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Physical Exam Indicated Cerclage in Twin pregnancy: a Randomized Controlled Trial

Amanda Roman, MD, Noelia Zork, MD, Sina Haeri, MD, MHSA, Corina N. Schoen,
MD, Gabriele Saccone, MD, Sarah Colihan, MD, Craig Zelig, MD, Alexis C.
Gimovsky, MD, Neil S. Seligman, MD, Fulvio Zullo, MD, Vincenzo Berghella, MD

PII: S0002-9378(20)30672-4
DOI: https://doi.org/10.1016/j.ajog.2020.06.047
Reference: YMOB 13338

To appear in: American Journal of Obstetrics and Gynecology

Received Date: 1 January 2020

Revised Date: 19 May 2020
Accepted Date: 22 June 2020

Please cite this article as: Roman A, Zork N, Haeri S, Schoen CN, Saccone G, Colihan S, Zelig C,
Gimovsky AC, Seligman NS, Zullo F, Berghella V, Physical Exam Indicated Cerclage in Twin pregnancy:
a Randomized Controlled Trial, American Journal of Obstetrics and Gynecology (2020), doi: https://
doi.org/10.1016/j.ajog.2020.06.047.

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1 Title: Physical Exam Indicated Cerclage in Twin pregnancy: a Randomized

2 Controlled Trial

3 Authors: Amanda ROMAN, MD (1), Noelia ZORK, MD (2), Sina HAERI, MD, MHSA

4 (3), Corina N. SCHOEN, MD (4) Gabriele SACCONE, MD (5), Sarah COLIHAN, MD (6),

5 Craig ZELIG, MD (6), Alexis C. GIMOVSKY, MD (7); Neil S. SELIGMAN, MD (8), Fulvio

6 ZULLO, MD, (5), Vincenzo BERGHELLA, MD (1)

7 (1) Maternal Fetal Medicine Division, Obstetrics and Gynecology Department, Sidney

8 Kimmel Medical College at Thomas Jefferson University, Philadelphia, PA, United

9 States.

10 (2) Maternal Fetal Medicine Division, Obstetrics and Gynecology Department, Columbia

11 University, New York, NY

12 (3) St David’s Women’s Center of Texas, Austin Maternal-Fetal Medicine, Austin, TX

13 (4) Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine,

14 University of Massachusetts-Baystate, Springfield, MA

15 (5) Department of Neuroscience, Reproductive Sciences and Dentistry, School of

16 Medicine, University of Naples Federico II, Naples, Italy

17 (6) Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine,

18 Albany Medical Center, Albany, NY

19 (7) Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine, The

20 George Washington University School of Medicine and Health Sciences, Washington,

21 DC
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22 (8) Department of Obstetrics and Gynecology, Division of Maternal-Fetal Medicine,

23 University of Rochester Medical Center, Rochester, NY

24 Disclosure: The authors report no conflicts of interest.

25 Financial Support: No external financial support was received for this study.

26 Data from this manuscript was presented at the 40th SMFM Meeting, February 3–8,

27 2020 at Grapevine, Texas,

28

29 Corresponding author

30 Amanda Roman, MD

31 Department of Obstetrics and Gynecology

32 Division of Maternal-Fetal Medicine

33 Thomas Jefferson University

34 833 Chestnut Street, First floor, Philadelphia, PA, 19107 USA

35 Email: amanda.roman@jefferson.edu

36 Phone: 215-955-9200

37 Fax: 215-955-5041

38

39 Word count

40 Abstract: 447

41 Main manuscript: 4013

42

43
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44 Condensation: Physical exam indicated cerclage, indomethacin, and antibiotics in

45 asymptomatic twin pregnancies <24 weeks significantly decreases preterm birth at all

46 evaluated gestational ages and perinatal mortality.

47 Short Title: Physical exam indicated cerclage in twin pregnancy decreases preterm

48 birth

49 AJOG at a Glance:

50 A. Why was the study conducted?

51 • To evaluate if physical exam indicated cerclage will decrease preterm birth

52 in twin pregnancy

53 B. What are the key findings?

54 • Physical exam indicated cerclage in twins significantly decreases preterm

55 birth at all gestational age cutoff studied and decreases perinatal morbidity

56 and mortality

57 C. What does this study add to what is already known?

58 • This study is the first randomized controlled trial evaluating physical exam

59 indicated cerclage in twins.

60

61 Clinical trials registration number: NCT02490384

62 First registered: July 3, 2015

63 First patient enrolled: July 25, 2015

64 https://clinicaltrials.gov/ct2/show/NCT02490384?cond=twin+cerclage&draw=2&rank=5

65
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66 ABSTRACT

67 Background: Twin pregnancies with dilated cervix in the second trimester are at

68 increased risk of pregnancy loss and early preterm birth; currently there is no proven

69 therapy to prevent preterm birth in this group of women.

70 Objective: To determine if physical exam indicated cerclage reduces the incidence of

71 preterm birth in asymptomatic women with twin gestations and cervical dilation

72 diagnosed before 24 weeks of pregnancy.

