Louise Ghesquière

Julie Demetz

Philippe Dufour

Sandrine Depret

Charles Garabedian

Damien Subtil

Louise Ghesquiere
email: louise.ghesquiere@chru-lille.fr

Type of breech presentation and prognosis for delivery

Keywords:

Background: Although its role in the prognosis for delivery remains controversial, the type of breech is sometimes taken into account in the decision about mode of delivery. Objective of our study was to compare maternal and neonatal morbidity for trial of vaginal delivery according to the type of breech (complete or frank).

Material and method:

Single-center retrospective study of women with trials of vaginal delivery of a singleton fetus in breech presentation at of after 37 weeks of gestation. Neonatal status was assessed by the composite variable of the Term Breech Trial, first considered alone, and then with the addition of a 5-min Apgar score < 7 or a neonatal arterial pH < 7.0.

Results: Of the 495 trials of vaginal delivery during the study period, approximately one third of them were complete breech (35.8%) and two thirds frank (64.2%). The frequency of cesareans during labor was similar regardless of the type of breech (16.4 for complete vs 12.6% for frank, p=0.24), nor did neonatal morbidity differ (1.7 for complete vs 4.1% for frank, p=0.15). On the other hand, cord prolapse occurred almost exclusively in complete breech presentations (4.5 vs 0.3%, p<0.01), and prognosis was good in all cases. Complete breech presentations were also associated with more frequent use of forceps to the after-coming head (16.2 vs 9.7%, p<0.05). Finally, there were non significant difference between the two types of breech concerning severe acidosis but it seemed to have it more in frank breech (2.3 vs 1.2%, p=0.34).

Conclusion:

Among women eligible for vaginal delivery, the type of breech presentation (complete or frank) has little influence on delivery maternal and neonatal morbidity. The type of breech need not be taken into account in deciding the route of delivery.

Introduction

Although the Term Breech Trial published in 2000 [START_REF] Hannah | Planned caesarean section versus planned vaginal birth for breech presentation at term: a randomised multicentre trial. Term Breech Trial Collaborative Group[END_REF] deeply affected obstetricians' practices in delivering fetuses in breech presentation, countries with low perinatal risk levels generally agree that vaginal delivery is feasible when the appropriate safety conditions are met [START_REF]ACOG Committee Opinion No. 340. Mode of term singleton breech delivery[END_REF][START_REF]Management (Green-top 20b[END_REF][START_REF] Taylor | Is cesarean section indicated for breach presentation?[END_REF][START_REF]Management of Breech Presentation at Term australian guidelines -Ask.com Search[END_REF][START_REF] Kotaska | Vaginal delivery of breech presentation[END_REF]. Some guidelines include the type of breech presentation among these conditions (4,7), while others do not [START_REF]ACOG Committee Opinion No. 340. Mode of term singleton breech delivery[END_REF][START_REF]Management (Green-top 20b[END_REF][START_REF]Management of Breech Presentation at Term australian guidelines -Ask.com Search[END_REF][START_REF] Kotaska | Vaginal delivery of breech presentation[END_REF]. Different types of breech are described: frank breech and incomplete or complete breech. In a frank breech, the fetus has flexion of both hips, and the legs are straight with the feet near the fetal face. The complete breech has the fetus sitting with flexion of both hips and both legs in a tuck position. Finally, the incomplete breech can have any combination of one or both hips extended.

Studies on association of adverse perinatal and maternal morbidity and the posture of the fetal legs in vaginal breech births are scarce and fairly of methodologically inadequate [START_REF] Sentilhes | Breech presentation: Clinical practice guidelines from the French College of Gynaecologists and Obstetricians (CNGOF)[END_REF][START_REF] Collea | The randomized management of term frank breech presentation: a study of 208 cases[END_REF][START_REF] Gimovsky | Randomized management of the nonfrank breech presentation at term: a preliminary report[END_REF][START_REF] Christian | Vaginal breech delivery: a fiveyear prospective evaluation of a protocol using computed tomographic pelvimetry[END_REF][START_REF] Descargues | Influence of the type of breech presentation on delivery in selected primiparous women at term[END_REF]. These do not agree on maternal and neonatal morbidity according to the type of breech. Krause et al. reported a higher rate of short term neonatal morbidity in deliveries out of incomplete and complete breech presentation in a small cohort of 266 patients [START_REF] Krause | What effect does leg position in breech presentation have on mode of delivery and early neonatal morbidity?[END_REF]. The more recent studies are more reassuring [START_REF] De Leeuw | Indications for caesarean section in breech presentation[END_REF][START_REF] Golfier | Planned vaginal delivery versus elective caesarean section in singleton term breech presentation: a study of 1116 cases[END_REF][START_REF] Azria | Factors associated with adverse perinatal outcomes for term breech fetuses with planned vaginal delivery[END_REF][START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF]. These demonstrated no significant feature predictive of obstetrical and neonatal prognosis irrespective of the type of breech. A recent study provided evidence, that perinatal morbidity is not associated with the fetal leg posture in vaginally intended breech deliveries [START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF]. These studies accorded to say that cord prolaps were significantly more often in deliveries with babies in complete breech.

Additional studies are needed to promote safe vaginal delivery of the breech.

Consistent evidence is needed on the role of the fetal leg posture in vaginally intended breech deliveries at term. Because our maternity ward has fairly extensive experience with vaginal deliveries of breech presentations [START_REF] Michel | Evaluation of a decision protocol for type of delivery of infants in breech presentation at term[END_REF], we conducted a study intended to compare delivery maternal morbidity and neonatal morbidity according to the type of breech presentation.

Material and methods

This retrospective study in a level 3 university hospital examined the outcome of trials of vaginal delivery of singleton fetuses in breech presentation at or after 37 weeks of gestation. Women were included in the study if they underwent a trial of vaginal delivery for a fetus in breech presentation between 1 January 2008 and 31 December 2017. The study excluded women with a termination of pregnancy, multiple pregnancy, delivery before 37 weeks of gestation, or in utero fetal death, as well as those with a cesarean before labor, severe prenatal malformation, or unknown type of breech presentation.

Principal objective was to compare delivery maternal morbidity and neonatal morbidity for trial vaginal delivery according to the type of breech presentation: frank or complete. During the study period, we developed and then systematically applied a protocol to select among the women with fetuses in breech presentation those eligible for trial of vaginal delivery, that is, those for whom vaginal delivery appeared safe [START_REF] Michel | Evaluation of a decision protocol for type of delivery of infants in breech presentation at term[END_REF]. Briefly, external cephalic version was offered between 36 +0 and 37 weeks +3days to women with such fetuses. If version failed, pelvimetric measurements by computed tomography and fetal ultrasound were performed to check for primary hyperextension of the fetal head and to compare maternal pelvic measurements with the fetal biparietal diameter and estimated fetal weight.

Women were eligible for trial of vaginal delivery if three conditions were met: i. median conjugate minus biparietal diameter ≥ 15 mm, ii. median transverse diameter minus biparietal diameter ≥ 25 mm, iii. Interspinous diameter minus biparietal diameter ≥ 0 mm. An other condition to accept vaginal delivery was the estimated fetal weight had to be less than 3800g for primiparas women. There was no limit of fetal weight for multiparas women. This decision did not take into consideration the type of breech, previous cesarean, or uterine malformation. Women considered eligible were informed about the conditions of vaginal breech delivery (presence of obstetrical, pediatric, and anesthesiology teams in the delivery room) and of the advantages and risks of both planned vaginal and cesarean deliveries in this situation. When the measurements were unfavorable or the woman refused a trial of vaginal delivery, a cesarean was planned at around 39 weeks. If the biparietal diameter measurements were more than 7 days old at the onset of labor, a new ultrasound measurement was taken to verify the criteria described above. Excepting these differences, the management of labor was identical to this of cephalic fetuses.

Induction and labor management for breech presentation was the same that cephalic presentation. All inductions were possible: balloon, prostaglandine, oxytocine. During labor, oxytocin protocol was the same as cephalic presentation and followed guidelines [START_REF] Dupont | Oxytocin administration during spontaneous labour: Guidelines for clinical practice. Guidelines short text[END_REF]. In case of two vaginal touch same, oxytocine could be used and artificial rupture could be realized. Artificial rupture in complete breech was to medical appreciated. Cervical dystocia was defined by three hours of same vaginal touch despite regular contractions and artificial rupture. In complete dilatation, two hours were possible before begin expulsive efforts. This retrospective study is based on the medical files of parturients and newborns in our hospital. It was approved by the ethics committee for gynecology and obstetrics (CEROG). The assessment of obstetric morbidity considered the following variables: cervical dystocia, defined by three essentially unchanged cervical dilatation measurements, each an hour apart, cord prolapse, cesarean delivery during labor, forceps to the after-coming head, obstetrical anal sphincter injuries (OASIS) (≥ 3rd degree), post-partum hemorrhage ≥ 500 ml and ≥ 1000 ml. To assess neonatal morbidity, we studied birth weight, biparietal diameter, and umbilical artery pH measured at birth, as well as the newborn's condition, transfer to intensive care, and mortality. Neonatal acidosis was considered severe when the pH ≤ 7.0, and moderate at ≤ 7.15. A composite variable matching that used in the Term Breech Trial (and named the TBT variable here) and then in subsequent publications included the following criteria: 5-minute Apgar score < 4, basal skull fracture, intracerebral or subdural hematoma, intraventricular hemorrhage, lesions of the spine marrow or brachial plexus or a peripheral nerve, length of NICU hospitalization > 4 days, intubation > 24 h, parenteral or gastric feeding > 4 days, convulsions persisting > 24 h, death during labor, or neonatal death [START_REF] Hannah | Planned caesarean section versus planned vaginal birth for breech presentation at term: a randomised multicentre trial. Term Breech Trial Collaborative Group[END_REF][START_REF] Michel | Evaluation of a decision protocol for type of delivery of infants in breech presentation at term[END_REF][START_REF] Roman | Planned vaginal delivery of fetuses in breech presentation at term: prenatal determinants predictive of elevated risk of cesarean delivery during labor[END_REF]. A second and broader composite variable specific to our study (labelled neonatal composite endpoint) was also created by adding to the TBT criteria above a 5-min Apgar score < 7 or severe neonatal acidosis (pH<7.0). Finally, neonatal mortality was defined as death during the first 28 days of life.

