihood of an actual premature delivery is unknown, and most hospitalized women will not deliver before 37 weeks gestation. Threatened preterm delivery is the primary cause of hospitalization during pregnancy and the leading cause of medicalized in utero transfers. Accurate risk identification would help reduce unnecessary hospitalizations and in utero transfers for women who are actually at low risk of preterm birth.

Certain authors have tried to develop risk scores to predict premature delivery. [START_REF] Honest | The accuracy of risk scores in predicting preterm birth--a systematic review[END_REF] The likelihood ratios vary widely among studies, and the global accuracy of these risk scores is fairly low. Recently, Allouche et al. developed a nomogram allowing to estimate the individual risk of preterm delivery for women transferred for threatened preterm delivery through perinatal care networks. [START_REF] Allouche | Development and validation of nomograms for predicting preterm delivery[END_REF] This score was designed to predict delivery within 48 hours and before 32 weeks gestation. It includes the following elements: Preterm PROM, sonographic cervical length, gestational age at transfer, uterine contractions requiring tocolysis, multiple pregnancy, and vaginal bleeding. Its accuracy was retrospectively validated in a cohort of 169 women transferred for threatened preterm delivery to a tertiary care center but was not validated in a secondary care center. This issue is particularly relevant to avoid unnecessarily referring women to tertiary care. The discrimination and calibration of the score were good (concordance index of 0.73 and 0.72, respectively). The purpose of the present study was to validate this nomogram for use as a diagnostic tool to support clinical decision-making in a secondary care center.

Material and methods

Study population

The cohort included 137 women hospitalized for threatened premature delivery (painful and regular uterine contractions and/or cervix modifications) between 22 and 32 weeks gestation in a French secondary care center (Hôpital Tenon, Assistance Publique -Hôpitaux de Paris) between September 2009 and September 2011.

Before hospitalization, an initial emergency maternal and fetal assessment was performed to evaluate the risk of preterm delivery (clinical examination, cardiotocography and ultrasound). Contractions were defined as deflections from a clear baseline, with a rounded peak lasting 40 to 120 seconds. For each patient, cervical length was measured by transvaginal sonography.

All women received standard care based on national guidelines. [START_REF] College | French Clinical Guidelines for the management of Threatened Preterm Delivery with intact membranes[END_REF] There was no difference in the care given in the previous study as national guidelines had already been issued. Provided care may have included tocolysis (calcic inhibitors: nicardipine or nifedipine; oxytocin receptor antagonist: atosiban), antenatal steroids (betamethasone: two intramuscular injections of 12 mg 24 hours apart) and antibiotics when preterm premature rupture of membranes (PROM) was diagnosed.

PROM was defined as a fluid leak through the cervix before labor. In uncertain cases, an insulin-like growth factor binding protein-1 analysis was performed (Actim PROM®, Medix Biochemica, Kauniainen, Finland). Based on the clinical probability of delivery before 32 weeks, an in utero transfer to a tertiary care center was decided by the medical team. For each woman included in the study, the following data were prospectively collected: individual demographic and obstetric data (age, parity, obstetric history, and characteristics of this pregnancy), clinical characteristics at time of enrolment (vaginal bleeding, uterine contractions requiring tocolysis, and/or preterm PROM), sonographic measurement of cervical length, delivery within 48 hours of admission and delivery before 32 weeks gestation. Study exclusion criteria included prior maternal and/or fetal disease responsible for the transfer (i.e., intrauterine growth restriction, hypertension, and non-reassuring fetal heart rate). For each woman, the individual probability of preterm delivery within 48 hours and before 32 weeks gestation was calculated using the previously published nomogram available at http://www.perinatology.com/calculators/TRANSFER.htm. [START_REF] Allouche | Development and validation of nomograms for predicting preterm delivery[END_REF] Preterm PROM, sonographic cervical length, gestational age at transfer, uterine contractions requiring tocolysis, multiple pregnancy, and vaginal bleeding are associated with the obstetric outcome and are used in the calculation of the probability of preterm delivery. In utero transfers were not performed according to calculated risks. Risk calculation was retrospectively performed. In utero transfers were requested by clinicians based on clinical and ultrasound features. Women were retrospectively allocated to the following groups according to the medical decision: "transfer group" (in utero transfer to a tertiary care unit) and "no transfer group" (no in utero transfer).

The primary aim was to validate previously published nomograms to predict the individual probability of preterm birth before transfer for threatened preterm delivery from a secondary to a tertiary care unit. The secondary endpoint was to set the threshold for deciding whether an in utero transfer is needed.

Statistical analysis

All data were entered into a prospectively collected database. Nonparametric Mann-Whitney and Fisher's exact tests were used to compare quantitative and qualitative data, respectively; p <.05 was considered to be significant.

The model performance was quantified as previously described with respect to discrimination and calibration. The discrimination (i.e., whether the relative ranking of individual predictions is in the correct order) was quantified using the area under the receiver operating characteristic curve (ROC AUC) or the concordance index, which is similar to the ROC AUC but is appropriate for censored data. The concordance index is the probability that given two randomly selected patients, the patient with the worse outcome prediction will in fact have a worse outcome. The concordance index ranges from 0 to 1, with 1 indicating perfect concordance, 0.5 indicating no better concordance than chance, and 0 indicating perfect discordance. We used the bootstrapping method (200 repetitions) to obtain relatively unbiased estimates. The calibration (i.e., alignment between observed outcome frequencies and predicted probabilities) was studied with graphical representations of the relationship between the observed outcome frequencies and the predicted probabilities (calibration curves). These curves present the grouped proportions vs. the average predicted probability in groups defined by quantiles.

To determine the best preterm risk threshold (minimization of false-negative and false-positive rates), two statistical tools were used: Youden's index and MinROCdist. Youden's index is the threshold that maximizes the sum of sensitivity and specificity (Y=Se+Sp-1); it minimizes the average error rate for positive observations and the error rate for negative observations. MinROCdist is a cut-off that minimizes the distance between the ROC plot and the upper left corner of the unit square.

To calculate the power of the test (necessary to estimate sample size effect), we considered that individual predictions of delivery rates were constant as the null hypothesis. Based on our sample size, we estimated a proportion of .05 delivery in the no transfer group and .2 in the transfer group (based on observed data) : power = .78.

All the analyses were performed using the R package with the rms, Hmisc, pROC and PresenceAbsence libraries (http://lib.stat.cmu.edu/R/CRAN/).

Results

Study population

Between September 2009 and September 2011, 137 eligible women were enrolled in the study. An in utero transfer to a tertiary level care unit was decided upon for 56 women (41%) (Figure 1). Two women declined to be transferred and delivered within 48 hours of admission. About half of the women were nulliparous, and most of the pregnancies were singletons (Table 1). As expected, twin pregnancies were more frequently included in the "transfer group" (11 (20%) in the "transfer group" versus 3 (4%) in the "no transfer group", p<.005). Women with a history of late miscarriage and/or prophylactic cerclage were more likely to be transferred, whereas a history of previous spontaneous birth before 32 weeks did not seem to impact the decision regarding in utero transfer. At admission, there were no statistical differences between the groups in terms of uterine contractions, preterm PROM or vaginal bleeding. Cervical length at time of admission was significantly shorter in the "transfer group" (16.1 mm ± 12.0) than in the "no transfer group" (25.5 mm ± 8.1; p<.001). Nonparametric Mann-Whitney and Fisher's exact tests were used to compare the quantitative and qualitative data between "transfer group" and "no transfer group", respectively. NS, nonsignificant; PROM, premature rupture of membranes; SD, standard deviation.

Prediction of the probability of preterm delivery within 48 hours

None of the women in the "no transfer group" delivered within 48 hours of admission (Figure 1 and Table 2A), whereas nine (16%) women in the "transfer group" delivered within 48 hours. None of the women included in the study delivered prematurely for iatrogenic reasons. According to the nomogram, the average likelihood estimation of very preterm delivery within 48 hours was 10% in the whole population, 19% in the "transfer group" versus 5% (p<.0001) in the "no transfer group" (Table 2B). When applied to our secondary care unit cohort, the prediction model had a ROC AUC of 0.98 (95% CI: 0.95-1.00) (Figure 2A). The calibration was assessed graphically using the unreliability index (Figure 2B). There was a slight difference between the observed and predicted probabilities (p=.03); we observed an overestimation of the risk of preterm delivery within 48 hours in the whole cohort, but the calibration was excellent for the transferred women (Supplemental

Prediction of the probability of preterm delivery before 32 weeks gestation

None of the women in the "no transfer group" delivered before 32 weeks gestation after admission (Figure 1 and Table 2A), whereas 12 (22%) women in the "transfer group" delivered before 32 weeks. According to the nomogram, the average likelihood estimation of preterm delivery before 32 weeks was 28% in the whole population, 41% in the "transfer group" versus 19% (p<.0001) in the "no transfer group" (Table 2B).