73 Study design: Multicenter, parallel-group, open-label, randomized controlled trial of

74 women with twin pregnancy, and asymptomatic cervical dilation from 1-5 cm between

75 16 0/7 to 23 6/7 weeks were enrolled from 7/2015 to 7/2019 in 8 centers. Eligible

76 women were randomized in a 1:1 ratio to either cerclage or no cerclage. We excluded

77 women with: monochorionic-monoamniotic pregnancy, selective fetal growth restriction,

78 twin-twin transfusion syndrome, major fetal malformation, known genetic anomaly,

79 placenta previa, signs of labor, or clinical chorioamnionitis. The primary outcome was

80 the incidence of preterm birth <34 weeks. Secondary outcomes were preterm birth <32,

81 <28 and <24 weeks, interval from diagnosis to delivery, and perinatal mortality. Data

82 were analyzed as intention to treat.

83 Results: After an interim analysis was performed, the Data Safety Monitoring Board

84 recommended stopping the trial due to significant decrease of perinatal mortality in the

85 cerclage group. We randomized 34 women, four were excluded due to expired informed

86 consent. Seventeen women were randomized to physical exam indicated cerclage and

87 13 women to no cerclage. Four women randomized to cerclage did not receive the
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88 surgical procedure, while no women in the no cerclage group received cerclage.

89 Maternal demographics were not significantly different. All women in the cerclage group

90 also received indomethacin and antibiotics. When comparing cerclage vs no cerclage

91 group, the incidence of preterm birth <34 weeks was significantly decreased: 12/17

92 (70%) vs 13/13 (100%) (RR: 0.71 95% CI 0.52-0.96), as well as preterm birth <32

93 weeks 11/17 (64.7%) vs 13/13 (100%) (RR: 0.65 95% CI 0.46-0.92), preterm birth <28

94 weeks 7/17 (41%) vs 11/13 (84%) (RR: 0.49 95% CI 0.26-0.89) and preterm birth <24

95 weeks 5/17 (30%) vs 11/13 (84%) (RR: 0.35 95% CI 0.16-0.75). The mean gestational

96 age at delivery was later: 29.05±1.7 vs. 22.5±3.9 weeks (p<0.01); the mean interval

97 from diagnosis of cervical dilation to delivery was longer: 8.3±5.8 vs. 2.9±3.0 weeks

98 (p=0.02). Perinatal mortality was also significantly reduced in the cerclage group 6/34

99 (17.6%) vs 20/26 (77%), (RR: 0.22 95% CI 0.1-0.5).

100 Conclusion: A combination of physical exam indicated cerclage, indomethacin, and

101 antibiotics in asymptomatic twin pregnancies before 24 weeks significantly decreases

102 preterm birth at all evaluated gestational ages. Most importantly, cerclage in this

103 population is associated with a 50% decrease in very early preterm birth <28 weeks and

104 with a 78% decrease in perinatal mortality.

105 Keywords: Physical exam indicated cerclage, twins, cerclage, cervical dilation, preterm

106 birth, perinatal mortality.

107

108

109
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110 INTRODUCTION

111 Twins pregnancies represent a small proportion of annual births in the United States but

112 account for 20% of preterm births. The 2017 twin birth rate in the United States was

113 33.3 twins per 1,000 births but 59.4% of twin pregnancies delivered before 37 weeks,

114 and 9.45% before 32 weeks, compared with singleton pregnancies, of whom 8.13%

115 delivered before 37 weeks and 2.12% before 32 weeks1. Twins are also at increased

116 risk for low birth-weight (LBW), have five times higher risk of early neonatal and infant

117 death and complications related to prematurity and LBW.1, 2

118 Singleton pregnancies with cervical dilation of 1 cm or more in the second trimester are

119 associated with a poor prognosis. Greater than 90% will result in pretem birth (PTB),

120 regardless of history of PTB, cervical length, obstetric history, or other PTB risk

121 factors.3-7 Physical exam indicated cerclage, previously called at times rescue,

122 emergency, or urgent cerclage, is defined as a cerclage placed in the presence of 1 cm

123 or more cervical dilation detected on physical examination (speculum or digital exam).8,
9
124 Placement of cerclage in patients with singleton gestations who have cervical dilation

125 in the second trimester has been shown to decrease incidences of PTB and perinatal

126 mortality.3-7, 9-11

127 In twins, the data on the efficacy of physical exam indicated cerclage is limited. The only

128 published randomized controlled trial (RCT) evaluating cerclage for cervical dilation

129 compared 13 women who received cerclage, indomethacin and bed rest, and 10

130 women who received bed rest-only; all women received antibiotics. This RCT included 7

131 twin pregnancies, 3 in the cerclage and 4 in the bed rest group. This RCT showed

132 overall a significant decrease in PTB < 34 weeks of gestation and longer latency interval
 7

133 from diagnosis to delivery (by 30 days). However, outcomes of twin pregnancies were

134 not reported separately.3

135 Multiple case series and cohorts studies on physical exam indicated cerclage in twins or

136 twins compared with singleton pregnancies have been published.12 The three

137 retrospective case-control studies in twins, where physical exam indicated cerclage was

138 compared with no cerclage,12-14 showed a significant decrease in spontaneous PTB

139 <34, and <32 weeks, two of the studies also had significant spontaneous PTB < 28

140 weeks12, 13 and one of them showed a decrease in the incidence of perinatal mortality.12

141 Our aim was to evaluate if physical exam indicated cerclage would reduce the rate of

142 spontaneous PTB before 34 weeks’ gestation and adverse perinatal outcomes in

143 women with twin pregnancy, and asymptomatic cervical dilation from 1-5 cm before 24

144 weeks when compared to no cerclage in a randomized controlled trial.