The data were recorded and analyzed with Epi Info software (Version 3.1, Epidata Association, Denmark). Comparisons between qualitative data used the Chi-2 test or Fisher's exact test, when the number of individuals was low. The Kruskal-Wallis nonparametric test was used to compare means. Percentages are reported in parentheses, and means are reported with the standard deviation of the distribution.

Differences were considered significant if p < 0.05. When a significant difference was found for any criterion, a subgroup analysis by parity was conducted to determine if parity (nulliparous/multiparous) affected the criterion. Last, odds ratio (OR) and 95% confidence interval were calculated for the risk of cesarean delivery during labor according to type of breech.

Results

The study period saw a total of 45 669 deliveries in our maternity ward (Figure 1). In all, 1319 (3.4%) women had singleton pregnancies in breech presentation at or after 37 weeks of gestation. We excluded 28 cases (2.1%) in which severe prenatal malformations might have influenced the prognosis or neonatal management (heart defects: n=7, uropathies: n=5, gastrointestinal abnormalities: n=5, nervous system abnormalities: n=4, other n=7). Our study therefore concerned 1276 women. Of the 511 women eligible to planned vaginal delivery (40.0%), 16 were secondarily excluded: the type of breech was footling or kneeling in 3 cases (0.6%) and unspecified in 13 (2.5%).

Of the 495 women in the study, the presentation was complete breech for approximately one third (n=177, 35.8%) and frank for two thirds (n=318, 64.2%).

Table 1 reports the variations in the frequency of complete breech presentations according to the patients' characteristics. This frequency was not associated with maternal age. On the other hand, complete breech presentations were more frequent in multiparous than nulliparous women, as well as in those with a body mass index (BMI) of 25 or higher. The frequency of complete breech presentations was not associated with known uterine malformations, uterine scars, or the results of the assessment described above comparing fetal and pelvic measurements. Gestational age at delivery was around 39 weeks for all women (Table 2). The frequency of cervical dystocia was similar for both principal types of breech presentation, as was the rate of cesareans during labor (16.4 for complete vs 12.6% for frank, OR=1.4 [0.8-2.4]). Inversely, cord prolapse occurred almost exclusively in complete breech cases, and the difference was highly significant (4.5 for complete vs 0.3% for frank, p=0.002). In case of cord prolapse, all neonatal outcomes were good with Apgar score=10 at 5 minutes, no severe acidosis and no transfers in NICU. All patients had cesarean section. Use of forceps for the after-coming head was significantly more frequent in the complete breech presentations (16.2% for complete vs 9.7% for frank p=0.049). OASIS were almost exclusively observed in complete breech births in nulliparas and were significantly associated with them (4.1% for complete vs 0.4% for frank, p=0.008).

Table 3 summarizes the data for neonatal outcome according to type of breech. The groups did not differ for neonatal measurements. Concerning umbilical artery pH, in frank breech newborns of nulliparous women had more often pH in moderate acidosis (< 7.15) with 32.2% for frank and 15.1% for complete, p=0.006. There were no significant difference between the two types of breech concerning severe acidosis but it seemed to have it more in frank breech (2.3% vs 1.2%, p=0.34). The frequency of severe neonatal acidosis (pH < 7.0), neonatal transfer, and the composite variables we studied -TBT and neonatal composite variables -did not differ by type of breech. About 1.7% (3/177) of the newborns in complete breech presentations met at least one criterion for the neonatal composite endpoint, and 4.1% of those in frank breech presentation (13/318) (p=0.15). About different variables of TBT and neonatal composite endpoint, in complete breech, two newborns had Apgar score<7 and 2 had severe acidosis. In frank breech, 5 had Apgar score<7, 7 had severe acidosis, 2 had brachial plexus and one was transferred to NICU more 4 days with an intubation more 24 hours. Six infants were transferred to our neonatal intensive care unit; all from the frank breech group (0.0 vs 1.9%, p=0.07). The reasons for neonatals transfers were: two severe acidosis in fetal growth restriction contexts, two fetal growth restrictions with postnatal hypoglycaemia and respiratory distress, one foeto-maternal alloimmunization and one fetal pneumothorax. Finally, no baby died.

Discussion

The latest French recommendations are reassuring regarding intented vaginal breech deliveries. They did not find an increase in neonatal morbidity whatever the type of breech but the rate of cesarean section would be increased in complete breech, with a low level of evidence. The choice of delivery route must be shared by the patient and the obstetricians. A good knowledge of maternal and neonatal morbidity according the type of breech seem essential to properly guide patients in their choice.

Few studies in the literature have compared maternal and neonatal morbidity in vaginal delivery according to the type of breech [START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF]. Our study shown that these differ little according to complete or frank breech presentation. The rate of cesareans during labor was similar according to type of breech. There were nonetheless several differences that may further our knowledge of these deliveries. Cord prolapse, forceps, and OASIS are more frequent in complete breech presentations. About neonatal morbidity, there were no difference concerning severe acidosis, TBT composite variable and neonatal composite variable. There were more transfers in NICU and more moderate acidosis in frank breech.

Our study found approximately two thirds (64.2 %) of planned vaginal deliveries were frank breech, consistent with the proportion found by most authors who have studied it [START_REF] Hannah | Planned caesarean section versus planned vaginal birth for breech presentation at term: a randomised multicentre trial. Term Breech Trial Collaborative Group[END_REF][START_REF] Gimovsky | Randomized management of the nonfrank breech presentation at term: a preliminary report[END_REF][START_REF] Descargues | Influence of the type of breech presentation on delivery in selected primiparous women at term[END_REF][START_REF] Broche | Obstetric and perinatal outcomes of a disreputable presentation: the nonfrank breech[END_REF][START_REF] Krause | What effect does leg position in breech presentation have on mode of delivery and early neonatal morbidity?[END_REF][START_REF] De Leeuw | Indications for caesarean section in breech presentation[END_REF][START_REF] Golfier | Planned vaginal delivery versus elective caesarean section in singleton term breech presentation: a study of 1116 cases[END_REF][START_REF] Azria | Factors associated with adverse perinatal outcomes for term breech fetuses with planned vaginal delivery[END_REF][START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF][START_REF] Michel | Evaluation of a decision protocol for type of delivery of infants in breech presentation at term[END_REF][START_REF] Roman | Planned vaginal delivery of fetuses in breech presentation at term: prenatal determinants predictive of elevated risk of cesarean delivery during labor[END_REF][START_REF] Goffinet | Is planned vaginal delivery for breech presentation at term still an option? Results of an observational prospective survey in France and Belgium[END_REF]. We observed frank breech presentations significantly more frequently in nulliparous patients. Although the uterus of these patients is often thought to be cylindrical and tonic. Inversely, the uterus of multiparas is thought to be larger and less tonic and thus to promote, to a slight extent, complete breech presentations. Broche DE et al. in 2005 published a serie of 198 complete breech in which nulliparas are less than 25%. So, non frank breech are far more frequent in multiparas [START_REF] Broche | Obstetric and perinatal outcomes of a disreputable presentation: the nonfrank breech[END_REF].

Our study indicated that the frequency of cesarean deliveries during labor is not influenced by whether the breech is complete or frank. The literature on this topic is discordant. Several authors reported that a complete breech presentation is significantly associated with an increased risk of cesarean delivery during labor [START_REF] Krause | What effect does leg position in breech presentation have on mode of delivery and early neonatal morbidity?[END_REF][START_REF] Golfier | Planned vaginal delivery versus elective caesarean section in singleton term breech presentation: a study of 1116 cases[END_REF]. Inversely and like us, de Leeuw et al found no significant increase in the risk of cesarean delivery during labor in complete breech presentations [START_REF] De Leeuw | Indications for caesarean section in breech presentation[END_REF]. In contrast, Jennewein et al found no significant decrease in the risk of cesarean delivery during labor in complete breech presentations [START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF]. Our rate of cesarean was low in contrast to literature (Table 4), probably because in our center all obstetricians are trained to practice vaginal delivery in case of breech presentation. The non-significant increase in the risk of cesareans during labor for complete breech presentations was almost totally due to the cases of cord prolapse, all of which led to cesareans. That was, this increase for the complete breech cases in our study was approximately 4% (16.4 vs 12.6%), which almost exactly matches the percentage of cord prolapse observed in that type of presentation (4.5%). Among these studies, Jennewein and us are the only studies whose main objective was to compare maternal and neonatal morbidity according to the type of seat. Even if a lack of power is possible in our study, it does not seem, in regards to these two studies, have significant difference concerning the rate of cesarean section according to the type of seat. Also, our study showed a significant increase in the risk of cord prolapse in complete breech presentations. Breech presentation is reported to be a risk factor for cord prolapses [START_REF] Gimovsky | Randomized management of the nonfrank breech presentation at term: a preliminary report[END_REF][START_REF] De Leeuw | Indications for caesarean section in breech presentation[END_REF][START_REF] Golfier | Planned vaginal delivery versus elective caesarean section in singleton term breech presentation: a study of 1116 cases[END_REF][START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF][START_REF] Cheng | Breech delivery at term: a critical review of the literature[END_REF]. In a meta-analysis of 24 publications bringing together more than 7800 women with trials of vaginal delivery for breech presentation, Cheng et al found an overall rate of cord prolapse of 7.4% in breech presentations, with a frequency very clearly higher for complete (5-10%) compared with frank (0-2%) breech presentations [START_REF] Cheng | Breech delivery at term: a critical review of the literature[END_REF]. This difference in risk may well be related to the crosslegged position of the fetus in this type of presentation, for a loop might passe between or around the fetus's legs. In our study, all incidents of cord prolapse were followed by cesarean deliveries, and neonatal status was excellent in all cases. Even if it was not our practice, vaginal delivery is possible in cord prolapse. Gannard-Pechin et al. published a serie of cord prolapse and they concluded that cord prolapse in breech delivery could be safer and quicker managed vaginally (23).