When applied to our secondary care unit cohort, the prediction model had a ROC AUC of 0.94 (95% CI: 0.89-0.99) (Figure 3A). There was a significant difference between the observed and predicted probabilities (p<.001); we also noticed an overestimation of the risk of preterm delivery before 32 weeks in the whole cohort (Figure 3B), but the calibration was excellent for the transferred women

(Supplemental Figure B).

Preterm birth risk within 48 hours: optimal threshold determination

We determined the optimal threshold for the risk of preterm delivery within 48 hours because of its particular relevance in emergency obstetrical care. According to the MinROCdist (Figure 4A) and Youden's index (Figure 4B) calculations, the optimal threshold for considering an in utero transfer is 0.16 when looking at the risk of preterm delivery within 48 hours. With a threshold of 0.16, the sensitivity and specificity were 0.89 (95% CI: 0.52-0.98) and 0.90 (95% CI: 0.83-0.94), respectively (Figure 5).

Discussion

Very preterm birth is the main factor associated with perinatal mortality and severe morbidity, accounting for more than 50% of perinatal deaths. [START_REF] Goffinet | The value of intravaginal ultrasonography of the cervix uteri for evaluation of the risk of premature labor[END_REF][START_REF] Goldenberg | Epidemiology and causes of preterm birth[END_REF][START_REF] Ancel | Epidemiology of preterm births[END_REF][START_REF] Ancel | Survival and Morbidity of Preterm Children Born at 22 Through 34 Weeks' Gestation in France in 2011: Results of the EPIPAGE-2 Cohort Study[END_REF] The identification of women at risk for very preterm delivery among those presenting symptoms of preterm labor relies on obstetric history and an assessment of clinical and ultrasound features (uterine contractions, cervical length, and preterm PROM). [START_REF] Goldenberg | Epidemiology and causes of preterm birth[END_REF] We observed an excellent ROC AUC of 0.98 (95% CI: 0.95-1.00) for the 48-hour risk nomogram. It was previously assessed in a tertiary care center, and we performed an independent validation of this prediction model based on the combination of multiple routine clinical and sonographic variables with excellent discrimination. We noticed a higher ROC AUC in our study than in previously published cohort: 0.73 (95% CI: 0.66-0.80). [START_REF] Allouche | Development and validation of nomograms for predicting preterm delivery[END_REF] It could be mostly explained by lower prevalence rates of preterm births in secondary care centers than in tertiary care centers. Accordingly, it improves negative predictive value and thus ROC AUC.

In our study, in utero transfers were not performed based on calculated risks.

Preterm PROM is a major argument in favour of a decision of in utero transfer to a tertiary care center. This main risk factor of preterm birth is an integral part of both nomograms with a relative weight of approximately 20 percent of the overall risk. [START_REF] Allouche | Development and validation of nomograms for predicting preterm delivery[END_REF] Therefore, its impact has necessarily been taken into account regardless of preterm PROM rate in the validation set.

Correct prediction of very preterm birth would be useful if it enabled interventions to reduce mortality and morbidity associated with preterm birth. 13 Nevertheless, women may be hospitalized and receive unnecessary treatments. We observed that most admitted, but not transferred, women did not deliver during the very preterm period (81/137), but the positive predictive value (PPV) was low. Conversely, other women may receive tocolytic therapy rather late, and the decision to conduct an in utero transfer may be made too late (risk of outborn birth). The assessment of women at risk for very preterm labor must be accurate and objective. Our study demonstrated the relevance and ease of use of a nomogram to predict the individual risk of very preterm delivery within 48 hours after admission for threatened preterm delivery in a secondary care unit before 32 weeks of gestation. As expected, predicting a delivery within 48 hours was more accurate than predicting delivery before 32 weeks of gestation because it is a more immediate event. Indeed, we noticed excellent discrimination and correct calibration of this model based on routine clinical variables and cervical ultrasonography, thereby offering an independent validation of this previously published tool. We decided to promote this prediction model based on universally utilized and affordable criteria to simplify clinical decision-making. Some of the weaknesses in our findings relate directly to the retrospective nature of the study; nevertheless, we emphasize the comprehensiveness of the reported data.

This study highlights the need to select high-risk women for transfer to a tertiary care unit, but The selection of a preterm risk threshold remains crucial to avoid providing unnecessary care. Retrospectively, the threshold of our cohort was implicitly established at 0.05. A threshold of 0.16 would help minimize the risk of outborn deliveries with an excellent negative predictive value (NPV) of 0.99 (21 in utero transfers for eight preterm deliveries and 116 decisions not to transfer for one outborn preterm delivery within 48 hours). The actual benefits of risk scoring for the prevention of preterm birth are unknown. 13 Although many risk scores have been published over the past 30 years, no randomized controlled interventional trials have been conducted. Indeed, we must determine the optimal threshold to avoid unnecessary transfers of women who will not delivery prematurely to a tertiary unit.

We highlight the difficulty to establish an accurate diagnosis of threatened preterm delivery (44/56 women of the "transfer group" delivered after 32 weeks and 23/56 delivered after 37 weeks). In our cohort, a cervical length of less than 25 mm has been observed in more than 60% of women. In symptomatic patients, cervical length is used in routine to predict the risk of preterm delivery. Boots and colleagues provided a meta-analysis which evaluated the diagnostic accuracy of different morphological tests or biomarkers for the prediction of preterm delivery. Cervical length diagnostic accuracy had been evaluated : ROC AUC for the prediction of preterm delivery within 48 hours in symptomatic patients was 0.90 (95% CI 0.88-0.93) when used alone. [START_REF] Boots | The short-term prediction of preterm birth: a systematic review and diagnostic metaanalysis[END_REF] Although shortened cervical length is a major marker of preterm delivery in symptomatic women, one could argue that the fetal fibronectin test has not been implemented in combination with cervical length in evaluating preterm delivery risk. [START_REF] Sotiriadis | Transvaginal cervical length measurement for prediction of preterm birth in women with threatened preterm labor: a meta-analysis[END_REF] Indeed, the sensitivity of the fetal fibronectin test is higher when combined with cervical sonography. [START_REF] Van Baaren | Predictive value of cervical length measurement and fibronectin testing in threatened preterm labor[END_REF][START_REF] Wax | Biophysical and biochemical screening for the risk of preterm labor[END_REF] Recently, Kuhrt and colleagues established a predictive model that included quantitative fFN and history of preterm birth and/or preterm PROM with an accurate risk calculation in both symptomatic and asymptomatic women. [START_REF] Kuhrt | Development and validation of a tool incorporating cervical length and quantitative fetal fibronectin to predict spontaneous preterm birth in asymptomatic high-risk women[END_REF] In a similar way, two modified prediction models based on Allouche and colleagues' work were published. [START_REF] Mailath-Pokorny | Individualized assessment of preterm birth risk using two modified prediction models[END_REF] The authors pointed out the great accuracy of fetal fibronectin test, however the 48 hours modified prediction model did not take it into account. There was a high representation of admitted women with a gestational age of less than 24 weeks (24% of the cohort). Fetal fibronectin detection before 24 weeks must be use with caution because of its moderate value in predicting very preterm spontaneous birth: it could be partially explained by the physiological presence of fFN in cervicovaginal fluid in patients at less than 24 weeks. [START_REF] Lowe | Prospective randomized controlled trial of fetal fibronectin on preterm labor management in a tertiary care center[END_REF] New cervicovaginal fluid biomarkers have been tested; differentially expressed proteins were identified by proteomic analysis in symptomatic women using enzyme linked immunosorbent assays. Albumin and vitamin D-binding protein were differentially expressed in cervicovaginal fluid from women who delivered prematurely compared with those who delivered at term. Moreover, the combination of these two proteins was more accurate than fetal fibronectin at predicting preterm delivery in symptomatic women.