145

146 MATHERIALS AND METHODS

147 Study design and participants

148 This multicenter, parallel group, open-label, randomized controlled trial was performed

149 by a consortium of 8 clinical centers between July 2015 and July 2019. The Institutional

150 Review Board (IRB) approved this study at each center, and all participants were

151 provided with and signed written informed consent. This trial was registered as a

152 randomized clinical trial (NCT02490384; clinicaltrials.gov). This trial had no external

153 funding. Eligible participants were women between 18 - 50 years of age carrying a
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154 diamniotic twin gestation with asymptomatic cervical dilation from 1 to 5 cm and/or

155 visible membranes by pelvic exam or speculum exam between 16 0/7 to 23 6/7 weeks.

156 Women were identified either by transvaginal ultrasound cervical length (TVUCL) at the

157 time of the anatomy scan or by pelvic examination during prenatal visits. We excluded

158 women with monochorionic-monoamniotic pregnancy, selective fetal growth restriction,

159 twin-twin transfusion syndrome, major fetal malformation or known genetic anomaly,

160 placenta previa, regular painful contractions with cervical changes over 12 hours of

161 observation, active bleeding or clinical chorioamnionitis at the time of diagnosis, cervical

162 dilation 6 cm or more, amniotic membranes prolapsed beyond external os into the

163 vagina, unable to visualize cervical tissue, fetal reduction after 14 weeks from higher-

164 order multiples, ruptured membranes prior to randomization, and cerclage already in

165 place.

166 All pregnancies were dated by crown-rump length during the first trimester, and

167 chorionicity was determined. Before randomization, they underwent anatomy scan for

168 both twins by a certified sonographer or Maternal-Fetal Medicine specialist. Cervical

169 dilation was determined by pelvic exam and/or speculum exam between 16 0/7 and 23

170 6/7 weeks and confirmed to have a dilated cervix from 1-5 cm by the primary

171 investigator at each site. Premature preterm rupture of the membranes (PPROM)

172 defined as rupture of membranes that occurred prior of labor and delivery but not

173 intrapartum. PPROM was diagnosed by visualization of amniotic fluid on sterile

174 speculum exam, a positive Nitrazine test and/or a ferning pattern on microscopy. Active

175 labor was defined as the presence of regular uterine contractions 3 or more in 10

176 minutes with cervical change. Subclinical chorioamnionitis was identified by

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177 amniocentesis of the presenting twin with: 1) presence of any bacteria on amniotic fluid

178 Gram stain, 2) amniotic fluid leukocyte count ≥ 6 leukocytes per high-power field or >30

179 cells/mm3, 3) amniotic fluid glucose concentration ≤15 mg/dL; or by positive amniotic

180 fluid culture. Clinical chorioamnionitis was defined as: maternal fever ≥38°C (≥100.4° F)

181 plus 1 of the following: maternal tachycardia (>100 beats/min), fetal tachycardia, (>160

182 beats/min), marked leukocytosis (>15,000 cells/mm3), uterine tenderness, or foul odor

183 of the amniotic fluid.15 Amniocentesis was offered to all patients before randomization;

184 however, it was not a prerequisite for enrollment. Patients who declined amniocentesis

185 were observed for approximately 12 hours for signs of labor, bleeding, or infection, and

186 those who remained stable were offered randomization. At the time of pelvic exam,

187 specimens for gonorrhea, chlamydia, group B streptococcus (GBS), bacterial vaginosis

188 and trichomonas’s were collected. Gonorrhea and chlamydia were identified by

189 polymerase chain reaction (PCR). The diagnosis of bacterial vaginosis was done by

190 Amsel criteria,16 (at least three criteria must be present): 1) homogeneous, thin, grayish-

191 white discharge that smoothly coats the vaginal walls, 2) vaginal pH >4.5, 3) positive

192 whiff-amine test, defined as the presence of a fishy odor when a drop of 10% potassium

193 hydroxide (KOH) is added to a sample of vaginal discharge and 4) clue cells on saline

194 wet mount (vaginal epithelial cells studded with adherent coccobacilli at the edge of the

195 cell in at least 20% of the epithelial cells on wet mount). Trichomonas were identified on

196 wet mount. Urinary tract infections (UTI) were diagnosed by positive urine culture. All

197 detected infections were treated. Initiation of indomethacin during the observation

198 period was acceptable. After delivery, the placentas were sent to pathology. Histologic

199 chorioamnionitis was defined by the presence of neutrophils that marginated into the
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200 placental chorionic plate; funisitis was defined by the presence of neutrophils in the

201 umbilical cord vessel wall.17

202 Randomization and masking

203 Eligible participants were randomly allocated in a 1:1 ratio by blocks of 4 to either

204 cerclage or no cerclage using a computer-based platform programmed in REDCap

205 (Research Electronic Data Capture) hosted at Thomas Jefferson University.18, 19 The

206 sequence of randomization was set by a third party. Principal investigators at each site

207 were masked to the allocation of patients; institutions were only able to see their own

208 patients. Due to small sample size of the study, enrollment was not stratified by

209 institution or any of the maternal characteristics.