Forceps were applied to the after-coming head significantly more frequently in fetuses in complete breech presentations (16.2%). We have not found this result elsewhere in the literature. Jennewein et al in their study found an elevated rate for assistance head delivery and more in complete breech (45.7 for complete vs 35.5% for frank, p=0.001) but they not detailed rate of forceps [START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF]. Descargues et al found that Mauriceau's maneuver was performed more often for complete breech infants [START_REF] Descargues | Influence of the type of breech presentation on delivery in selected primiparous women at term[END_REF]. This result is similar to us because in our center we don't do Mauriceau's maneuver but forceps. This finding points suggests that delivering the head may be more difficult in these cases. Jennewein et al had hypothesis that these might be explained by a shorter second stage of labor in complete breech cases caused by early manipulation. So baby not having enough time to rotate adequately in the pelvis leading to more obstetrical interventions and ultimately to assisted arm and head delivery [START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF]. Our higher forceps rate could explain why our OASIS rate was also higher in contrast to the literature [START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF].

Type of breech was not associated with the risk of severe neonatal morbidity, regardless of the composite variable we used to define it (TBT composite variable, Apgar score < 7 and severe acidosis). No neonatal death was observed. This result is similar to earlier published data. First, De Leuw et al studied neonatal morbidity and mortality according to type of breech in women eligible for a trial of vaginal delivery [START_REF] De Leeuw | Indications for caesarean section in breech presentation[END_REF]. Three groups were defined according to birth weight (500-1499 g; 1500-2499 g, and ≥ 2500 g). No statistically significant difference was found for the neonatal outcome according to type of breech or mode of delivery in any of the three weight categories [START_REF] Broche | Obstetric and perinatal outcomes of a disreputable presentation: the nonfrank breech[END_REF]. Golfier et al defined neonatal distress at 5 minutes of life by a 5-min Apgar score < 7 or the need for resuscitation in the delivery room and found no significant difference according to type of breech for neonatal distress at 5 minutes [START_REF] Golfier | Planned vaginal delivery versus elective caesarean section in singleton term breech presentation: a study of 1116 cases[END_REF]. Jennewein et al found no difference in the neonatal morbidity rates between frank and complete breech in intented vaginal deliveries [START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF]. Finally, Azria et al, studying neonatal prognosis in 2502 trials of vaginal delivery after 37 weeks, found four factors associated with death or poor neonatal condition: gestational age ≤ 38 weeks, birth weight < 10 th percentile, mother's geographic origin, and size (small) of the maternity ward [START_REF] Azria | Factors associated with adverse perinatal outcomes for term breech fetuses with planned vaginal delivery[END_REF]. In this large-scale study, the type of breech was not associated with the risk of poor neonatal condition.

It seemed to have more transfers in NICU in frank breech (1.9 for frank vs 0% for complete, p=0.07). Likewise, the risk of severe acidosis (pH<7.0) was similar regardless of the type of breech but it seemed to have it more in frank breech (2.3 in frank vs 1.2% in complete, p=0.34). We found more pH<7.15 in frank breech. Descargues et al observed the same trend among 146 nulliparous women with trials of vaginal delivery: they found frank breech presentation to be associated with a slight excess rate (on the borderline of statistical significance) of cases with pH < 7.20, but no excess of infants in poor neonatal condition [START_REF] Descargues | Influence of the type of breech presentation on delivery in selected primiparous women at term[END_REF]. We therefore hypothesize that the frank breech presentation may be accompanied -in nulliparous women -by a risk of moderate acidosis and transfer in NICU but without more severe inconsequential.

Finally, our study, based on a large series in a center with obstetricians trained in vaginal breech delivery, confirmed that maternal and neonatal prognosis in cases of trial of vaginal delivery do not differ much according to the type of breech. This finding is consistent with those of older less supported studies [START_REF] Collea | The randomized management of term frank breech presentation: a study of 208 cases[END_REF][START_REF] Gimovsky | Randomized management of the nonfrank breech presentation at term: a preliminary report[END_REF][START_REF] Christian | Vaginal breech delivery: a fiveyear prospective evaluation of a protocol using computed tomographic pelvimetry[END_REF] as well as newer studies that are consistent with one another [START_REF] Broche | Obstetric and perinatal outcomes of a disreputable presentation: the nonfrank breech[END_REF][START_REF] De Leeuw | Indications for caesarean section in breech presentation[END_REF][START_REF] Golfier | Planned vaginal delivery versus elective caesarean section in singleton term breech presentation: a study of 1116 cases[END_REF][START_REF] Azria | Factors associated with adverse perinatal outcomes for term breech fetuses with planned vaginal delivery[END_REF][START_REF] Jennewein | The influence of the fetal leg position on the outcome in vaginally intended deliveries out of breech presentation at term -A FRABAT prospective cohort study[END_REF][START_REF] Michel | Evaluation of a decision protocol for type of delivery of infants in breech presentation at term[END_REF]. Even if it is a large database, some results may be no significant due to a lack of power. This is more of a retrospective study. To date, no prospective study has specifically compared breech delivery according to the type of breech.

Conclusion:

Like the 2020 French guidelines [START_REF] Sentilhes | Breech presentation: Clinical practice guidelines from the French College of Gynaecologists and Obstetricians (CNGOF)[END_REF], our study indicated that the type of breech didn't appear to be a particularly relevant criterion for determining mode of delivery of a fetus in breech presentation. We conclude that the type of breech should not be used as a criterion for determining eligibility for trial of vaginal delivery in cases of breech presentation at term.

Figure 1 .

 1 Figure 1. Flow Chart

Table 1 . Frequency of complete breech according to pregnancy characteristics

 1 BMI, body mass index. **see Material and Methods. °For one patient we didn't have age and for 4 patients we didn't have pelvic cephalic comparaison. The table is read line by line. Results are in n/N (%) (complete breech / all breech). p is significant if <0.05

	Frequency of Complete	p
	breech / All Breech
	n/N (%)

*

Table 2 . Maternal outcome according to type of breech

 2 PROM = premature rupture of the membranes. **These items are just for vaginal delivery. °Obstetrical anal sphincter injuries. The table is read line by line. Results are in mean +/-standard deviation or in n (%). p is significant if <0.05

	Complete N=177	Frank N=318	p
	n (%)	n (%)

*

Table 3 . Neonatal outcome according to type of breech

 3 * NICU = neonatal intensive care unit. *** Neonatal composite variable = TBT composite variable or pH<7 or 5-min Apgar score < 7. (1): see Material and methods. The table is read line by line. Results are in n (%) or in mean +/-standard deviation. p is significant if <0.05

	Complete N=177	Frank N=318	p
	n (%)	n (%)

* BPD = Biparietal diameter. *

Table 4 . Risk of cesarean delivery during labor according to type of breech. Analysis of the literature.

 4 ° Reference. * OR significant. Results are in n/N (%)

	Publications	Complete	Frank	Odds Ratio
		n/N (%)	n/N (%)


MD Vincent Balaya
email: vincent.balayagouraya@aphp.fr

Fabien Guimiot

Matthieu Bruzzi

Salma El Batti

Alexis Guedon

Martin Lhuaire

Jean-Marc Chevallier

Richard Douard

Jean-François Uhl

Feasibility of a fetal anatomy 3D atlas by computer-assisted anatomic dissection

Keywords: "Anatomy", "Computer-assisted anatomic dissection", "Embryology", "Fetal pelvis", "Fetal brain", "Fetus", "Morphogenesis"

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

Introduction

According to the French Public Health Institute, the congenital defects were reported to 3.4% of newborns [START_REF]malformations-congenitales[END_REF]. Knowledge of normal human development is of paramount importance for prenatal diagnosis of congenital malformations and may also help understanding surgical anatomy in adult [START_REF] Baskin | Anatomical studies of the human clitoris[END_REF][START_REF] Kraima | Lymphatic drainage pathways from the cervix uteri: implications for radical hysterectomy?[END_REF].

Current resources on human development are usually based on textbooks and precursive embryologist work on animal models. Moreover, teaching embryologic development to medical students remained a pedagogic challenge due to the lack of subjects and the complexity of each mechanisms [START_REF] Abid | Traditional versus threedimensional teaching of peritoneal embryogenesis: a comparative prospective study[END_REF][START_REF] Arraez-Aybar | A computerised technique for morphometry and 3D reconstruction of embryological structures[END_REF].

The Computer-Assisted Anatomic Dissection (CAAD) is an original method of virtual dissection which consists in a combination of immunohistochemically staining with 3D reconstruction of serial histologic sections or radiologic slices. The 3D reconstruction provides a better understanding of complex anatomic structures and clarifies their relationship with neighbouring organs. The CAAD may overcome the limitations imposed by classical anatomic dissection and provides an interesting approach for describing the fetal anatomy.

The aim of this study was to describe the CAAD applied to fetal anatomy and to assess the feasibility of a 3D fetal anatomy model.

Material and Methods

3D modelization were focused on fetal brain, pelvic innervation and lower limbs vascularisation. These 3 regions were chosen to fully describe each type of anatomical structure and specific technique of 3D modelisation: bones and muscles were described on lower limbs whereas vessels and nerves were described on lower limbs and pelvis. Description of venous network is of clinical interest for diagnosis and surgical treatment of congenital vascular malformations [START_REF] Lee | New approaches to the treatment of congenital vascular malformations (CVMs)--a single centre experience[END_REF][START_REF] Paolacci | Vascular anomalies: molecular bases, genetic testing and therapeutic approaches[END_REF]. Knowledge of pelvic innervation has a surgical impact in nerve-sparing technique for rectal and gynecologic cancer surgeries [START_REF] Peschaud | Preservation of genital innervation in women during total mesorectal excision: which anterior plane?[END_REF][START_REF] Balaya | Anatomical consideration for the technique of nerve-sparing during radical hysterectomy for cervical cancer[END_REF]. Based on magnetic resonance imaging (MRI), fetal brain was modelised to describe the gyration process and because of its clinical interest in prenatal diagnosis of brain development disorders.