Nomograms facilitate decision-making, but this prediction score should be implemented after a cost-effectiveness analysis is performed to determine whether the routine assessment of the risk of premature birth in cases of threatened preterm labor is a cost-effective strategy. [START_REF] Honest | Screening to prevent spontaneous preterm birth: systematic reviews of accuracy and effectiveness literature with economic modelling[END_REF][START_REF] Bhutta | Can available interventions end preventable deaths in mothers, newborn babies, and stillbirths, and at what cost?[END_REF] Werner and colleagues conducted an analysis among asymptomatic women; routine cervical length measurements were performed with or without daily vaginal progesterone supplementation. [START_REF] Werner | Universal cervical-length screening to prevent preterm birth: a cost-effectiveness analysis[END_REF] The authors demonstrated that a screening program with appropriate interventions to reduce preterm birth would be cost-effective and cost-saving. These data were consistent with another previously published study. [START_REF] Cahill | Universal cervical length screening and treatment with vaginal progesterone to prevent preterm birth: a decision and economic analysis[END_REF] Nevertheless, no one has evaluated the cost-effectiveness of this strategy in symptomatic women or of the use of a screening tool for determining premature birth risk and its impact on the in utero transfer decision. A well-designed prospective randomized controlled clinical trial is needed to confirm these risk score results, including an evaluation of maternal well-being and a cost-effective analysis.

List of Abbreviation

PROM: preterm premature rupture of membranes ROC AUC: receiver operating characteristic curve SD: standard deviation

Figure 1 .

 1 Figure 1. Flow chart

Figure

Figure 2 .Fig 2A ,

 22A Figure 2. Accuracy and calibration of the prediction model for delivery within 48 hours of admission A B

Figure 3 .Fig 3A ,

 33A Figure 3. Accuracy and calibration of the prediction model for delivery before 32 weeks after admission A B

Figure 4 .

 4 Figure 4. Preterm birth risk within 48 hours: optimal threshold determination A B

Figure 5 .

 5 Figure 5. Accuracy of the prediction model of delivery within 48 hours after admission according to the threshold A B

Table 1 .

 1 Characteristics of the women in the panel and their pregnancies

	Characteristics	Whole population	Transfer group	No transfer group	p-value
		(n = 137)	(n = 56)	(n = 81)
	Age, mean ± SD	29.9 ± 5.9	29.0 ± 5.6	30.6 ± 6.1	NS
	Gestational age at inclusion,
	mean in wk ± SD	28.4 ± 2.8	27.3 ± 2.8	29.2 ± 2.5	< .0001
	< 24 wk, n (%)	10 (7)	6 (11)	4 (5)
	24-28 wk, n (%)	44 (32)	23 (41)	21 (26)	< .05
	28-32 wk, n (%)	83 (61)	27 (48)	56 (69)
	Nulliparous, n (%)	64 (47)	25 (44)	39 (48)	NS
	Singleton/twins, n (%)	123 (90) / 14 (10)	45 (80) / 11 (20)	78 (96) / 3 (4)	< .005
	Previous spontaneous birth
	<32 wk, n (%)	12 (9)	5 (9)	7 (9)	NS
	Previous late miscarriage
	between 14 and 24 wk, n (%)	9 (7)	8 (14)	1 (1)	< .05
	Prophylactic cerclage, n (%)	11 (8)	8 (9)	3 (37)	< .05
	Clinical and ultrasound features at admission
	Uterine contractions, n (%)	111 (81)	49 (87)	62 (76)	NS
	Preterm PROM, n (%)	11 (8)	7 (13)	4 (5)	NS
	Vaginal bleeding, n (%)	3 (2)	2 (3)	1 (3)	NS
	Cervical length, mean in mm ±
	SD	21.6 ± 10.9	16.1 ± 12.0	25.5 ± 8.1	< .0001
	<15 mm, n (%)	28 (20)	24 (43)	4 (5)
	<25 mm, n (%)	52 (38)	8 (14)	44 (54)	< .001
	≥25 mm, n (%)	57 (42)	24 (43)	33 (41)

Table 2 .

 2 Actual delivery and risk of preterm delivery according to the nomograms, within 48 hours and before 32 weeks gestation

	A
	Delivery within 48 hours and before 32 weeks gestation after admission according to
	each group
			Delivery <48 h	p-value	Delivery <32 wk	p-value
			Yes	No		Yes	No
	Whole		9 (7)	128 (93)		12 (9)	125 (91)
	population,	n
	(%)
	Transfer group,	9 (16)	47 (84)		12 (22)	44 (78)
	n (%) No transfer group, n (%)		0 (0)	81 (100)	< .001	0 (0)	81 (100)	< .001
	B

Acknowledgements

We thank all the families who participated in the study. We thank Caroline Roque for language editing

Funding

None.

Availability of data and materials

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study was approved by the CNIL (Commission Nationale de l′Informatique et des Libertes, French law no. 78 -17). All data were de-identified to ensure patient privacy and confidentiality. Oral informed consents have been obtained from the participants. According to the French law, written informed consent is not required for this observational retrospective study.

Consent to publish

Not applicable

Competing interests

None.

Authors' Contributions

AJV analyzed the data and wrote the manuscript. BM collected and analyzed the data. MB, ED and FR participated in the analysis of the data. RR conceived the study, analyzed the data and wrote the manuscript.


Elise Davoine

MD Ludivine Dion

Krystel Nyangoh Timoh

Eugenie Beraud

MD Patrick Tas

MD Aude Tavenard

Bruno Laviolle

PhD Christophe Perrin

MD Fabrice Foucher

MD Jean Levêque

PhD Vincent Lavoué

Predictive factors associated with involved margins in breast cancer treated with neoadjuvant chemotherapy followed by breast-conserving therapy Running title: involved margins in breast-conserving therapy after neoadjuvant chemotherapy

Keywords:

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Breast-Conserving Therapy (BCT), including lumpectomy and sentinel lymph node surgery followed by radiation therapy, is the treatment of choice for early-stage breast cancer, i.e. tumor sizes smaller than 3 cm [START_REF] Rouzier | Breast-conserving surgery after neoadjuvant anthracycline-based chemotherapy for large breast tumors[END_REF]. For larger tumor sizes, mastectomy is the gold standard.

Oncoplastic surgery can change this dogma, combining the principle of cancer resection with plastic surgery. It can allow Breast-Conserving Surgery (BCS) in larger tumors but this is not always feasible. Since the 1980's, neoadjuvant chemotherapy (NAC) has been developed to reduce tumor size. It can allow BCS in patients who otherwise would have required mastectomy. Randomized trials have shown the same efficiency for adjuvant and neoadjuvant chemotherapy in terms of survival [START_REF] Fisher | Effect of preoperative chemotherapy on local-regional disease in women with operable breast cancer: findings from National Surgical Adjuvant Breast and Bowel Project B-18[END_REF][START_REF] Scholl | Neoadjuvant versus adjuvant chemotherapy in premenopausal patients with tumours considered too large for breast conserving surgery: preliminary results of a randomised trial: S6[END_REF][START_REF] Rastogi | Preoperative chemotherapy: updates of National Surgical Adjuvant Breast and Bowel Project Protocols B-18 and B-27[END_REF][START_REF] Kaufmann | Recommendations from an international expert panel on the use of neoadjuvant (primary) systemic treatment of operable breast cancer: an update[END_REF]. Likewise, several trials have shown similar overall survival between mastectomy after NAC and BCT after NAC [START_REF] Rouzier | Breast-conserving surgery after neoadjuvant anthracycline-based chemotherapy for large breast tumors[END_REF][START_REF] Bonadonna | Primary chemotherapy to avoid mastectomy in tumors with diameters of three centimeters or more[END_REF][START_REF] Brain | Long-term prognostic and predictive factors in 107 stage II/III breast cancer patients treated with anthracycline-based neoadjuvant chemotherapy[END_REF][START_REF] Calais | Conservative treatment feasibility with induction chemotherapy, surgery, and radiotherapy for patients with breast carcinoma larger than 3 cm[END_REF][START_REF] Mieog | Neoadjuvant chemotherapy for operable breast cancer[END_REF]. After neoadjuvant chemotherapy, BCS is performed in 17% to 85% of cases [START_REF] Beriwal | Breast-conserving therapy after neoadjuvant chemotherapy: long-term results[END_REF] depending on the histological subtype. BCT allows oncologic treatment to be undertaken with an acceptable cosmetic outcome. This is easier for women to accept as it is less of a psychological trauma [START_REF] Maguire | Assessing clinical competence[END_REF][START_REF] Fallowfield | Psychological outcomes of different treatment policies in women with early breast cancer outside a clinical trial[END_REF].