210 Interventions

211 Physical exam indicated cerclage was to be placed by a trained physician in the

212 operating room under anesthesia (regional or general at the discretion of the

213 anesthesiologist). Amnioreduction at the time of the amniocentesis, use of

214 indomethacin, antibiotics before and/or after the cerclage, surgical technique, selection

215 of sutures, additional tocolysis, vaginal progesterone, admission to the hospital for

216 observation, or maternal physical activity after randomization were at the discretion of

217 the physician performing the cerclage.

218 Parcipants follow-up

219 Women on both arms cerclage and no cerclage were observed in the hospital until they

220 were stable for discharge, and preterm labor precautions were given. Readmission to
 11

221 the hospital per physician discretion. Antenatal steroids were offered to all women ≥ 23

222 weeks, and a second course of steroids was offered if delivery was considered

223 imminent ≥ 2 weeks after the first course. Prenatal care continued according to the

224 local guidelines, there were no other study-specific recommendations for pregnancy

225 care.

226 Women were contacted monthly either by phone or in person at the time of their

227 prenatal visit. Consents for release of medical information were signed at the time of

228 randomization to gather information pertinent to the study in case of admissions to other

229 institutions. Fetal growth evaluations were done every 3 to 4 weeks; estimated fetal

230 weight calculations were based on the Hadlock, et al. growth curve.20 Patients

231 diagnosed with intrauterine growth restriction (IUGR) in one or both twins were followed

232 by biweekly non-stress test, weekly umbilical artery Doppler and maximal vertical

233 pocket on each side of the membrane. The primary care provider removed the cerclage

234 during the 36th week of gestation or sooner in the presence of active vaginal bleeding,

235 labor, PPROM, or membranes prolapsed through the suture. Delivery time and mode of

236 delivery were per obstetrical indications. After delivery, all neonates were followed until

237 they were discharged home.

238 Primary and secondary outcomes

239 The primary outcome was the incidence of spontaneous PTB <34 weeks of gestation.

240 Secondary maternal outcomes included: incidence of spontaneous PTB <32, <28, and

241 <24 weeks gestation, PPROM at <34 weeks gestation, GA at delivery, interval between

242 diagnosis and delivery, and birth weight. Secondary neonatal outcomes include: Apgar
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243 score at 5 minutes, admission to neonatal intensive care unit (NICU), neonatal death,

244 length of stay (LOS) in the NICU until discharge home, and composite neonatal adverse

245 outcome, defined as at least 1 of the following: respiratory distress syndrome (RDS)

246 (ventilator support, intubation, continuous positive airway pressure [CPAP], or use of

247 surfactant), intraventricular hemorrhage (IVH) grade 3 and 4 as determined by cranial

248 ultrasound or computed tomography (CT) according to the Papile classification,21

249 necrotizing enterocolitis (NEC) grade 3 and 4 requiring surgery,22 proven sepsis

250 (clinically ill infant with suspected infection plus positive blood, cerebrospinal fluid [CSF],

251 or catheterized/ suprapubic urine culture or cardiovascular collapse or unequivocal x-ray

252 finding), or retinopathy of prematurity (ROP) requiring laser treatment.23 Significant

253 adverse maternal effects included: intraoperative rupture of membranes, clinical

254 chorioamnionitis, postpartum hemorrhage, cervical laceration or tear, and uterine

255 rupture.

256 Sample Size Calculation

257 Calculation of sample size was based on a reduction in the incidence of PTB < 34

258 weeks from 80% in the no cerclage group to 40% in the cerclage group, with a power of

259 80%.12 To detect this difference at a significance level of 5%, we calculated 46 subjects

260 with 23 subjects in each arm, plus 10% for loss of follow up. A total sample size of 52

261 subjects was needed.

262 It was planned that the Data and Safety Monitoring Board (DSMB) would review data

263 relevant to safety (not efficacy) after approximately 50% of the subjects had delivered

264 and the DSMB would provide a recommendation as to whether the study should
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265 continue without modification of the protocol or informed consent. Pre-specified

266 stopping rules were: significantly different maternal sepsis with admission to the

267 intensive care unit, maternal death, maternal bleeding requiring blood transfusion, or

268 fetal or neonatal mortality associated with the cerclage placement when compared with

269 no cerclage. Adverse events were reported to the Human Research Protection Office

270 and the Institutional Review Board (IRB) as they occurred.

271 Statistical analysis

272 Statistical analysis was conducted using Statistical Package for Social Sciences (SPSS)

273 version. 22 (IBM Inc., Armonk, NY, USA). Data are shown as mean ± standard

274 deviation (SD) or number (percentage). Univariate comparisons of dichotomous data

275 were performed with the use of the chi-square test with continuity correction.