Study population

According to a specific protocol, this study conformed to that required examination and authorization of the French Biomedecine Agency (Articles L. 1241-5 and R. 1241-20 of the French Code of Public Health) and was recorded under reference PFS14-017. A consent form for fetal autopsy was signed by the parents (Articles. L. 1232-2, L. 1241-6, and L. 1241-5 of the French Code of Public Health).

The gestational age of each fetus was determined by the Crown-Rump Length (CRL) measurement at the first-trimester ultrasound. The dating was confirmed during fetal autopsy by the measure of the fetal heel-to-toe length, the fetal aspect and the estimation of organ maturation at autopsy [START_REF] Hern | Correlation of fetal age and measurements between 10 and 26 weeks of gestation[END_REF].

For the pelvis and lower limbs study, fetal subjects came from legal abortion, medical pregnancy termination and late miscarriage. For the brain study, MRI were performed in utero or post-mortem after late miscarriage. On macroscopic examination, fetuses were free of abdominal, pelvic or limb malformations and were not macerated. In the region under examination, no prior pathology was report.

Study protocol

Macroscopic dissection and immunohistochemistry

The whole pelvis, containing all the intra pelvic organs and surrounding pelvic bones, were completely dissected from the sacral promontory to the pubic arch. The limbs and legs were cut at the level of the coxo-femoral articulation.

Firstly, the specimens were fixed in formalin for 48-72 h. During 7 to 10 days, EDTA were used to decalcify the bones. Due to knee angulation, 2 or 3 intra-specimen straight needles were added in sagittal axe to facilitate superposing the histological slides in the subsequent alignment step for lower limbs. Then, the whole pelvis was transversally cut in block of 5 mm each and the whole limb in block of 10 mm each. These blocks were inserted in baskets of 40 x 25 x 10 mm and were embedded with paraffin. A binocular microscope was used to orient the limb perpendicular to the cutting plane of the block. Thirdly, series of transverse cross-section of 5 μm thickness using a microtome every 100 μm of interval were made on each block. Each block was divided in ribbons which were constituted by 5 consecutive slices for the pelvis and 10 consecutive slices for the limb.

A total number of 2000 of slices was obtained for each limb and 133 to 575 slices for each pelvis depending on the age and size of each specimen. For the pelvis, a conventional histological examination with HES was performed on the 1st and the 5th slice in order to distinguish muscular from connective tissue, an immunostaining was done on the 2 nd and 3rd slice and the last one was unstained and stocked at 4°C for further examination. For the limbs, the 1st slice was stained using HES, the 2 nd slice was stained using Masson Trichrome, the 3rd and 4th slices were stained using Protein S100 and the six unstained slices of each series of ten were stored at 4°C. Slices numbering was done in the order they were cut.

Immunolabelling was done by using anti-Protein S100 Protein S100 is a marker of cells from neuro-ectodermic origin that specifically identifies the nucleus and cytoplasm of Schwann cells in formalin-fixed paraffin-embedded nerve tissue [START_REF] Stefansson | Distribution of S-100 protein outside the central nervous system[END_REF]. This immunolabeling detects the distribution of nerve fibers and nerve endings. D2-40 is a marker of endothelial cells that we used for identifying lymphatic and vascular structure.

Fetal brain MRI protocol

Fetal brain MRI was performed on post-mortem or in-utero. Among the fetal brain MRI examinations performed, 5 fetuses were selected with a gestational age of 19 to 37 WG. All fetuses presented normal findings on sonographic brain examinations. In 2 cases, MRI was performed in postmortem just after late miscarriage and macroscopic examinations did not reveal any abnormalities. In 3 cases, MRI imaging was performed in utero within standardized exploration protocols for fetal indications secondary to maternal toxoplasma primo-infection. MR images were acquired using a 1-T superconducting magnet (Panorama 1.0T, Philips ® ) and slices were always orthogonal to the fetal brain. Two types of sequences were used: gradient-echo T2weighted sequences were performed for post-mortem brain MRI with parameters of 1500/250 (TR/TE), a flip angle of 90, a slice thickness 2 mm and gradient-echo T2-weighted sequences were performed for inutero brain MRI with parameters of 15000/120 (TR/TE), a flip angle of 90, a slice thickness 4 mm. We considered that a sulcus appeared as a clear indentation at the surface of the brain.

Conception and images treatment

Stained and immunolabelled histologic slices were digitalized at a resolution of 9,600 dots/inch. These high-resolution details permitted to analyse very fine structure under appropriate high magnifications (x4 to x20). Given that the low interval of 5 μm between the slices was insignificant, sections were almost at the same level and so stackable. Each digitalised section was saved in JPEG format and were edited with Adobe Photoshop CC ® (V2014.2.1 Adobe systems, San Jose, USA) The contrast and the brightness of each digitalised slice were adjusted as their alignment. The alignment process is essential because a misalignment between the slices leads to a progressive drift of the serial section stack and alter the 3D reconstruction with an undented and "zigzag" aspect. The alignment axis was defined by selected anatomical landmarks such as the vertebral column, spinal cord and iliac bones for the pelvis and the nerve stained by antibodies anti PS-100 for the limbs. The alignment was realised by superimposing slice by slice using a semi-transparent layer and a combination of translation and rotation, the preceding image serving as a reference for the next one (Supplementary material 1).

The MRI slices were extracted by using the software Osirix ® Lite (V11.0.3, Bernex Switzerland) and files were also converted in JPEG format. The alignment process was automatically done, and contrast or brightness adjustment were unnecessary.

Three-dimensional reconstruction

To perform the 3D reconstruction, the software WinSurf ® (version 4.3) was used by manual segmentation and building 3D vectorial models. The 3D scale parameters were setup. The "slice width'' in a X and Y plane and the ''slice thickness'' in the Z scale were determined. The slice numbers interval used in the labeling (''slice interval'') was recorded. The 3D reconstruction consisted in the following steps:

(1) Main anatomical structures are manually outlined (Supplementary material 2)

(2) Geometrical alignment to adjust the contours of stacked points.

(3) Converting the series of point into polygons called splines (wire-framed object).

(4) Modeling of the surfaces by meshing the splines and smoothing the contours of the object reconstructed from points (surface rendering)

(5) Applying a realistic texture on the surface and finally getting and a 3D realistic model (Texturing process). ( 6) 3D interactive visualization of the reconstructed structures.

The 3D model can be mobilized in the x-, y-and z-axes. The topography of the different organs, nerves and vessels can be analysed by transparency as an animated motion picture (Supplementary material 3).

The whole process is summarised in Figure 1.

Results

CAAD was performed on 10 fetuses between 13 and 37 weeks of gestation (WG). Different structures were modelised as following: pelvis from 3 fetuses of 13, 15 and 24 WG, lower limbs from 2 fetuses, 14 and 15 WG and brain from 5 fetuses aged between 19 and 37 WG. Fetal population characteristics are presented in Table 1.

CAAD of fetal pelvic innervation

The superior hypogastric plexus (SHP) was in front of the body of L5 for the fetus 1 (13 WG) and in front of the promontory for the two others (15 and 24 WG) as adult. Underneath the promontory, the SHP caudally and laterally divided into two hypogastric nerves. Hypogastric nerves (HN) descended along the lateral side of the rectum, then ran postero-medially to the ureter until the superior angle of the Inferior Hypogastric Plexus (IHP) (Figure 2). Pelvic splanchnic nerves (PSN) emerged from ventral rami of S2, S3

and S4 and sacral splanchnic nerve (SSN) came from sympathetic trunk of S2, S3 and S4, reaching the dorsal edge of the IHP. Fibers from HN, SSN and PSN joined to constitute the IHP. The 3D modelisation highlighted that IHP has a triangular form with a posterior base and an anterior inferior top and was located in a sagittal plane. The IHP appeared on the sections between the second and fifth sacral vertebrae and extended from the anterolateral face of the rectum, passed lateral to the cervix and the vaginal fornix, and attempts to the lateral vaginal wall and the base of the bladder. Efferences of the IHP were constituted by three main plexuses: vesical plexus, vagino-rectal plexus and inferior rectal plexus.

CAAD of fetal Lower limbs venous system

In the 3D model of the right limb of the 15 weeks old fetus, the femoral vein followed the artery inside the femoral canal and was formed by a plexus of 2-3 veins (diameter from 200 to 300 μm) much smaller than the axial vein. The axial vein ran along the sciatic nerve at the level of the thigh and reached a diameter of 700 μm. The axial vein joined the deep femoral vein at the root of the limb. At the calf level, the sural nerve travelled along the small saphenous vein. Furthermore, the saphenous nerve was close to the great saphenous vein in the middle portion of the knee and the leg. As a general rule, the large diameter of all the nerves, in particular the two plantar nerves on the sole were observed.

Similarly, the study of the left lower limb of the 14 weeks old fetus put the stress on a large axial vein (diameter = 500 μm) following the sciatic nerve at the level of the thigh. In contrast, only a small plexus of 3-4 veins (diameter from 100 to 200 μm) constituted the femoral vein traveling with the femoral artery inside the femoral canal. On the opposite limb, the same elements were found, namely 2 veins versus 1 artery forming the deep vascular pedicle (Figure 3).

CAAD of fetal brain

The sequential identification of each brain sulci was noted. At 19 WG, the fetal brain appeared smooth with a few gyri but the Sylvian fissure, the interhemispheric and parieto-occipital sulci were already visualized (Figure 4). At 22 WG, the Sylvian fissure was deeply dug. The calcarine fissure, the cingular and marginal sulci were individualized. At 30 WG, the superior and inferior frontal sulci, the superior temporal sulci and precentral and postcentral sulci were identified. At 37 WG, the operculization of the sylvian fissure was achieved and insular and cingular gyri were constituted. Superior and inferior temporal sulci and occipitotemporal sulcus were also individualised (Figure 5). Lateral ventricles had the same aspects as found in adults. However, due to MRI low resolution, secondary and tertiary sulci were not precisely seen.