However, some studies have shown specific factors to be associated with BCT failure, such as initial clinical tumor size > 50 mm, tumor size after NAC > 30 mm [START_REF] Rouzier | Breast-conserving surgery after neoadjuvant anthracycline-based chemotherapy for large breast tumors[END_REF][START_REF] Buchholz | Statement of the science concerning locoregional treatments after preoperative chemotherapy for breast cancer: a National Cancer Institute conference[END_REF], multifocality and lobular histology [START_REF] Rouzier | Breast-conserving surgery after neoadjuvant anthracycline-based chemotherapy for large breast tumors[END_REF][START_REF] Boughey | Neoadjuvant chemotherapy in invasive lobular carcinoma may not improve rates of breast conservation[END_REF][START_REF] Loibl | Surgical procedures after neoadjuvant chemotherapy in operable breast cancer: results of the GEPARDUO trial[END_REF].

In BCS, involved margins are correlated with high locoregional relapse (LRR), increasing rates by 2 or 3 times [START_REF] Ananthakrishnan | Optimizing surgical margins in breast conservation[END_REF][START_REF] Corsi | Preoperative localization and surgical margins in conservative breast surgery[END_REF][START_REF] Houssami | Meta-analysis of the impact of surgical margins on local recurrence in women with earlystage invasive breast cancer treated with breast-conserving therapy[END_REF][START_REF] Krekel | Intraoperative ultrasound guidance for palpable breast cancer excision (COBALT trial): a multicentre, randomised controlled trial[END_REF][START_REF] Morrow | Surgical margins in lumpectomy for breast cancer-bigger is not better[END_REF][START_REF] Nadrljanski | MRI in the evaluation of breast cancer patient response to neoadjuvant chemotherapy: predictive factors for breast conservative surgery[END_REF][START_REF] Behm | Surgical margins and risk of locoregional recurrence in invasive breast cancer: an analysis of 10-year data from the Breast Cancer Treatment Quality Assurance Project[END_REF]. Recent trials show narrow margins to be sufficient. Studies or meta-analysis have shown the same LRR rates for margins of 1 mm and 5 mm [START_REF] Houssami | Meta-analysis of the impact of surgical margins on local recurrence in women with earlystage invasive breast cancer treated with breast-conserving therapy[END_REF] [START_REF] Morrow | Standard for breast conservation therapy in the management of invasive breast carcinoma[END_REF][START_REF] Park | Outcome at 8 years after breast-conserving surgery and radiation therapy for invasive breast cancer: influence of margin status and systemic therapy on local recurrence[END_REF], and a report from the annual Meeting of the American Society of Breast Surgeons [START_REF] Harness | Margins: A Status Report from the Annual Meeting of the American Society of Breast Surgeons[END_REF] concluded that "no ink on tumor" is an adequate surgical margin for invasive breast cancer.

On the other hand, to achieve a good cosmetic outcome, most of the healthy tissue should be preserved. For BCS, the surgical dilemma is between tumor-free margins for optimum oncologic results and no tumor-free tissue removal for optimum cosmetic results.

Patients and Methods

Patients and study design

Between January 2008 and December 2013, we conducted a retrospective study of patients who had BCS after NAC in Eugène Marquis breast cancer center.

The patients were selected using the center's software. Inclusion criteria were BCS after NAC. Exclusion criteria were radical mastectomy after NAC, multifocal tumor and metastatic disease.

Neoadjuvant chemotherapy, surgical procedure and pathologic assessment

Six cycles of anthracycline-and taxol-based neoadjuvant chemotherapy were administered to all patients. Trastuzumab was also given for HER2-positive tumors. Wires or marker clips were placed before the first chemotherapy cycle. Breast tumor response after chemotherapy was assessed by clinical examination, mammography, ultrasonography and MRI. RECIST criteria [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF] were used to assess tumor response according to which mastectomy or BCT was chosen. Surgery was performed 4 weeks after completion of chemotherapy.

In the case of no clinical or radiological tumor signal after chemotherapy, a radiological examination was performed the day before the procedure using the hook wire left attached to the clip or wire marker. Four experienced breast surgeons performed BCT with axillary lymphadenectomy. The operative specimen was localized in the 3 spatial planes by the surgeon and referred to the pathologist who dried it with absorbent paper. A numbered glass slide was applied to each surface of interest (superior, inferior, anterior-posterior, lateral) after gently pressing the specimen. The slides were room-dried and stained with toluidine blue prior to interpretation. The results reported to the surgeon were expressed as: (a) acellular slides corresponding to healthy tissue (shown by a lack of normal epithelial cell desquamation); (b) presence of benign cells (macrophages, columnar or apocrine metaplasia cells); or (c) suspect positive slide in the presence of malignant cells. The precise site of the lesions was deduced from their location on the slide with anatomical mapping to the specimen to guide resection. In the case of resection, also guided by the surgeon, the cytological procedure was reproduced until standardization of the imprint cytology technique was achieved.

Histological examination was performed after fixing and embedding in paraffin. The specimen was inked with different colors (1 color per specimen surface), then sectioned into 3-mm slices in the frontal plane to enable us to best analyze the 4 surfaces of interest. The inked resection margins were analyzed in 3-mm slices perpendicular to the lumpectomy bed.

Data collection

Relevant data was collected. This included personal data such as age, weight, cup size, menopausal status, medical and family history, method of diagnosis (clinical or imaging and biopsy), tumor size and node status determined by clinical examination and imaging before and after NAC. Data on hormone status including ER/RP and HER2 status were also collected, as well as antigen Ki-67. Tumors were classified as defined by the 2011 St Gallen consensus [START_REF] Goldhirsch | Strategies for subtypes--dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer[END_REF] (luminal A: ER+, RP+, HER2 negative, Ki-67<14%; luminal B: ER+, RP+, HER2positive or HER2negative, Ki-67>14%; basal like: ER-, RP-, HER2-; HER2 positive: ER-, RP-, HER2 positive). Scarff-Bloom and Richardson (SBR) grading on biopsy before NAC was also recorded. For surgical specimens (post NAC), ductal carcinoma in situ (DCIS) or invasive residual tumor, node assessment and margin status were recorded. The number of procedures and patient status (survival and recurrence diagnosis) at last follow-up were also recorded.

Outcome measure

The outcome measure was tumor involvement on margin assessment. Tumor-involved margins were defined by tumor cells on ink for invasive cancer and tumor-free margins < 2mm for DCIS.

Statistical analysis

In the univariate analysis, continuous variables were compared using the Wilcoxon test, and qualitative variables were compared with Fisher's exact test or the Chi-square test. For continuous variables, cutoff was determined by a standard value in the literature or the ROC curve (value with best sensitivity and specificity). Finally, a logistic regression model was used to identify the risk factors associated with tumor-involved margins in a multivariate analysis. The variables found to have a p-value < 0.1 in the univariate analysis were used in the model. For all the analyses, p < 0.05 was considered to be statistically significant.

Results

Population characteristics and tumor response after NAC

Ninety-seven patients were included in the study (Figure 1). The median age of patients was 46 years old and the body mass index (BMI) median was 24 . For eighty patients (82.5%) the lesion was diagnosed by palpation. The initial average tumor size was 47.8 mm [+/-18.6]. Forty percent of the tumors were localized in the supero-external breast quadrant. Patient and tumor characteristics are provided in Table 1.

After neoadjuvant chemotherapy and before breast-conserving surgery, clinical and radiological tumor assessment showed a normal clinical breast examination in 59 patients (60%) and a complete response in 22% of patients assessed by mammography and 27.3% of patients assessed by MRI. On MRI evaluation, the scores for complete response and partial tumor response were 27.3% and 48% of patients respectively (data on 25 cases) according to RECIST criteria.

According to the pathologic assessment of the surgical specimens, a pCR was observed in 27 patients (27.8%). For a further 4 patients, no invasive residue was found, but persistent DCIS was observed.

Predictive factors for positive margins

Twelve patients (12.4%) had no tumor-free surgical margins on final histology. The method of diagnosis (screening or clinical), method of pre-surgical identification and surgical techniques (small or large lumpectomy, round block procedures) were not significantly associated with positive margins. Preoperative data associated with margin status are shown in Tables 2 and3. Postoperative data are shown in Table 4. The result of the multivariate analysis using pre-and post-operative data is shown in Table 5.