276 Comparisons between groups were performed with the use of the T-test to test group

277 means by assuming equal within-group variances for parametric data, and with the use

278 of Wilcoxon and Mann-Whitney tests for nonparametric data. The primary analysis was

279 an intention to treat comparison of the treatment assigned at randomization. The

280 incidence of primary and secondary outcomes were quantified by the crude relative ratio

281 (RR) or mean difference with 95% of confidence interval (CI). In addition to standard

282 logistic regression analysis, in which each fetus was treated as an independent unit, we

283 used a generalized model-model approach in which each twin pair was a cluster unit for

284 outcomes of neonates born alive. For these outcomes, intracluster correlation

285 coefficient was also estimated. ICC was calculated using the two-way mixed model with

286 95% CI. Risk of PTB was assessed with the use of Kaplan-Meier analysis, in which

287 gestational age (GA) was time scale, and delivery the event. Log-Rank Hazard Ratio
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288 was estimated. For outcomes in which cerclage had a statistically significant beneficial

289 or harmful effect number needed to treat (NNT) was calculated. P value <0.05 was

290 considered statistically significant. This trial was reported following the CONSORT

291 guidelines.24

292

293 RESULTS

294 Trial participants

295 A total of 70 women were assessed for eligibility between July 2015 and July 2019

296 (Figure 1). Of these, 14 women had an exclusion criteria and 22 declined participation,

297 34 women provided informed consent and were randomized to either cerclage (n=18) or

298 from analysis when their consent form was identified to be expired, and a new consent

299 was not available due to lapse in the IRB documents renewal at two enrolling sites (1

300 woman in the cerclage group and 3 women in the no cerclage group). Therefore a total

301 of 30 women were included in the final analysis (17 cerclage and 13 no cerclage). Four

302 women assigned to cerclage did not receive the surgical procedure: 2 of them due to

303 contraindications to the procedure, which occurred soon after randomization (rupture of

304 amniotic membranes, and vaginal bleeding), one due to friable cervix and the cerclage

305 placement was suspended, while one declined cerclage after being randomized. All four

306 patients were included in the intention to treat analysis. At the interim analysis

307 performed with 30 enrolled women (58% of the sample size), the DSMB recommended

308 stopping the trial due to significant decrease of perinatal mortality in the cerclage group.
 15

309 Participant demographic characteristics were similar for each group (Table 1).

310 Gestational age at diagnosis and randomization was the same. Most twin gestations

311 were diamniotic-dichorionic 28 (93.3%); assisted reproductive technology was used by

312 11 (36.6%); 6 women (20%) had a history of singleton PTB; 24 (80%) women had one

313 or more TVUCL done as part of the screening for preterm birth; of them, 20 (66.6%) had

314 TVUCL ≤ 25 mm and 7 (23.3%) had TVUCL ≤15mm, while 19 (63.3%) received vaginal

315 progesterone due to the finding of short cervix. All 24 women subsequently were

316 identified as having a dilated cervix. The other 10 women were identified with dilated

317 cervix during a pelvic examination indicated by other symptoms (pain, pressure, or

318 vaginal discharge) but considered not in active labor. Four women (13%) agreed and

319 had an amniocentesis before randomization, and all were negative for intra-amniotic

320 infection. UTI was identified in 4 (13.3%) women; cervico-vaginal evaluation identified

321 bacterial vaginosis in 3 (10%) and chlamydia in 1 (3%), all patients received appropriate

322 treatment. Cerclage placement was successful in 13 of 14 women. McDonald cerclage

323 with one suture was done on all patients. Manipulation of the amniotic membrane was

324 required on 9/14 (64%) with either Foley balloon or sponge. Mersilene™ tape was used

325 on 8 women, Mersilene™ suture in 2, Ethibond™ in 3 and Prolene™ in 1 woman. All

326 women who underwent cerclage, received antibiotics (12 cephalosporin, 1 clindamycin-

327 gentamycin and 1 azithromycin) and indomethacin (50-100mg loading dose followed by

328 25-50 mg every 6 hours for 48 hours). Intraoperative complications was present in only

329 one case when physical exam indicated cerclage was attempted but the cervix was

330 more effaced and friable than expected, therefore the procedure was stopped and

331 placement was considered unsuccessful. No serious adverse events were identified.
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332 Primary and secondary outcomes

333 The primary outcome of spontaneous PTB <34 weeks of gestation was observed in

334 12/17 (70%) of women in the cerclage group vs 13/13 (100%) in the no cerclage group

335 (RR, 0.7, 95% CI, 0.46–0.96; p=0.05). Secondary outcomes of spontaneous PTB <32,

336 <28, and <24 weeks of gestation were also significantly decreased in the cerclage

337 group (Table 2). There were no cases of medically indicated PTB. The latency period

338 from diagnosis to delivery was significantly prolonged in the cerclage group by mean

339 difference of 5.6 (2.0-9.3) weeks (p=0.02). The cumulative percentage of participants

340 who did not give birth preterm was significantly higher in the cerclage group than in the

341 no cerclage group (Log-Rank Hazard Ratio 0.33 [95%CI 0.13-0.80], p<0.001), (Figure

342 2). While more women in the cerclage delivered between 23 0/7 to 27 6/7 weeks: 5/17

343 (29.4%) vs. 1/13 (7.7%), this difference was not significantly different, p=0.2. Overall

344 perinatal mortality was observed in 6/34 (17.6%) of cerclage group versus 20/26 (77%)