Discussion

In this study, we described fetal anatomy 3D modelization by CAAD technique and we highlighted that a fetal 3D anatomy was feasible. Fetal specimen present numerous practical advantages compared to adult for studying very fine structure. The size of the fetus allows to analyse a whole anatomical structure in one histological slice and immunolabelling is easier with automates. The CAAD is a fully reliable and less onerous technique for microdissection. 3D reconstruction by CAAD permits to precisely localize very thin structures such as nerve fibers of 25 μm-diameter and their relationships thanks to highresolution digitalization system that allows X40 optical zoom. With the increasing of data processor capabilities, the current power of computer and the development of new software, complex anatomical structures and regions can be easily reconstructed in 3D for anatomical and clinical studies. This modelization reflects the true anatomy and each structure can be analysed one by one or simultaneously by mobilization in the three axes, from different angles and opacity adjustment allowing a virtual dissection.

After eight weeks of gestation, corresponding to Carnegie stage 23, pelvic innervation is mature and does not undergo fundamental changes during growth in fetal and post fetal periods [START_REF] Fritsch | Topography of the pelvic autonomic nerves in human fetuses between 21-29 weeks of gestation[END_REF][START_REF] Arango-Toro | Development of the pelvic plexus in human embryos and fetuses and its relationship with the pelvic viscera[END_REF]. This difference of vertebral level is due to the difference in age between the different fetuses. Initially the IHP is a ganglionic mass that comes from the pre-aortic plexus at Carnegie stage 16 and then migrate progressively in an inferior direction to reach its final position [START_REF] Arango-Toro | Development of the pelvic plexus in human embryos and fetuses and its relationship with the pelvic viscera[END_REF]. We found that the female IHP had a triangular form with a posterior base and an anterior inferior top as described by Arango-Toro and Hounnou [START_REF] Arango-Toro | Development of the pelvic plexus in human embryos and fetuses and its relationship with the pelvic viscera[END_REF][START_REF] Hounnou | Morphometry by computerized three-dimensional reconstruction of the hypogastric plexus of a human fetus[END_REF] whereas Alsaid et al. described the IHP as a quadrangular shape [START_REF] Alsaid | Coexistence of adrenergic and cholinergic nerves in the inferior hypogastric plexus: anatomical and immunohistochemical study with 3D reconstruction in human male fetus[END_REF]. The 3D modelization of pelvic innervation in fetus has been used to determine the anatomic risk-zone of pelvic nerve-injury in rectal and gynecologic surgery [START_REF] Moszkowicz | Where does pelvic nerve injury occur during rectal surgery for cancer?[END_REF][START_REF] Balaya | Pelvic nerve injury during radical hysterectomy for cervical cancer: key anatomical zone[END_REF].

In the lower limbs, a large axial vein was highlighted adjacent and parallel to the sciatic nerve. This vein appeared bigger than the plexiform femoral vein and should be considered as the principal vessel of the thigh at the end of organogenesis. In most of cases in adult, this large vein becomes nonetheless hypoplastic being limited to a small venous arcade contrary to the femoral vein, located with the artery and the femoral nerve inside the femoral canal, which becomes the main vein of the limb. These findings are concordant with the "angio-guiding nerves" concept which states that there is an interaction between angiogenesis and neurogenesis [START_REF] Uhl | Embryology and three-dimensional anatomy of the superficial venous system of the lower limbs[END_REF] and may help identifying at the doppler ultrasound the highrisk zones for the treatment of chronic venous disease [START_REF] Uhl | Anatomy and embryology of the small saphenous vein: nerve relationships and implications for treatment[END_REF].

According to a specific chronology determined by pathologic analysis, gyri and sulci reflect the fetal brain maturity [START_REF] Fees-Higgins | Le developpement du cerveau foetal humain=[END_REF][START_REF] Larroche | The development of the central nervous system during intrauterine life[END_REF]. Our results were concordant to those found in the literature, but our small number of subjects did not permit to extrapole them. Moreover, the small number of slices limited the delineation of tertiary sulci. In utero fetal imaging raises some difficulties especially due to artefacts linked to natural living fetal movement during acquisition. Due to MRI low-resolution, intracerebral structures such as thalami or callosum corpus were not described. To highlight brain vascularisation in post-mortem subjects, gadolinium injection through the umbilical cord may be helpful. We feel that 1T MRI would not be an option for earlier stages of fetal development and more efficient MRI would provide better results as described for heart anatomy with a 4.7 T research MRI [START_REF] Blondiaux | Evaluation of septal insertion of atrioventricular valves in fetuses by postmortem 4.7 Tesla cardiac MRI: A feasibility study[END_REF]. Appropriate post-mortem analysis, even if this process requires a long time to achieve, emerges as the best solution for a fine analysis of the brain structures. An alternative way to improve this modelisation could be to digitalize existing collection of fetal brain specimens or to initiate collaborative work with prenatal diagnosis centers with a high level of expertise in the fetal brain.

From a technical point of view, getting slices perpendicular to the main axis of the structures is the best way for an anatomical reconstruction by making the slices alignment easier. Due to the loss orientation after slices cutting, one of the main technical problem is the manual realignment process. The transverse cross-section realized with the microtome can result in distortion, detachment or narrowing of the different slice obtained. To overcome this problem, fiducial landmarks included in the paraffin block may be used such as straight needles. The Winsurf ® software has an alignment function but restricted to object by object and does not permit a global alignment process. A great improvement of this procedure could be an automatic alignment that prevents progressive sliding of the pile of sections and is more precise, but it is rarely feasible because of the lack of contrast between the different tissues. Manual contouring is a step which is highly time-consuming. 20 structures were rebuilt for each specimen on each slice, such as more than 10.000 surrounded cycles were realized. Although high resolution slices were obtained, manual contouring is a subjective process that requires an anatomical expertise and accuracy to build a valid 3D reconstruction. Moreover, some structure cannot be reconstructed such as the ligament because of the high density of adipose tissue or as pineal gland due to low resolution of MRI. To resolve these inconvenient, informatic algorithms should be developed to automatically recognize and surround these anatomic structures but it necessitates an adequate density gradient between the surrounding tissues. However, this technique is precise and could be used easily by non-professional computer users such as surgeon or radiologists. This technique could hardly be used in everyday practice and should remain in research area.

The major limitation of our study was the small number of specimens which cannot take in account interindividual variations and reduces the extrapolation of our results. This low number was mainly due to legal restrictions and difficulties to obtain specimen. Several inclusions were not possible due to an absence of signature of consent (or to a refusal of consent) for fetal autopsy, mostly because of the absence of appropriate information to the parents. The delivery time of specimen to the fetal pathology department exceeded sometimes 24 hours causing a maceration of tissue and thus immunolabelling could not be performed any more.

The 3D modeling by CAAD represents a useful anatomical educational tool for medical students [START_REF] Uhl | 3D modeling in the field of morphology: methods, interest and results[END_REF][START_REF] Uhl | Three-dimensional reconstruction of urogenital tract from Visible Korean Human[END_REF]. These 3D tools assets could be used on graphic tablet as a new teaching way of Anatomy and embryology complementary to cadaver dissection and blackboard chalk drawings. For the study of organogenesis, each embryonic stage could be reconstructed in three dimensions as a dynamic process (4). The 3D modelization is a consistent method of teaching because these data can be saved and can constitute electronic atlases available online through institution site. To assess the impact of such 3D model as a pedagogic tool, a controlled study should be performed among medical students [START_REF] Balaya | 3D modeling of the female pelvis by Computer-Assisted Anatomical Dissection: Applications and perspectives[END_REF].

CAAD technique is an original method that provides an accurate 3D reconstruction of fetal anatomy for lower limbs and pelvis but has to be improved for brain model since midline structures were not amendable for analysis. These data may be useful for comparison between normal and pathologic fetal anatomy. This 3D model constitutes an innovative tool for the teaching of fetal anatomy. Modelisation of other fetal structures such as the heart or the liver remained uncharted territories and might bring new challenges. The present results are encouraging but require to be confirmed using a larger series of specimens at different stages of embryonic and fetal development.

Figures

Figure 1 .

 1 Figure 1. General workflow of the Computer-Assisted Anatomical dissection process.

Figure 2 .

 2 Figure 2. 3D reconstruction of a fetus of 13 weeks of gestation. Anterior view. The Superior HypogastricPlexus is located in front of L5 and is higher than its usual localization. Uterus and bladder are hidden.

Figure 3 .

 3 Figure 3. Three-dimensional reconstruction of a 15 weeks old fetal right lower limb with muscle (a) and without muscles (b). 1 sciatic nerve, 2 axial vein, 3 femoral vein and artery, 4 great saphenous vein, 5 saphenous nerve, 6 small saphenous vein, 7 sural nerve, 8b fibular nerve, 8 tibial nerve, 9 medial plantar nerve, 10 lateral plantar nerve, 11 posterior tibial artery and two veins, 12 tendon of the hallux flexor longus, 13 flexor digitorum longus, 14 posterior tibial tendon, 15 tendons of peronaeus longus and brevis

Figure 4 .Figure 5 .

 45 Figure 4. Three-dimensional reconstruction of a fetal brain of 22 weeks of gestation -A. Right lateral view -B. Left lateral view -C. Antero-inferior view -D. Superior view -E. Posterior view. -F. Right lateral view with lateral ventricle -G. Antero-inferior view with lateral ventricle. The yellow arrow corresponds to the lateral scissure and the blue arrow corresponds to the calcarine scissure. Ventricles are blue

TableTable 1 .

 1 Population study characteristics.

Table 1 .