We also developed a multivariate model including only preoperative variables (predictors) of positive margins: echo T0, SBR grade, and continuous Ki-67 content. The data independently associated with positive margins were the initial ultrasound tumor size (T ≤ 27 mm) (p = 0.0456) and a low-grade vs high-grade SBR (p = 0.0097).

Surgical re-excision

Sixteen patients, i.e. 16.5% of the study population, had one or two re-excision procedures.

Fifteen patients (15.4%) had two operative procedures (initial BCT and re-excision) and one patient had three operative procedures (initial BCT, second BCT and a final mastectomy). For patients with two procedures: 12 patients out of 15 had repeat surgery due to positive surgical margins (11 patients had a second BCT and one patient had a mastectomy) and 3 patients out of 15 due to poor cosmetic results, abscess after conservative surgery or at the patient's request after learning of her BRCA1 mutation. Finally, there were 5 patients with secondary mastectomies (5.1%), but only 2 out of 5 for tumor-involved margins.

Survival

The average follow-up was 2.84 years [+/-1.12]. The rate of recurrence-free survival was 99% at 2 years and 85% at 5 years. The 2-year and 5-year overall survival rates were 95% and 77%, respectively (Figure 2).

Discussion

This study identified three factors independently correlated with positive margins after neoadjuvant chemotherapy: preoperative sonography size, low SBR grade and DCIS on pathologic surgical specimen analysis. Two factors relate to preoperative data and one to postoperative data. To our knowledge, ours was the first study to show criteria independently correlated with margin status in cases of NAC with BCT. One of the aims of NAC is to allow conservative surgery for cosmetic reasons. Narrow margins are important to maintain breast appearance while removing the whole tumor. However, if there are involved margins, additional breast surgery is necessary and it is correlated with worse cosmetic results, psychological trauma, high costs and subsequent radiation therapy [START_REF] Mullen | Involved anterior margins after breast conserving surgery: is reexcision required? European journal of surgical oncology[END_REF]. Surgeons need to know whether to remove more tissue or perform radical mastectomy in order to avoid a second surgical procedure.

Chemotherapy is more effective against tumors with high cell division potential. Several trials have shown that a high grade (SBR 3) is associated with better pCR rates [START_REF] Mauriac | Neoadjuvant chemotherapy for operable breast carcinoma larger than 3 cm: a unicentre randomized trial with a 124-month median follow-up. Institut Bergonie Bordeaux Groupe Sein (IBBGS)[END_REF][START_REF] Aas | Predictive value of tumour cell proliferation in locally advanced breast cancer treated with neoadjuvant chemotherapy[END_REF][START_REF] Amat | Scarff-Bloom-Richardson (SBR) grading: a pleiotropic marker of chemosensitivity in invasive ductal breast carcinomas treated by neoadjuvant chemotherapy[END_REF][START_REF] Dieras | Randomized parallel study of doxorubicin plus paclitaxel and doxorubicin plus cyclophosphamide as neoadjuvant treatment of patients with breast cancer[END_REF].

Conversely, low-grade tumor size decreases less after NAC, which increases the risk of positive margins in the case of BCT, as shown in our study. Forty percent of tumors in our study have an initial US tumor size < 27 mm but this represents 87.5% of tumors with positive margins. There are no other studies published with which to compare our results. However, large tumors (independently of histological subtype) seem to decrease to multifocal and patch-like lesions, and this increases the risk of involved margins [START_REF] Wang | Shrink pattern of breast cancer after neoadjuvant chemotherapy and its correlation with clinical pathological factors[END_REF]. On the other hand, luminal tumors are initially smaller, and trials have shown that NAC is less efficient [START_REF] Sanchez-Munoz | The role of immunohistochemistry in breast cancer patients treated with neoadjuvant chemotherapy: an old tool with an enduring prognostic value[END_REF].

Therefore, the residual tumor may be large, increasing the risk of positive margins. In our study, the luminal subtype seemed more associated with positive margins (58 vs 34%, p=0.13) and with an initial tumor size < 27 mm (55 vs 35%, p=0.16). This finding could explain why "smaller" tumors are associated with positive margins. Many studies have shown that DCIS is a risk factor for positive margins in BCT as a first treatment step. We showed that it was also a risk factor in BCT after NAC. The presence of DCIS is generally not considered in the case of NAC as studies tend to focus on pCR. Future studies could be performed to assess DCIS preoperatively. It is noteworthy that preoperative MRI is not efficient for detecting DCIS [START_REF] Fumagalli | A common language in neoadjuvant breast cancer clinical trials: proposals for standard definitions and endpoints[END_REF][START_REF] Partridge | Accuracy of MR imaging for revealing residual breast cancer in patients who have undergone neoadjuvant chemotherapy[END_REF] and DCIS is not palpable.

One limitation of this study is its retrospective nature. Therefore, given the inherent bias of the study design and the absence of other data confirming our results, we stress that our data should be interpreted with caution and be confirmed by prospective multi-institutional studies.

In fact, in the absence of level I evidence the evaluation of prospective series, as they evolve, is crucial. Furthermore, the low number of included patients and the low number of patients with involved margins were also others weakness of present study. Nevertheless, our results are consistent with other studies. Our pCR rate is 27.8%, in agreement with the rate observed in the Beriwal [START_REF] Beriwal | Breast-conserving therapy after neoadjuvant chemotherapy: long-term results[END_REF][START_REF] Komenaka | Preoperative chemotherapy for operable breast cancer improves surgical outcomes in the community hospital setting[END_REF] and Komekana trials (24-25%) [START_REF] Beriwal | Breast-conserving therapy after neoadjuvant chemotherapy: long-term results[END_REF]. After NAC, the BCT rate varies between 17 and 85% [START_REF] Beriwal | Breast-conserving therapy after neoadjuvant chemotherapy: long-term results[END_REF], which is consistent with the 60% in our study. The re-excision rate was 16.5% in our study, consistent with the literature [START_REF] Morrow | Surgical margins in lumpectomy for breast cancer-bigger is not better[END_REF] which showed a rate of between 12.4% [START_REF] Loibl | Surgical procedures after neoadjuvant chemotherapy in operable breast cancer: results of the GEPARDUO trial[END_REF] and 18% [START_REF] Komenaka | Preoperative chemotherapy for operable breast cancer improves surgical outcomes in the community hospital setting[END_REF]. Finally, our secondary mastectomy rate was 5.1% in line with previous published data which showed 9% [START_REF] Waljee | Predictors of re-excision among women undergoing breast-conserving surgery for cancer[END_REF]. Thus, our findings could be used in practice due to the lack of other available data on this key aspect of margin status after NAC and BCS.

Future prospective evaluation of the use of predictive criteria of tumor-involved margins as a way of reducing re-excision rates is required. Besides, preoperative detection of DCIS could increase negative margin rates, using wider BCT. As seen previously, MRI is the gold standard for detecting residual tumors after NCT but it failed to detect DCIS [START_REF] Fumagalli | A common language in neoadjuvant breast cancer clinical trials: proposals for standard definitions and endpoints[END_REF][START_REF] Partridge | Accuracy of MR imaging for revealing residual breast cancer in patients who have undergone neoadjuvant chemotherapy[END_REF]. In his meta-analysis, Marinovich shows that MRI is better for detecting pCR when the finding is "no invasive residue" as opposed to "no invasive ductal tumor + DCIS" [START_REF] Marinovich | Meta-analysis of magnetic resonance imaging in detecting residual breast cancer after neoadjuvant therapy[END_REF]. Sardenelli's trial shows that the DCIS detection rate is 35% in mammography, 46% in MRI and 54% in clinical examination [START_REF] Sardanelli | What is the sensitivity of mammography and dynamic MR imaging for DCIS if the whole-breast histopathology is used as a reference standard?[END_REF]. The use of a second biopsy after NAC, in order to detect DCIS, could improve treatment. The completion of a second series of biopsies after NAC is not currently offered. The main pitfall of performing a biopsy to detect DCIS is that false-negative results may be obtained due to the site being missed. However, if the presence of DCIS is identified by biopsy, it increases the risk of extensive component DCIS [START_REF] Dzierzanowski | Ductal carcinoma in situ in core biopsies containing invasive breast cancer: correlation with extensive intraductal component and lumpectomy margins[END_REF][START_REF] Jimenez | Clinicopathologic significance of ductal carcinoma in situ in breast core needle biopsies with invasive cancer[END_REF], [START_REF] Mai | Predictive value of extent and grade of ductal carcinoma in situ in radiologically guided core biopsy for the status of margins in lumpectomy specimens[END_REF]. Detection prior to surgery may allow the surgeon to perform wide surgery to achieve tumor-free margins. To determine whether implementation of this second set of biopsies would improve surgical outcome, a prospective evaluation should be undertaken.