345 in the no cerclage group RR 0.23 (0.11-0.49), p< 0.0001. There were no cases of fetal

346 demise. All perinatal mortality cases were associated with delivery < 24 weeks, (Table

347 2). Delivery prior to 23 weeks was seen in 2/17 (11.7%) women in the cerclage group

348 and 10/13 (76.9%) in the no cerclage group, all neonates died secondary to extreme

349 prematurity and received palliative care. Two women (11.7%) in the cerclage group

350 delivered between 23 0/7 and 23 6/7 weeks, they were born alive but one twin of each

351 women died in the first 48 hours (one secondary to sepsis and the other due to

352 respiratory failure). There were no significant differences in other neonatal outcomes,

353 but the trial was not powered for these outcomes (Table 3).

354
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355 COMMENT

356 Principal Findings

357 In women with twin pregnancies and asymptomatic cervical dilation from 1 to 4 cm

358 before 24 weeks, a combination of physical exam indicated cerclage, indomethacin, and

359 antibiotics significantly decreases the incidence of spontaneous PTB at all evaluated

360 GA providing a longer latency period from diagnosis to delivery by 5.6 weeks. Most

361 importantly, cerclage in these population was associated with a 50% decrease in very

362 early PTB <28 weeks, and a 78% decrease in perinatal mortality.

363 Results

364 Twin pregnancy with asymptomatic cervical dilation before 24 weeks offers a formidable

365 challenge for obstetricians. Physical exam indicated cerclage in twins with dilated cervix

366 before 24 weeks has been evaluated in only three retrospective studies in twins with no

367 cerclage as a control group, all of them favored cerclage placement. Our results are

368 similar to these studies. Roman et al, published in 201612 was the first case control

369 study of twins with cervical dilation ≥ 1 cm before 24 weeks from 6 different institutions

370 in the United States and one in Italy. The study showed that the combination of physical

371 exam indicated cerclage, indomethacin, and antibiotics were associated with a

372 significant prolongation of latency from diagnosis to delivery by 6.7 weeks, decreased

373 incidence of spontaneous PTB at any given GA by 80-90%, decreased perinatal

374 mortality by 76% and improved perinatal outcome when compared with no cerclage.

375 Abbasi et al.13 published a retrospective cohort study conducted at a single institution in

376 Toronto. This study included women with twin pregnancies, dilated cervix, and intact
 18

377 membranes <25 weeks of gestation, 27 women had cerclage compared to 9 women

378 managed expectantly. Compared with the subjects enrolled in this study, their cohort

379 had more advanced GA and cervical dilation at diagnosis as 16 (44%) of them were ≥ 3

380 cm at diagnosis. Additionally, antibiotics, indomethacin or progesterone were not given

381 in either group. Women in the cerclage group experienced longer latency from

382 presentation to delivery (7.3±5.5 versus 0.5±0.3 weeks, p<.001) and a lower incidence

383 of PTB before 34, 32 and 28 weeks, while all women in the no cerclage group delivered

384 <28 weeks. The overall neonatal survival at discharge was 38/54 (70.4%) in the

385 cerclage group versus 10/18 (55.6%) in the no cerclage group (p=0.2). Han et al.14

386 published a cohort study from two hospitals in California that included twins with cervical

387 dilation > 1cm; the study included 54 women who received physical exam indicated

388 cerclage, and 17 women no cerclage. Maternal demographics were not presented

389 separately limiting comparison of the groups. Nonetheless, the use of cerclage was

390 associated with a significant decrease in the odds of PTB <32, <34, and <36 weeks.

391 Physical exam indicated cerclage in our study had no intraoperative complications; in

392 one case, the surgeon was unable to place the cerclage due to effaced and friable

393 cervix. Larger cohorts in singleton pregnancies have informed a 10-20% risk of

394 intraoperative rupture of the membranes, cervical laceration, and bleeding during the

395 procedure.25

396 Strengths and Limitations

397 The strengths of our study: this is the first randomized study enrolling only twin

398 pregnancies with dilated cervix to cerclage or no cerclage. Expectant management with
 19

399 no cerclage is the current standard of care for these women. There are no prior

400 dedicated RCTs in this population. Despite small sample size, we were able to show a

401 significant benefit to physical exam indicated cerclage. In our RCT, physical exam

402 indicated cerclage was associated with decreased perinatal mortality and overall

403 decreased in preterm birth at all gestational ages, however with the potential of more

404 early preterm deliveries. Some of the limitations include: small sample size as the

405 enrollment did not reach the intended 52 cases. The DSMB considered that it was

406 unethical to continue the study due to the considerable perinatal mortality in one of the

407 arms: 76.9% versus 17.6% (p<0.0001) and requested to unmask the arms of the study.