 1 Population study characteristics. MTP: Medical pregnancy termination

		Fetus 1	Fetus 2	Fetus 3	Fetus 4	Fetus 5	Fetus 6	Fetus 7	Fetus 8	Fetus 9	Fetus 10
	Age (WG)	14	15	13	24	15	19	22	30	36	37
	Gender	Male	Male	Female	Female	Female	Male	Female	Male	Male	Male
	Anatomic region	Lower limb	Lower limb	Pelvis	Pelvis	Pelvis	Brain	Brain	Brain	Brain	Brain
	Origin	Legal abortion	Late miscarriage	Legal abortion	MPT	MPT	post mortem MRI	post mortem MRI	in utero MRI	in utero MRI	in utero MRI
	Crown-Rump Length (mm)	90	105	69	205	110	-	-	-	-	-
					Hydrocephaly	Malformative
	Associated malformation	None	None	None	Myelomening-	cardiopathy	None	None	None	None	None
					ocele	Cleft palate
	Total number of slices	1800	2000	133	575	320	50	161	50	55	50
	Number of HES stained slices	360	400	133	230	132	-	-	-	-	-
	Number of
	immunolabeled	360	400	0	230	132	-	-	-	-	-
	slices

Declaration of interest: None


Alexis Hermida

MD Mustafa Zaitouni

MD Momar Diouf

Pierre-Marc Lallemand

MD Floriane Fay

MD Otilia Buiciuc

MD Maciej Kubala

MD, pHD Jean-Sylvain Hermida

Long-term incidence and predictive factors of thromboembolic events after a cryoballoon ablation for atrial fibrillation

Keywords: thromboembolic event, cryoballoon pulmonary vein isolation, CHA2DS2-VASc Score

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

INTRODUCTION

Periprocedural rates of stroke/transient ischemic attack (TIA) appear to be comparable between cryoballoon ablation and radiofrequency (RF) catheter ablation for atrial fibrillation (AF), ranging from 0.3 to 0.5%. Long-term annual rates of stroke/TIA after RF catheter ablation for AF are well documented and range from 0.13%/year to 2.10%/year [START_REF] Chao | CHADS2 and CHA2DS2-VASc Scores in the Prediction of Clinical Outcomes in Patients With Atrial Fibrillation After Catheter Ablation[END_REF][START_REF] Hunter | Maintenance of sinus rhythm with an ablation strategy in patients with atrial fibrillation is associated with a lower risk of stroke and death[END_REF][START_REF] Lin | Successful catheter ablation reduces the risk of cardiovascular events in atrial fibrillation patients with CHA2DS2-VASc risk score of 1 and higher[END_REF][START_REF] Bunch | Atrial fibrillation ablation patients have long-term stroke rates similar to patients without atrial fibrillation regardless of CHADS2 score[END_REF][START_REF] Noseworthy | Risk of stroke after catheter ablation versus cardioversion for atrial fibrillation: A propensity-matched study of 24,244 patients[END_REF][START_REF] Friberg | Catheter ablation for atrial fibrillation is associated with lower incidence of stroke and death: data from Swedish health registries[END_REF][START_REF] Saliba | Catheter ablation of atrial fibrillation is associated with reduced risk of stroke and mortality: A propensity score-matched analysis[END_REF][START_REF] Uma | CAABL-AF (California Study of Ablation for Atrial Fibrillation)[END_REF][START_REF] Kim | Ischemic stroke risk during long-term follow up in patients with successful catheter ablation for atrial fibrillation in Korea[END_REF] . However, this rate has not been reported for cryoballoon pulmonary vein isolation (cryo-PVI). In addition, systemic embolisms (acute limb ischemia, renal infarction, etc.) are rarely taken into account to quantify the ischemic risk after AF ablation.

Studies have shown that it is safe for low-risk patients for stroke/TIA to stop oral anticoagulant therapy (OAT) when the sinus rhythm is maintained after catheter ablation for AF. [START_REF] Hakan | Risk of Thromboembolic Events After Percutaneous Left Atrial Radiofrequency Ablation of Atrial Fibrillation[END_REF][START_REF] Nademanee | Clinical outcomes of catheter substrate ablation for high-risk patients with atrial fibrillation[END_REF][START_REF] Themistoclakis | The risk of thromboembolism and need for oral anticoagulation after successful atrial fibrillation ablation[END_REF][START_REF] Yagishita | Incidence of Late Thromboembolic Events After Catheter Ablation of Atrial Fibrillation[END_REF][START_REF] Saad | Very low risk of thromboembolic events in patients undergoing successful catheter ablation of atrial fibrillation with a CHADS2 score ≤3: a long-term outcome study[END_REF][START_REF] Hussein | Natural History and Long-Term Outcomes of Ablated Atrial Fibrillation[END_REF][START_REF] Riley | Risk of stroke or transient ischemic attack after atrial fibrillation ablation with oral anticoagulant use guided by ECG monitoring and pulse assessment[END_REF][START_REF] Karasoy | Oral anticoagulation therapy after radiofrequency ablation of atrial fibrillation and the risk of thromboembolism and serious bleeding: long-term followup in nationwide cohort of Denmark[END_REF][START_REF] Yang | The safety of discontinuation of oral anticoagulation therapy after apparently successful atrial fibrillation ablation: a report from the Chinese Atrial Fibrillation Registry study[END_REF] Several authors have stated that the CHA2DS2-VASc score does not predict the stroke/TIA risk after RF catheter ablation for AF. [START_REF] Kochhäuser | Risk of Stroke and Recurrence After AF Ablation in Patients With an Initial Event-Free Period of 12 Months[END_REF][START_REF] Liang | The anatomic impact of sequential, additional, ostial radiofrequency ablation following pulmonary vein cryoisolation[END_REF] We aimed to evaluate the long-term incidence and identify the predictive factors of thromboembolic events (TEs) after one cryo-PVI procedure.

METHODS

Study population

We prospectively included all consecutive patients with paroxysmal or persistent AF undergoing cryo-PVI at the Amiens-Picardie University Hospital between November 2012 and October 2017. Patients provided their signed informed consent to participate in the registry and the study was approved by the local ethics committee (PI2020_843_0056). All patients underwent preprocedural cardiac computed tomography to determine the left atrial volume and assess the pulmonary venous anatomy. The left atrial area and left ventricular ejection fraction were determined by transthoracic echocardiography prior to the cryo-PVI.

AF was classified according to the 2016 ESC Guidelines for the management of atrial fibrillation. 21 Paroxysmal AF was defined as AF that terminates spontaneously or with intervention within seven days of onset. Persistent AF was defined as continuous AF that is sustained beyond seven days, including episodes that were terminated by cardioversion.

Patients with continuous atrial fibrillation lasting > 1 year (long-standing persistent atrial fibrillation) were also included.

Cryoballoon ablation

Cryoballoon ablations were preceded by at least three weeks of OAT and contrast-enhanced multi-slice computed tomography (CT) of the left atrium. The cryoballoon ablation procedure is described in detail elsewhere. 22 Briefly, left atrial access was obtained after fluoroscopyguided transseptal puncture (SL1, St. Jude Medical, St. Paul, MN, USA) under conscious sedation. The sheath was flushed continuously with heparinized saline. One 4-min application was performed with a second-generation cryoballoon (28 or 23mm Advance balloon, Cryocath, Medtronic, MN, USA). An Achieve (Medtronic) intraluminal catheter was used for all procedures, allowing real-time visualization of the atriovenous block during cryoapplication. A second 4-min cryoapplication was performed if there was persistent atriovenous conduction to achieve a complete block. During cryoablation in the right pulmonary veins, a quadripolar catheter was used to continuously pace the phrenic nerve adjacent to the superior vena cava. Cryoablation was stopped immediately if there was loss of diaphragmatic contraction to avoid subsequent right phrenic nerve palsy.

Follow-up

After ablation, antiarrhythmic drugs were maintained for at least the three-month blanking period. Anticoagulants were continued for the first three months and can be stopped when the risk of thromboembolism is low, the risk of bleeding high, or according to the decision of the referring cardiologist. Patients were followed-up at our institution at 3, 6, and 12 months, with a Holter-monitoring recording at three months and one year. After the first year, patients were followed by the patient's general practitioners and cardiologists. Subsequent information was obtained by phone and/or e-mail.

Recurrence of atrial arrhythmia was defined as an episode of AF or left atrial flutter documented by electrocardiogram (ECG) or Holter-monitoring, occurring after the threemonth blanking period and lasting > 30 s.

The status of the anticoagulation therapy was determined at the time of the TE, the time of the first redo procedure for patients with a recurrence of atrial arrhythmia, or at the last follow up for patients without an atrial arrhythmia recurrence, as appropriate.

The indications for OAT were classified according to the recommendations of the 2016 ESC guideline on AF. 21

Assessment of ischemic events

We collected data on all ischemic events (stroke, TIA, and systemic embolism) that occurred at least 72 h after the procedure. We thus searched for these events in all electronic files of the patients during the entire period of their follow-up and interviewed their general practitioner and/or cardiologist.

Ischemic stroke was defined as an episode of neurological dysfunction caused by focal cerebral, spinal, or retinal infarction. [START_REF] Sacco | An Updated Definition of Stroke for the 21st Century: A Statement for Healthcare Professionals From the American Heart Association/American Stroke Association[END_REF] TIA was defined as a transient episode of neurological dysfunction caused by focal brain, spinal cord, or retinal ischemia, without acute infarction. [START_REF] Donald | Definition and Evaluation of Transient Ischemic Attack[END_REF] Cerebral magnetic resonance imaging (MRI) or cerebral CT were used to diagnose the ischemic stroke/TIA. Systemic ischemia was defined as an acute loss of perfusion of a limb or organ, diagnosed clinically, with CT angiography of a peripheral artery and eventually by ascertainment using open surgical or endovascular procedures.

A thromboembolic mechanism was suspected based on specific elements, including the radiological presentation, transthoracic echocardiography (TTE) and transesophageal echocardiography (TEE) data, and carotid imaging.

Statistical analysis

The primary endpoint was the occurrence of a TE after one cryo-PVI procedure. The occurrence of a TE for patients who did not experience such an event was censored at the time of the first redo procedure for patients who had a redo procedure or at the last follow up.

The secondary endpoint was the time to first atrial arrhythmia recurrence, defined as the time from ablation to the first atrial arrhythmia recurrence. Time to first recurrence was censored at the last follow-up for patients in sinus rhythm.

Continuous variables, expressed as the median (interquartile Q1-Q3 range) or mean ± standard deviation, were compared using an unpaired t-test, Mann Whitney U-test, or Kruskal-Wallis test, as appropriate. Categorical variables are presented as absolute and relative frequencies and were compared using the Chi 2 or Fisher exact tests. Categorical variables are expressed as a percentage and were compared using Fisher's exact test.