Conclusion

Our study showed that 3 factors: initial US tumor size < 27 mm, SBR grade 1-2 and presence of DCIS after NAC independently correlated with positive margins. To our knowledge, there is no other study reporting on margin status in the context of NAC plus BCT. These criteria could help surgeons to optimize BCT and reduce second surgery rates. A prospective randomized trial assessing the benefit of a surgical biopsy after neoadjuvant chemotherapy should be performed to investigate the presence or absence of DCIS and its correlation with involved margins.

Figure 1 -

 1 Figure 1-Inclusion flow chart and number of second surgery procedures

Figure 2 -

 2 Figure 2-overall survival curve

Table 1 -Patient and tumor characteristics

 1

	Variable	N/NB (%)
	Menopausal status
	Yes	68/97 (70.1)
	Hormonal therapy
	Yes	06/97 (06.2)
	Personal history of breast
	disease
	Benign	18/97 (18.6)
	Malignant	02/97 (02.0)
	Family history
	First degree	15/97 (15.5)
	≥ Second degree	26/97 (26.8)
	Histological type
	Invasive Ductal Carcinoma	87/97 (89.7)
	Invasive Ductal Carcinoma plus	06/97 (06.2)
	DCIS	04/97 (04.1)
	Invasive Lobular Carcinoma
	Initial tumor stage
	T1	01/97 (1.0)
	T2	48/97 (49.5)
	T3	42/97 (43.3)
	T4	06/97 (06.2)
	Initial clinical axillary node
	status
	N0	42/97 (43.3)
	N1	55/97 (55.7)
	DCIS: Ductal Carcinoma in situ

Table 2 -Preoperative clinical data and radiological findings associated with tumor-involved margins

 2

	Variable	N0/total (%)	Unadjusted OR [95% CI]	p value
	Initial size
	≤ 45mm	04/53 (7.5%)	1
	> 45mm	05/34(14.7%)	2.72 [0.66 -13.20]	0.13
	Size after NAC
	≤11 mm	09/71 (12.7%)	1
	>11 mm	03/26 (11.5%)	0.9 [0.14 -4.04]	1.00
	Initial US size
	≤ 27mm	07/32 (21.9%)	1
	> 27mm	01/48 (2.1%)	0.08 [0 -0.06]	0.0059
	US size after NAC
	≤ 18mm	07/55 (12.7%)	1
	>18mm	05/34 (14.7%)	1.18 [0.27 -4.79]	1.00
	Initial MRI size
	≤ 36mm	02/25 (08%)	1
	>36mm	03/11 (27.3%)	4.31 [0.4 -57.70]	0.15
			0.99 [0.94 -1.05]	0.97
	US: Ultrasound; NAC: Neoadjuvant chemotherapy

Few studies so far have investigated predictive margin status in BCT after NAC. For successful BCS with optimum cosmetic results, the surgeon has to carefully plan the procedure and determine whether a small or large amount of breast tissue should be removed.

This study sought to identify predictive factors of involved surgical margins in BCS after NAC to help guide the surgical procedure.

Conflict of interest:

The authors have no conflict of interest to declare.


Jean-Christophe Gris
email: jean.christophe.gris@chu-nimes.fr

Sylvie Bouvier

Éva Cochery-Nouvellon

Éric Mercier

Ève Mousty

Antonia Pérez-Martin

The role of haemostasis in placenta-mediated complications

Introduction

Placenta-mediated pregnancy complications (PMPCs) are a group of diverse clinically defi ned late outcomes that include preeclampsia (PE), placental abruption, late pregnancy loss, and birth of a smallfor-gestational age infant (SGA). These common complications cause signifi cant maternal and neonatal morbidity and mortality. Their name implies a central role of the placenta in their pathophysiology. Their occurrence announces an increase in their incidences during the following pregnancies, and an increased cardiovascular risk throughout life.

The most studied and investigated type is PE, which is characterised by new-onset hypertension and proteinuria at >20 weeks of gestation. In the absence of proteinuria, diagnosis requires the presence of hypertension together with evidence of systemic disease. PE occurs in 1-8% of pregnant women (a range of prevalence related to variability in the risk factors of pregnant women from one country to another) and aff ects 1% of the general European population, including 1.5% of nulliparas. PE causes substantial maternal and perinatal morbidity, is the second cause of maternal mortality worldwide, and is one of the fi ve leading causes of maternal mortality in the developed world. Several eff orts to reduce recurrent PE have been implemented in clinical practice in the past decades but reported recurrence rates still range between 20% and 30%. PE still escapes to molecular annotations needed by contemporary precision medicine, limiting the accuracy of therapeutic developments. PE can occur in women with hydatiform moles: a placenta, but not the fetus, is thus mandatory.

Early reports in the early 1950s recognised an association between PE and coagulation activation [START_REF] Butler | The relationship of disorders of the blood-clotting mechanism to toxemia of pregnancy and the value of heparin in therapy[END_REF]. Increased thrombin generation, the end-stage central marker of coagulation activation, was later on described in preeclamptic women, both at onset of the disease [START_REF] Van Wijk | Enhanced coagulation activation in preeclampsia: the role of APC resistance, microparticles and other plasma constituents[END_REF] but also far from pregnancy [START_REF] Hamad | Increased thrombin generation in women with a history of preeclampsia[END_REF]. Strongest expressions of the thrombin receptor, the protease-activated receptor-1 (PAR-1), were evidenced in villous trophoblasts and stromal endothelial cells of placentas from pregnancies complicated by preterm PE [START_REF] Erez | Over-expression of the thrombin receptor (PAR-1) in the placenta in preeclampsia: a mechanism for the intersection of coagulation and infl ammation[END_REF]. This reinforces the data showing PAR-1 bound thrombin increasing the production of the soluble isoforms of the vascular endothelial growth factor receptor 1 (sFlt-1) by trophoblasts in vitro [START_REF] Huang | Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia[END_REF], sFlt-1 driving the systemic maternal syndrome of PE.

The activation of the haemostatic system might thus be a cornerstone in the pathophysiology of PE.

Heterogeneity of placenta-mediated pregnancy complications: the example of preeclampsia

The heterogeneity of diseases is one of the confounding variables explaining the failure of intervention studies to modify their incidence by acting on one of their components. Normal pregnancy is associated with an increasing state of activation of the haemostatic system. This activation state is excessive in women with placenta-mediated pregnancy complications (PMPCs), including preeclampsia (PE). Platelet activation plays a crucial pathophysiological role in PE. The very early activation of coagulation in the intervillous space is mandatory for placental growth and morphogenesis but its excesses and/or inadequate control may participate to the emergence of the trophoblastic phenotype of PE. Extracellular vesicles, of endothelial but also of trophoblastic origin, can favour key cellular reactions of preeclampsia, acting as proactive cofactors. The understanding of this intricate relationship between haemostasis activation and PMPCs may provide interesting keys for new pathophysiological therapeutic developments.

S11

Preeclampsia is ordinary described as a two-stage disease with a pre-clinical stage characterised by a defi ciency of the remodelling the terminal portion of the spiral uterine arteries by invading trophoblasts, between weeks 8 and 18 [START_REF] Redman | Latest advances in understanding preeclampsia[END_REF]. The consequence being placental underperfusion and oxidative stress, which generates placenta-derived circulating mediators prone to induce the maternal systemic vascular syndrome of PE after 20 weeks, to summarise: a nitric oxide (NO)deprived syndrome inducing a constriction of the peripheral vessels.