408 The primary and secondary outcomes were also significantly different, favoring the

409 cerclage group. Due to early termination of the study, we were unable to perform

410 additional sub analysis based on the degree of cervical dilation, GA before and after 20

411 weeks, use of progesterone or surgical techniques. The incidence of this challenging

412 obstetrical problem is very low. Only 3% of all deliveries are twins, of which about 5%

413 will have TVUCL ≤15mm,26 and even fewer will be identified with asymptomatic cervical

414 dilation ≥ 1cm before 24 weeks. The challenge of studying this clinical question is

415 highlighted by the fact that enrollment and randomization of 34 women was over a

416 period of 4 years at 8 academic centers in 8 different cities. The demographic

417 characteristics were not significant different but some imbalance in the participant

418 characteristics were noticed suggesting bias in the intervention allocation as the

419 enrollment was not stratified by institution or any of the maternal characteristics (like

420 history of preterm birth, gestational age or cervical dilation). In addition, we

421 acknowledge that four women were excluded from the study due to expired consent.
 20

422 We did not renew IRB documentation on time to have a new consent. There is also a

423 discrepancy between groups in withdrawals from the study (1 of 18 in the cerclage

424 group [5.6%] vs 3 of 16 in the no cerclage group [18.8%]). The outcomes of these four

425 cases could have biased the outcome of the study.

426 The history of PTB in prior pregnancy was present in only 6 (20%) of our study group

427 while TVUCL ≤ 25mm preceded cervical dilation in 20/24 (83.3%) evaluated women. It

428 has been established that cervical length <25 mm is the best predictor of PTB in twin

429 pregnancy,27 and the shorter the TVUCL and the earlier GA at presentation, the higher

430 the risk of PTB.28 Cervical changes are dynamic, in singleton pregnancy TVUCL may

431 present with further shortening as pregnancy progresses; this is the bases of the

432 recommendation of serial TVUCL in women with history of preterm birth.29 In women

433 without risk for preterm birth, 30% of women with TVUCL <11mm had cervical dilation >

434 1cm at pelvic exam.30 Serial cervical evaluation have shown cervical shortening in twins

435 as well,31 however there are no studies assessing how many of them will have cervical

436 dilation. We did not performed serial TVUCL evaluation in twins and we are not able to

437 provide the incidence of cervical dilation. While TVUCL is currently the best tool

438 available to screen for PTB in twin pregnancy, independent of other risk factors,

439 ACOG32 and SMFM still recommends against TVCL in twins as there are no current

440 proven therapies to offer and TVUCL should be reserved for randomized controlled.

441 Retrospective studies in singleton pregnancy recommend amniocentesis to rule out

442 subclinical intrauterine infection; amniocentesis is expected to select a better candidate

443 prior to physical exam indicated cerclage.33, 34 In our trial amniocentesis was not

444 mandatory as IRB found it coercive, only 4/30 (13.3%) participants agreed with the
 21

445 procedure. Assessment of amniotic fluid gram stain or amount of glucose in patients

446 prior to physical exam indicated cerclage have shown a positive predictive value of

447 47%, and a negative predictive value of 98% for intrauterine infection.35 Amniotic fluid

448 culture is considered the “gold standard” for diagnosis of infection, but cultures take

449 several days for final result and therefore limit the utility in clinical decision-making.

450 Multiple studies have reported an association between preterm labor/delivery and

451 various urogenital tract infections/colonization.36 The presence of cervico-vaginal

452 infections correlates with intra-amniotic infections and histologic chorioamnionitis,

453 considering the ascending infection and colonization the most probable pathway.37 In

454 our study only 4/30 (13.3%) had positive vaginal bacterial studies and 4/30 (13.3%)

455 were diagnosed with UTI. Our study had limited financial resources for additional

456 vaginal microbial studies or evaluation of cytokines in amniotic fluid or vagina, as we did

457 not have external funding; those studies included in our study were considered standard

458 of care per each institution.

459 While it was not prespecified in our RCT, all women who received physical exam

460 indicated cerclage also received indomethacin and antibiotics. This management is

461 similar to previous studies. The RCT by Althuisius et al. in 2003,3 used a combination of

462 indomethacin and antibiotics in all cases randomized to cerclage. In Roman et al.,12

463 retrospective case-control study of physical exam indicated cerclage in twins, the

464 majority of women received indomethacin 29 (76%) and prophylactic antibiotics 36

465 (94%) at the time of the cerclage placement. In a study by Miller et al.,38 a large

466 retrospective cohort of physical exam indicated cerclage in twins, 59 (57.3%) subjects

467 received indomethacin and 56 (54.3%) received antibiotics. Miller et al39 randomized
 22

468 women with singleton pregnancies undergoing physical exam indicated cerclage to

469 indometacin and antibiotics (n=26) or no additional treatment (n=24). The median

470 latency from cerclage placement to delivery, GA at delivery, and neonatal outcomes

471 were no different between groups, but post hoc analysis revealed a greater proportion

472 of pregnancies were prolonged by at least 28 days among women who received

473 indomethacin and perioperative antibiotics (24 [92.3%] vs 15 [62.5%], p=0.01). In our

474 trial, the combination of cerclage, indomethacin, and antibiotics significantly decreased

475 the PTB at different GA cut-offs, increased median latency from cerclage placement to

476 delivery, and decreased perinatal mortality. Finally, the numerous secondary endpoints

477 with no adjustment for multiple comparisons could have led to type 1 error.