Prognostic factors for the time to an ischemic event were assessed using a univariable Cox model. A multivariable Cox model adjusted for the CHA2DS2-VASc score was fitted after treating the OAT variable as a time-varying covariate (OAT discontinuation is a dynamic variable occurring during follow up). The first-order interaction between OAT and TE recurrence was evaluated to test either a differential effect of OAT discontinuation between patients who recurred and those who did not, or a differential effect of recurrence between patients who discontinued OAT and those who continued.

All tests were two-tailed and the limit for statistical significance was set at P < 0.05.

Statistical analysis was performed using SPSS for Windows software, version 22.0 (SPSS Inc., Chicago, IL, USA).

RESULTS

Patient characteristics (Table 1)

All eligible patients were included. In total, 450 patients were included (mean age = 60 ± 9 years, 78% men). Among them, 242 patients (54%) had paroxysmal AF, 160 (35%) were in persistent AF, and 48 (11%) had long-standing AF. Seventy-one percent had no structural heart disease. The mean CHA2DS2-VASc score was 1.6 ± 1.3 and the mean left atrial volume 142 ± 45 ml. Twenty-six patients (6%) had a history of stroke/TIA before the procedure.

Atrial arrhythmia recurrences

Four hundred and thirty-two (96%) patients completed at least one year of follow-up.

Eighteen patients were considered as lost of follow up. Two hundred and seventy patients (60%) had no recurrence of atrial arrhythmia after one cryo-PVI procedure, with a median follow-up of 26 months (IQR 39-16). The 24-months freedom from atrial arrhythmia recurrence probability was 59% [56%;65%].

OAT discontinuation (Supplementary Table 1)

OAT was discontinued for 75 patients (17%) and maintained for 375 (83%), who were essentially treated with a direct oral anticoagulant (234 patients; 62%).

Off-OAT patients were younger (54 years vs. 62 years, p < 0.001) and had a lower median CHA2DS2-VASc score (0 vs. 2, p < 0.001) than on-OAT patients. In addition, fewer off-OAT patients were initially in persistent AF (31% vs. 49%, p = 0.003) and they had a significantly lower incidence of prior stroke or TIA (0% vs. 7%, p = 0.01).

The reasons for discontinuation of OAT were the absence of an indication for long-term anticoagulation therapy (a CHA2DS2-VASc score = 0 for men or 1 for women, in the absence of another stroke risk factor) for 50 patients (67%); patient and/or physician choice, despite a class IIa indication for long-term anticoagulation therapy (CHA2DS2-VASc score = 1 for men or = 2 for women), for 14 patients (19%); patient and/or physician choice, despite a class I indication for long-term anticoagulation therapy (CHA2DS2-VASc score > 1 for men or > 2 for women), for six patients (8%); and bleeding, despite the presence of a class I or class IIa indication for anticoagulation therapy, for five patients (7%).

The median interval between the cryo-PVI procedure and OAT discontinuation was 299 days (IQR 523-135). The median duration of follow-up after the discontinuation of OAT was 472 days (IQR 837-148).

TEs during follow-up (Figure 1, Table 2) Six ischemic events, all considered to be of thromboembolic origin, occurred in six different patients, with a mean interval of 15 months after cryo-PVI. Stroke occurred in three patients, TIA in two, and systemic embolism, with a presentation of acute lower-limb ischemia, in one. The mean follow up of the entire population was 30 months. Therefore, the annualized rate of all TEs was 0.53%/year. Risk of TE at 24 months was estimated at 2% [0.4%;3.6%].

Detailed data for the six patients who experienced a TE are presented Table 2.

A thromboembolic origin was established by neurologists based on the absence of stenosis of the extracranial (patients 1 to 5) and intracranial arteries (patients 1, 2, 3 and 4). Patients 1 and 3 showed multiple spots of cerebral infarction. For patients 4 and 5, prior documented or reported recurrences of atrial arrhythmia was a supplementary argument. For patient 6, a thromboembolic origin was determined based on the typical aspect of a thrombus by arterial angiography and a documented AF recurrence one week before the event. Patients 2 and 6 underwent TEE, which did not show a thrombus.

Five patients had a class I indication for long-term OAT at the time of the event.

Among them, one (patient 4) was off OAT, despite the recurrence of atrial arrhythmia reported two months earlier (no ECG documentation). Two (patients 5 and 6) of the other four patients (on-OAT) had recurrences of atrial arrhythmia documented before the TE, and the cryo-PVI of two (patients 1 and 3) was considered to be successful at the 11-and 22month follow-ups, respectively.

One patient (patient 2) had a class IIa indication for long-term OAT at the time of the TE. He was off OAT and had no documented or patient-reported recurrence of atrial arrhythmia for the 24 months after cryo-PVI.

Among the four patients on OAT at the time of the event, all treated by direct oral anticoagulant, one was switched to vitamin K antagonist after the event (patient 1), one was switched to another direct oral anticoagulant (patient 5), one continued to use the same direct anticoagulant and underwent left atrial appendage occlusion (patient 6), and one died one week after the event (patient 3).

OAT was reintroduced for the two remaining patients (patients 2 and 4).

None of the patients experienced another TE during follow-up.

Predictive factors of TEs

The absence of OAT and the recurrence of atrial arrhythmia were not predictive of a TE after univariate analysis (Table 3). The CHA2DS2-VASc Score (HR = 1.76, CI [1.05;2.95], p = 0.0315) was the only predictive factor of a TE after univariate analysis.

After multivariate analysis, the CHA2DS2-VASc score (HR = 2.13, CI [1.39;3.26], p = 0.0005) was still a predictor of TE. The effect of discontinuing OAT did not significantly differ between patients who recurred and those who did not (p = 0.1642), based on the interaction between the discontinuation of OAT and recurrence. However, the effect of discontinuing OAT appeared to be stronger for patients who recurred (HR = 73.05, CI [5.58;956.90], p = 0.0011) than for those who did not (HR = 5.00, CI [0.48;51.66], p = 0.18). Moreover, the effect of recurrence was greater for patients who discontinued OAT (HR = 24.5, CI [1.30;464.01], p = 0.0329) than for those who continued treatment (HR = 1.66, CI [0.23;11.92], p = 0.6998).

After multivariate analysis without the two TIAs, the CHA2DS2-VASc score (HR = 2.22, CI [1.34;3.67], p=0.0018) was still a predictor of TE. The effect of discontinuing OAT did not significantly differ between patients who recurred and those who did not (p = 0.9516), based on the interaction between the discontinuation of OAT and recurrence.

We also performed the multivariate analysis with CHA2DS2-VASc score as a binary variable. The CHA2DS2-VASc score ≥ 3 (HR = 15.71, CI [2.12;116.36], p=0.0070) was a predictor of TE. The effect of discontinuing OAT did not significantly differ between patients who recurred and those who did not (p = 0.1739), based on the interaction between the discontinuation of OAT and recurrence.

Three additional TEs (two stroke, one acute lower-limb ischemia) occurred after the second procedure. All patients had class I indication for long term OAT and were on OAT. All patients had recurrences of atrial arrhythmia documented before or at the time of the TE. We decided to verify our results by adding these TEs. After multivariate analysis, the CHA2DS2-VASc score (HR = 1.63, CI [1.24;2.14], p=0.0004) was a predictor of TE. The effect of discontinuing OAT did not significantly differ between patients who recurred and those who did not (p = 0.5595), based on the interaction between the discontinuation of OAT and recurrence. However, the effect of discontinuing OAT appeared to be stronger for patients who recurred (HR = 6.03, CI [0.51;70.62], p=0.2867) than for those who did not (HR = 2.25, CI [0.23;21.88], p=0.4829). Moreover, the effect of recurrence was greater for patients who discontinued OAT (HR = 6.77, CI [0.34;134.98], p = 0.2101) than for those who continued treatment (HR = 2.53, CI [0.51; 12.70], p = 0.4208).

DISCUSSION

Our study provides two important findings. First, the long-term rate of a TE after one cryo-PVI procedure was 0.53%/year. Second, the CHA2DS2-VASc score is the only independent predictor of TE.

Long-term rates of TEs after catheter ablation of AF

There are no data concerning the long-term rates of TEs after cryo-PVI. Existing published studies include patients treated by RF-PVI or a mixture of patients treated by RF-or cryo-PVI.

It was important to obtain specific data about the TE risk after cryo-PVI, as it has been demonstrated that the overall lesion width is significantly wider with the cryoballoon than with RF. [START_REF] Kurose | The lesion characteristics assessed by LGE-MRI after the cryoballoon ablation and conventional radiofrequency ablation[END_REF] We could have thus hypothesized that left atrium contractility would be more impaired after cryo-PVI, with a potentially greater long-term incidence of TE.

Published studies should be compared with caution, as they may include not only RF-PVI but other lesions in the left atrium. Sometimes, they include periprocedural events and not only those that are post-procedural. Studies also may not include all types of TE, such as systemic embolism, but only strokes.

Overall, our incidence of 0.53%/year with cryoballoon ablation is quite close to data obtained for RF catheter ablation (Supplementary Table 2). Strivatsa et al. [START_REF] Uma | CAABL-AF (California Study of Ablation for Atrial Fibrillation)[END_REF] found an incidence of 0.37%/year for stroke; Noseworthy et al. [START_REF] Noseworthy | Risk of stroke after catheter ablation versus cardioversion for atrial fibrillation: A propensity-matched study of 24,244 patients[END_REF] , 0.43%/year for stroke/TIA; Hunter et al. [START_REF] Hunter | Maintenance of sinus rhythm with an ablation strategy in patients with atrial fibrillation is associated with a lower risk of stroke and death[END_REF] , 0.5%/year for stroke/TIA; Lin et al. [START_REF] Lin | Successful catheter ablation reduces the risk of cardiovascular events in atrial fibrillation patients with CHA2DS2-VASc risk score of 1 and higher[END_REF] , 0.59%/year for stroke/TIA; Friberg et al. [START_REF] Friberg | Catheter ablation for atrial fibrillation is associated with lower incidence of stroke and death: data from Swedish health registries[END_REF] , 0.70%/year for stroke; and Chao et al. [START_REF] Chao | CHADS2 and CHA2DS2-VASc Scores in the Prediction of Clinical Outcomes in Patients With Atrial Fibrillation After Catheter Ablation[END_REF] , 0.87%/year for TE.