However, the scenario is currently validated for early-onset PE before 34 weeks, late onset PE having its own particularities. Lateonset PE is associated with low total vascular resistance and high cardiac output whereas only early-onset PE is associated with high total vascular resistance and low cardiac output [START_REF] Valensise | Early and late preeclampsia: two diff erent maternal hemodynamic states in the latent phase of the disease[END_REF]. Placental histopathology also diff ers. In comparison with gestational age-matched controls, placentas from early-onset PE are generally smaller whereas placentas from late-onset PE are generally larger. Placentas from earlyonset PE contain more infarcted areas and show a lower terminal villi volume whereas placentas from late-onset PE are globally normal but show increased arteriopathy [START_REF] Egbor | Morphometric placental villous and vascular abnormalities in early-and late-onset pre-eclampsia with and without fetal growth restriction[END_REF].

It is thus proposed a "placental PE" concept in which normal maternal vessels are modifi ed by an abnormal placenta (early-onset PE), and a "maternal PE" concept in which abnormal maternal vessels modify a normal placenta (late-onset PE) [START_REF] Staff | Redefi ning preeclampsia using placenta-derived biomarkers[END_REF]. However, the maternal systemic syndrome of PE is, in both cases, driven by the same placentaderived mediators, i.e. sFlt1 and soluble endoglin, with reduced levels of functional free PlGF [START_REF] Staff | Redefi ning preeclampsia using placenta-derived biomarkers[END_REF].

Platelets in placenta-mediated pregnancy complications

The decrease of circulating platelet numbers, with increased platelet volumes, can precede the onset of PE [START_REF] Hutt | Increased platelet volume and aggregation precede the onset of preeclampsia[END_REF]. Laboratory proofs of platelet activation can be evidenced weeks before any clinical symptomatology of PE [START_REF] Janes | Flow cytometric detection of activated platelets in pregnant women prior to the development of pre-eclampsia[END_REF]. These data suggest a function of platelet activation and platelet-derived mediators in PMPCS.

A study examined the localisation of leukocytes and platelets in early human placental tissues and the physiologic roles of plateletderived chemoattractants on extravillous trophoblasts (EVTs) invasion [START_REF] Sato | Platelet-derived soluble factors induce human extravillous trophoblast migration and diff erentiation: platelets are a possible regulator of trophoblast infi ltration into maternal spiral arteries[END_REF]. Maternal platelets were localised among endovascular trophoblasts within the lumen of spiral arteries. Maternal plateletderived soluble factors are suggested to regulate human SVTs diff erentiation, infi ltration and invasion into maternal spiral arteries.

Data accumulated on genetically modifi ed mouse strains, in embryo lacking thrombomodulin or its co-receptor endothelial protein C receptor in placental tissues, and in the hypomorphic thrombomodulin variant mouse model (TM Pro/Pro ) [START_REF] Sood | Fetal gene defects precipitate plateletmediated pregnancy failure in factor V Leiden mothers[END_REF] demonstrate that platelets can cause placental failure, the suggested detrimental function of platelets being independent from its thrombogenic capacity.

Increased platelet-monocyte aggregates are observed in PE [START_REF] Macey | Platelet activation and endogenous thrombin potential in pre-eclampsia[END_REF], activated platelets in PE can bind monocytes to generate sFlt-1, platelet-monocyte aggregates potentially contributing to endothelial dysfunction and systemic infl ammation commonly observed in PE [START_REF] Major | Synthesis of sFlt-1 by platelet-monocyte aggregates contributes to the pathogenesis of preeclampsia[END_REF].

Low-dose aspirin, the most widely used antiplatelet treatment during pregnancy, reduces the risk of preterm preeclampsia, but not of term preeclampsia, when it is initiated at ≤16 weeks of gestation at a daily dose of ≥100 mg [START_REF] Roberge | Aspirin for the prevention of preterm and term preeclampsia: systematic review and metaanalysis[END_REF], and may also decrease the risk of placental abruption.

Extracellular vesicles as modifying/reprograming circulating vectors

Extracellular vesicles (EV) are shed from a variety of cells and have a number of important physiological as well as pathological functions that have attracted increasing attention in vascular research.

It is now well established that the placenta releases fetalderived EVs into the maternal circulation during normal pregnancy, and that this shedding is altered in PE [START_REF] Knight | Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies[END_REF]. EV are released from the syncytiotrophoblast (ST) layer, which covers the surface of the placenta, directly into the maternal bloodstream where they can potentially interact with the endothelium, circulating immune cells and platelets. The release of these syncytiotrophoblast-derived EV in PE is elevated compared to healthy pregnancies, and the size, and the profi le of proteins present within EV in PE, is also signifi cantly altered.

Platelets can take up EV derived from ST in vitro [START_REF] Tannetta | Syncytiotrophoblast extracellular vesicles from pre-eclampsia placentas diff erentially aff ect platelet function[END_REF]; EV from normal placenta cause platelet activation and this activation capacity is increased by EVs from PE pregnancies-a fact which can be abolished by low-dose aspirin-placenta-derived mRNA and proteins being detectable in platelets from pregnant women.

Circulating EVs isolated from blood samples of patients with PE were shown to have varying eff ects according to gestational age and cell type [START_REF] Shomer | Microvesicles of women with gestational hypertension and preeclampsia aff ect human trophoblast fate and endothelial function[END_REF]. Their previous characterisation had shown that only one fi fth of them were negatively charged, thus prone to participate to any procoagulant activity; comparatively to normal pregnancies, they were enriched in platelet-derived, endothelial cell-derived, lymphocytederived, but not trophoblast-derived events. They contained higher levels of angiogenesis-modulating proteins and proinfl ammatory proteins, reduced early-stage trophoblast cell migration and induced higher levels of term trophoblast apoptosis, pointing to their role in trophoblast dysfunction related to PE. They also inhibited endothelial tube formation, pointing to their role in endothelial dysfunction related to PE. Therapy with low-molecular weight heparin LMWH aff ected patients' MV content, with higher levels of proangiogenic proteins, and normalised invasion, angiogenesis activity and survival on endothelial cells and trophoblasts cells in vivo [START_REF] Shomer | Microvesicles of pregnant women receiving low molecular weight heparin improve trophoblast function[END_REF].

A recent study showed that pregnant mice injected with in vitrogenerated, endothelial cell-derived procoagulant EVs, develop a PElike phenotype (elevated blood pressure, renal dysfunction, increased plasma sFlt-1) [START_REF] Kohli | Maternal extracellular vesicles and platelets promote preeclampsia via infl ammasome activation in trophoblasts[END_REF]. These endothelial EVs induced activation and accumulation of platelets within the placenta. EVs and platelets caused infl ammasome activation in trophoblast cells, with marked induction of NLRP3, cleaved Casp-1 and IL-1β. The use of NLRP3 defi cient mouse strains allowed establishing the central pathogenic function of the placental infl ammasome for the induction of the PE-like phenotype, placental dysfunction and embryonic demise. The platelet-derived, ATP-mediated signalling through the trophoblastic purinergic receptor P2X, leading to infl ammasome activation in trophoblasts, induced PE. Finally, human platelets with human endothelium-derived EVs induced ATP-dependent infl ammasome activation in human trophoblast cells and in placentae tissue samples obtained from women with PE. Infl ammasome inhibition using a P2X antagonist, or the anti-IL-1 anakinra, was protective. The capacity of enhancing infl ammasome activation was markedly reduced when using platelets obtained from healthy donors who had taken low-dose aspirin.

Focusing on EVs extruded from placental explants, EVs from PE placental explants activated endothelial cells in vitro, an eff ect which was part mediated by EVs-associated sFlt-1. Placental EVs may be thus one placental toxin/danger signal that contributes to the pathogenesis of preeclampsia, partially in a Flt-1-dependent manner [START_REF] Tong | Micro-and nano-vesicles from fi rst trimester human placentae carry Flt-1 and levels are increased in severe preeclampsia[END_REF]. Placental EVs can also transfer functional miRNAs to primary human endothelial cells [START_REF] Cronqvist | Syncytiotrophoblast derived extracellular vesicles transfer functional placental miRNAs to primary human endothelial cells[END_REF], including miR155 which inhibits eNOS expression in human endothelial cells.

Placental syncytiotrophoblastic EVs can also induce neutrophil extracellular traps NETs in isolated neutrophils. An increased presence of NETs directly in the intervillous space of pre-eclamptic placentae could be evidenced [START_REF] Gupta | Occurrence of neutrophil extracellular DNA traps (NETs) in pre-eclampsia: a link with elevated levels of cell-free DNA?[END_REF].