Only, Kim et al. [START_REF] Kim | Ischemic stroke risk during long-term follow up in patients with successful catheter ablation for atrial fibrillation in Korea[END_REF] found a lower incidence of stroke (0.13%/year), which may be explained by lower a CHADS2 score in their population.

Concerning documented recurrences of atrial arrhythmia, Riley et al. [START_REF] Riley | Risk of stroke or transient ischemic attack after atrial fibrillation ablation with oral anticoagulant use guided by ECG monitoring and pulse assessment[END_REF] found 75% of the patients in AF after an ischemic event. Kim et al. [START_REF] Kim | Ischemic stroke risk during long-term follow up in patients with successful catheter ablation for atrial fibrillation in Korea[END_REF] and Hunter et al. [START_REF] Hunter | Maintenance of sinus rhythm with an ablation strategy in patients with atrial fibrillation is associated with a lower risk of stroke and death[END_REF] found that being in sinus rhythm reduced the risk of stroke, after multivariate analysis. On the other hand, Kochhäuser et al. [START_REF] Kochhäuser | Risk of Stroke and Recurrence After AF Ablation in Patients With an Initial Event-Free Period of 12 Months[END_REF] found no difference in AF recurrence for patients, with or without stroke, and Liang et al. [START_REF] Liang | The anatomic impact of sequential, additional, ostial radiofrequency ablation following pulmonary vein cryoisolation[END_REF] found that AF recurrence was not a predictor of stroke/TIA. These two studies did not use Cox Models to reach their conclusions. In our study, the recurrence of atrial arrhythmia was not a predictive factor of TEs after multivariate analysis. However, we logically found a significant effect of recurrence for patients off OAT (HR = 24.5, CI [1.30;464.01], p = 0.0329).

Several studies have examined the role of the CHA2DS2-VASc score in the occurrence of TE. Kochhäuser et al. [START_REF] Kochhäuser | Risk of Stroke and Recurrence After AF Ablation in Patients With an Initial Event-Free Period of 12 Months[END_REF] find no difference in the CHA2DS2-VASc score for patients with or without stroke. Liang et al. [START_REF] Liang | The anatomic impact of sequential, additional, ostial radiofrequency ablation following pulmonary vein cryoisolation[END_REF] found that the CHA2DS2-VASc score did not predict stroke/TIA, in contrast to our results. In our study, all patients who experienced a TE had a class I or class IIa indication for long-term anticoagulation therapy and the CHA2DS2-VASc score was a predictive factor of a TE after multivariate analysis. This difference can be explained by the lack of statistical power (small number of events) and again by the different statistical tests used in these two studies. Conversely, Chao et al. [START_REF] Chao | CHADS2 and CHA2DS2-VASc Scores in the Prediction of Clinical Outcomes in Patients With Atrial Fibrillation After Catheter Ablation[END_REF] obtained a result similar to ours, with a hazard ratio of 1.69 (CI 1.32;2.17, p < 0.001) for each incremental increase of the CHA2DS2-VASc score predicting stroke/TIA. In the study of Kim et al. [START_REF] Kim | Ischemic stroke risk during long-term follow up in patients with successful catheter ablation for atrial fibrillation in Korea[END_REF] , the CHA2DS2-VASc score was a predictor of stroke after univariate analysis.

In conclusion, we propose that a lack of recurrence should not be considered in the decision to stop anticoagulation therapy for a particular individual. Indeed, it is difficult in real life to monitor the recurrence of AF as performed in certain studies. By contrast, the CHA2DS2-VASc score should be considered when deciding whether or not to stop anticoagulation therapy, as recommended in current guidelines. To go further, we showed that CHA2DS2-VASc score ≥ 3 could be a good cut-off to continue OAT after AF ablation.

Limitation and strengths

Our results are limited by the small number of TEs that occurred. However, this could also be seen as a strength, as it permitted a detailed description of these six events. We provide "safe" data.

We thoroughly researched the information concerning the occurrence of the TEs, both prospectively and retrospectively and are confident about full completeness.

Our protocol of atrial arrhythmia monitoring after cryo-PVI does not include continuous monitoring but it corresponds to real life, and our conclusions are thus applicable to daily practice. There were no defined instructions concerning OAT discontinuation in this study. Indeed, information provided about the patients who stopped or continued OAT should be considered with caution.

The multivariate analysis includes only 6 events. This exploratory analysis and its results should be confirmed on a larger number of events.

Figure 1 .

 1 Figure 1. Thromboembolic events according to the result of cryoablation, and anticoagulation status.

ACKNOWLEDGEMENTS

The authors thank Helene Duquenne for her help with data acquisition.

Conversely, Bunch et al. [START_REF] Bunch | Atrial fibrillation ablation patients have long-term stroke rates similar to patients without atrial fibrillation regardless of CHADS2 score[END_REF] included periprocedural events and found a higher incidence of stroke (1.4% at one year). The population studied by Saliba et al. [START_REF] Saliba | Catheter ablation of atrial fibrillation is associated with reduced risk of stroke and mortality: A propensity score-matched analysis[END_REF] had a high mean CHA2DS2-VASc score (3.6) and they logically found the highest rate of stroke/TIA of 2.10%/year. Finally, our incidence of 0.53%/year is much lower than would be expected for our population, for which the mean CHA2DS2-VASc score was 1.6. Indeed, the estimated stroke risk is 0.66% when the CHA2DS2-VASc score = 1 and 1.5% (0.93;2.21) when the CHA2DS2-VASc score = 2. [START_REF] Van Doorn | Predictive performance of the CHA2DS2-VASc rule in atrial fibrillation: a systematic review and meta-analysis[END_REF] Management of OAT after cryo-PVI European 21 and US guidelines state that it is necessary to continue long-term anticoagulation therapy after AF ablation based on the CHA2DS2-VASc score, irrespective of the atrial arrhythmia recurrence status.

Several observational studies have tried to demonstrate that it is safe to interrupt OAT if a stable sinus rhythm is proven, irrespective of the CHA2DS2-VASc score for some.

The literature clearly shows that the rate of off-OAT events for patients in supposed sinus rhythm is very low (Supplementary Table 3). [START_REF] Hakan | Risk of Thromboembolic Events After Percutaneous Left Atrial Radiofrequency Ablation of Atrial Fibrillation[END_REF][START_REF] Nademanee | Clinical outcomes of catheter substrate ablation for high-risk patients with atrial fibrillation[END_REF][START_REF] Themistoclakis | The risk of thromboembolism and need for oral anticoagulation after successful atrial fibrillation ablation[END_REF][START_REF] Yagishita | Incidence of Late Thromboembolic Events After Catheter Ablation of Atrial Fibrillation[END_REF][START_REF] Saad | Very low risk of thromboembolic events in patients undergoing successful catheter ablation of atrial fibrillation with a CHADS2 score ≤3: a long-term outcome study[END_REF][START_REF] Hussein | Natural History and Long-Term Outcomes of Ablated Atrial Fibrillation[END_REF][START_REF] Riley | Risk of stroke or transient ischemic attack after atrial fibrillation ablation with oral anticoagulant use guided by ECG monitoring and pulse assessment[END_REF][START_REF] Karasoy | Oral anticoagulation therapy after radiofrequency ablation of atrial fibrillation and the risk of thromboembolism and serious bleeding: long-term followup in nationwide cohort of Denmark[END_REF][START_REF] Yang | The safety of discontinuation of oral anticoagulation therapy after apparently successful atrial fibrillation ablation: a report from the Chinese Atrial Fibrillation Registry study[END_REF][START_REF] Liang | The anatomic impact of sequential, additional, ostial radiofrequency ablation following pulmonary vein cryoisolation[END_REF][START_REF] Kochhäuser | Risk of Stroke and Recurrence After AF Ablation in Patients With an Initial Event-Free Period of 12 Months[END_REF] Nademanee et al. [START_REF] Nademanee | Clinical outcomes of catheter substrate ablation for high-risk patients with atrial fibrillation[END_REF] , Themistoclakis et al. [START_REF] Themistoclakis | The risk of thromboembolism and need for oral anticoagulation after successful atrial fibrillation ablation[END_REF] , and Yang et al. [START_REF] Yang | The safety of discontinuation of oral anticoagulation therapy after apparently successful atrial fibrillation ablation: a report from the Chinese Atrial Fibrillation Registry study[END_REF] even found a lower rate for patients off OAT than for those on OAT.

Riley et al. [START_REF] Riley | Risk of stroke or transient ischemic attack after atrial fibrillation ablation with oral anticoagulant use guided by ECG monitoring and pulse assessment[END_REF] found no difference in stroke risk, regardless of the OAT status for patients with a CHADS2 = 1 or ≥ 2. Karasoy et al. [START_REF] Karasoy | Oral anticoagulation therapy after radiofrequency ablation of atrial fibrillation and the risk of thromboembolism and serious bleeding: long-term followup in nationwide cohort of Denmark[END_REF] found that the effect of OAT discontinuation was insignificant after multivariate analysis, as did Yang et al. [START_REF] Yang | The safety of discontinuation of oral anticoagulation therapy after apparently successful atrial fibrillation ablation: a report from the Chinese Atrial Fibrillation Registry study[END_REF] We obtained a similar result but, in our study, OAT discontinuation appeared to have a significant impact on TEs for patients who recurred (HR = 73.05, CI [5.58;956.90], p = 0.0011). The effect of OAT discontinuation on TEs for patients who did not recur was substantially lower (HR = 5.00, CI [0.48;51.66], p = 0.18).

CONCLUSION

The long-term rate of a TE after one cryo-PVI procedure was 0.53%/year, comparable to rates found after a RF AF ablation. Atrial arrhythmia recurrences, which are difficult to document, should not be considered in the decision to discontinue OAT, in contrast to the CHA2DS2-VASc score, which was the only predictive factor of a TE after multivariate analysis.