Coagulation in placenta-mediated pregnancy complications

Trophoblastic cells have the unique property to constitutively express tissue factor (TF), which is mandatory to placental development. A very initial activation of coagulation is therefore necessary for placental growth, mouse models showing that tissue factor-initiated S12 activation of the blood coagulation cascade at the feto-maternal interface allows activating trophoblastic cell surface bound protein C, which in turn can engage specifi c protease-activated receptors (PARs) and signaling pathways prone to support trophoblastic growth and diff erentiation [START_REF] Isermann | The thrombomodulin-protein C system is essential for the maintenance of pregnancy[END_REF]. Investigations performed at 8-14 gestational weeks from a huge series of 1220 consecutive Japanese women found low levels of the protein C cofactor protein S (PS), defi ned as a free PS antigen lower than the fi fth percentile (i.e. 23%), to be an independent risk factor for pre-eclampsia [START_REF] Ebina | Low levels of palsmaptotein S, protein C and factor XII during early pregnancy and adverse pregnancy outcomes[END_REF]. The study of changes of thrombin generation and activated protein C resistance in the fi rst 16 weeks of gestation in women with history of pre-eclampsia concluded that the pregnancy-related decrease of free PS levels and of free tissue factor pathway inhibitor (TFPI) attenuates the function of the protein C and TFPI systems and results in elevated thrombin generation and increased activated protein C resistance [START_REF] Tchaikovski | Role of protein S and tissue factor pathway inhibitor in the development of activated protein C resistance early in pregnancy in women with a history of preeclampsia[END_REF]. Apart from its natural anticoagulant property, activated protein C also has strong anti-infl ammatory, and anti-apoptotic properties, but also barrier stabilizing properties by binding directly to and activating Tie2, a transmembrane endothelial tyrosine kinase receptor, competing with the only known ligands of Tie2, the angiopoietins [START_REF] Minhas | Activated protein C binds directly to Tie2: possible benefi cial eff ects on endothelial barrier function[END_REF]. Activated protein C having protective eff ects in vascular leakage-related pathologies. Increased activated protein C resistance in pre-eclampsia may thus also play a role in the loss of the endothelial barrier integrity and in vascular leakage associated with the syndrome. An insuffi cient protein C system leading activated coagulation factors to engage deleterious alternative PARs, and to coagulate fi brinogen to fi brin, the subsequent formation of fi brin degradation products being pro-apoptotic. In normal pregnancies, trophoblastic TF procoagulant activity is thus partly held in check, as the syncytiotrophoblast adopts a pseudoendothelial phenotype through the expression of coagulation inhibitors [START_REF] Sood | Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells[END_REF]. This prevents TF-induced thrombin generation in normal pregnancies [START_REF] Sood | Fetomaternal cross talk in the placental vascular bed: control of coagulation by trophoblast cells[END_REF]. If coagulation-controlling systems are insuffi cient, placental failure develops with a fatal arrest of placental morphogenesis, in which platelets and PARs play a more prominent role than fi brin formation itself [START_REF] Sood | Maternal Par4 and platelets contribute to defective placenta formation in mouse embryos lacking thrombomodulin[END_REF].

Failure of trophoblasts to adopt an endothelial phenotype is associated with PE [START_REF] Zhou | Preeclampsia is associated with failure of human cytotrophoblasts to mimic a vascular adhesion phenotype. One cause of defective endovascular invasion in this syndrome?[END_REF]. Increased expression of TF in endothelial cells of the basal decidua is associated with severe PE and with bilateral notching of the uterine artery on ultrasound exams [START_REF] Di Paolo | Increased placental expression of tissue factor is associated with abnormal uterine and umbilical Doppler waveforms in severe preeclampsia with fetal growth restriction[END_REF]. Abnormal patterns of TF expression in the syncytiotrophoblast have been described in PE, with a reduction in TFPI-1 and TFPI-2, with higher plasma TF levels and lower plasma TFPI-1 and TFPI-2, higher placental TF gene and protein expression levels and lower TFPI-1 and TFPI-2 levels, and fi nally a signifi cant correlation between plasma and placental TF protein levels [START_REF] Teng | The relationship between plasma and placental tissue factor, and tissue factor pathway inhibitors in severe pre-eclampsia patients[END_REF]. The study of changes of thrombin generation in the fi rst 16 weeks of gestation in women with history of pre-eclampsia concluded that the pregnancy-related decrease of free tissue factor pathway inhibitor (TFPI) attenuates the function of the TFPI systems and results in elevated thrombin generation [START_REF] Tchaikovski | Role of protein S and tissue factor pathway inhibitor in the development of activated protein C resistance early in pregnancy in women with a history of preeclampsia[END_REF]. Low free TFPI plasma levels assayed apart from pregnancy were described to be risk factors for gestational vascular complications [START_REF] Ittel | Increased risk of gestational vascular complications in women with low free tissue factor pathway inhibitor plasma levels out of pregnancy[END_REF]. Increased TF activity was also evidenced in syncytiotrophoblast MVs released from PE placentae [START_REF] Gardiner | Syncytiotrophoblast microvesicles released from pre-eclampsia placentae exhibit increased tissue factor activity[END_REF]. An impaired balance between TF and TFPI, with TF predominance over TFPI, is thus associated with PE.

PAR-1 is overexpressed in villous trophoblasts of placentae from early-onset PE [START_REF] Erez | Over-expression of the thrombin receptor (PAR-1) in the placenta in preeclampsia: a mechanism for the intersection of coagulation and infl ammation[END_REF]. Excessive PAR-1 engagement by thrombin increases sFlt-1 secretion by extravillous trophoblasts, with a commitment of the PAR-1/NADPH oxidase/ROS signaling pathway [START_REF] Huang | Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia[END_REF], with parallel increased levels of fi brin generation markers [START_REF] De B Pinheiro | D-dimer in preeclampsia: systematic review and meta-analysis[END_REF]. A link thus emerges between increased placental TF expression, excessive thrombin generation, secondary PAR-1 engagement, abnormal levels of sFlt-1 secretion and systemic endothelial dysfunction in the mother. Focusing on thrombin generation within the intervillous space, placentas from early-onset PE contain more infarcted areas, with signifi cant perivillous fi brin deposition [START_REF] Egbor | Morphometric placental villous and vascular abnormalities in early-and late-onset pre-eclampsia with and without fetal growth restriction[END_REF]. The local circulatory insuffi ciency and consequent hypoxia may favour the expression of hypoxia-inducible factor 1-alpha HIF-1α, a transcription factor which persistent elevation in placenta has been described in women with PE, HIF-1α inducing the synthesis of sFlt-1, endoglin and endothelin-1, all PE contributors [38]. Hypoxic trophoblasts also secrete HMGB1 (highmobility group box 1), a proinfl ammatory protein, which emerges as a key factor in stimulating vesicle production in human umbilical vein endothelial cells [39]. The vesicles from HMGB1-stimulated human umbilical vein endothelial cells promote blood coagulation and neutrophil activation in vitro, injection of HMGB1 in pregnant mice increases plasma endothelial vesicles and promotes blood coagulation [39]. In pre-eclampsia women, elevated placental HMGB1 expression are detected in PE women and high levels of plasma HMGB1 correlate with increased plasma endothelial vesicles [39].

Conclusion

Placenta-mediated complications, among which pre-eclampsia, are associated with evidences of an excessive activation of the haemostatic system, both at the placental materno-fetal interface and at the maternal systemic level.

The very early activation of coagulation in the intervillous space is mandatory for placental growth and morphogenesis but its excesses and/or inadequate control may participate to the emergence of the trophoblastic phenotype of PE. Platelet activation plays a crucial pathophysiological role in PE. Extracellular vesicles, of endothelial but also of trophoblastic origin, can facilitate and amplify key cellular reactions of pre-eclampsia and act as proactive cofactors.

Low-dose aspirin reduces the risk of preterm pre-eclampsia and of placental abruption, but not of term pre-eclampsia, when it is initiated at ≤16 weeks of gestation at a daily dose of ≥100 mg.

The question is to know if increasing the antithrombotic pressure, according to modalities remaining to be defi ned, can make it possible to improve the prophylaxis of the PMPCs despite contradictory data on prophylactic doses of LMWH, or if it is time to introduce new therapeutic paradigms. In this view, inhibitors of trophoblastic infl ammasome signaling, and inhibitors of HMG-Co A reductase, deserve to be tested.

Confl ict of interest statement

J-CG received grants and research supports from Sanofi , Léo Pharma France, Stago, and Laboratoire Français de Biotechnologies (LFB).