478 Clinical implications

479 A prospective registry of twin pregnancies receiving physical exam indicated cerclage

480 would allow us to examine how differences in gestational age, cervical dilation, prolapse

481 of amniotic membranes, surgical technique, and association with other therapies like

482 progesterone supplementation may affect the prognosis and outcome. Prospective

483 studies on the incidence of twin diamniotic pregnancies with TVUCL < 25 mm before 24

484 weeks that presented posteriorly with dilated cervix and lastly prospective studies

485 evaluating therapies in diamniotic twins with short cervix. Our group is enrolling for an

486 RCT in twins with TVUCL <15mm comparing cerclage vs. no cerclage and vaginal

487 progesterone on both arms, NCT03340688.

488 Conclusion
 23

489 A combination of physical exam indicated cerclage, indomethacin, and antibiotics in

490 asymptomatic twin pregnancies before 24 weeks significantly decreases preterm birth

491 and perinatal mortality.

492

493 ACKNOWLEDGMENT

494 We thank the participant centers that screened for women with twin pregnancy at risk of

495 preterm birth but were unable to enroll patients: David McKenna, MD at Wright State

496 University, (Dayton, Ohio), Montse Palacio, MD at Hospital Clinic of Barcelona (Spain),

497 Federico Prefumo, MD at University of Brescia (Italy), Giuliana Simonazzi, MD at

498 University of Bologna (Italy), Patrycja Jarmużek, MD at University of Warsaw, (Poland),

499 Kate Pettit, MD at University of Virginia, Lone Hvidman, MD at Aarthus

500 Universitethospital, (Denmark) and Begona Martinez de Tejada, MD at University

501 Hospital of Geneva, (Switzerland).

502

503

504

505

506

507

508

509
 24

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632 TABLES

633 Table 1. Maternal Demographics

VARIABLE Physical exam

Indicated No
Cerclage Cerclage
n=17 n=13
Maternal Age (years) 31.6±4.4 28.2±5.1
Race
Caucasian 7 (41.2) 6 (46.2)
African-American 6 (35.3) 4 (30.8)
Hispanic 2 (11.8) 2 (15.4)
Asian/Indian 2 (11.8) 1 (7.7)
Nuliparity 11 (64.7) 9 (69.2)
Diamniotic-Dichorionic 16 (94.1) 12 (92.3)
Level of education
Incomplete high school 2 (11.8) 2 (15.4)
Completed high school 6 (35.3) 2 (15.4)
College/some college 9 (52.9) 9 (69.2)
Assisted Reproductive Technology 7 (41.2) 4 (30.8)
Prior PTB (16-36 weeks) 4 (23.5) 2 (15.4)
Prior cervical surgery (LEEP/CKC) 1 (5.9) 1 (7.7)
Smoking 1 (5.9) 0 (0)
BMI 27.8±4.4 30.0±8.6
BMI ≥ 30 4 (23.5) 6 (46.2)
CHTN 0 (0) 2 (15.4)
History of Preeclampsia 2 (11.8) 0 (0)
TVUCL done 14 (82.4) 10 (76.9)
TVUCL (<25 mm) 10/14 (71.4) 10/10 (100)
GA at randomization (weeks) 20.7±1.7 19.4±1.5
GA < 20 weeks 5 (29.4) 8 (61.5)
 28

Cervical dilation at diagnosis (cm) 1.8±0.8 2.1±0.7

1 cm 6 (35.3) 2 (15.4)
2 cm 9 (52.9) 7 (53.8)
3 cm 1 (5.9) 3 (23.1)
4 cm 1 (5.9) 1 (7.7)
Amniotic membrane prolapse
50% Into cervical canal 8 (47.1) 6 (46.1)
At external os 9 (52.9) 7 (53.8)
Amniocentesis 2 (11.8) 2 (15.4)
Vaginal progesterone 9 (52.9) 10 (76.9)
Indomethacin administration 14 (82.4) 4 (30.8)
Antibiotics at cerclage 14 (82.4) 0 (0)
Urinary infection 1 (5.9) 4 (30.8)
Bacterial vaginosis 2 (11.8) 1 (7.7)
Chlamydia 1 (5.9) 0 (0)
Gonorrhea 0 (0) 0 (0)
634
635 Variables described as mean ± standard deviation or frequencies (percentage).
636 GA: Gestational age
637 PTB: Preterm birth
638 CHTN: Chronic hypertension
639 BMI: Body mass index
640 TVUCL: Transvaginal ultrasound cervical length
641 LEEP: Loop electrosurgical excision procedure
642 CKC: Cold knife cone
643

644

645

646

647

648
 29

649 Table 2. Antepartum and delivery outcomes of twin pregnancies with dilated cervix

Physical
exam No
VARIABLE Indicated Cerclage RR (Cl, 95%) or NNT
p-value
Cerclage MD (Cl, 95%)
n=17 n=13
SPTB < 34 weeks 12 (70.6) 13 (100) 0.71 (0.52-0.96) 0.05 3.4

SPTB < 32 weeks 11 (64.7) 13 (100) 0.65 (0.46-0.92) 0.02 2.8

SPTB < 28 weeks 7 (41.2) 11(84.6) 0.49 (0.26-0.89) 0.02 2.3

SPTB < 24 weeks 5 (29.4) 11 (84.6) 0.35 (0.16-0.75) 0.004 1.8

Antepartum admission
9 (52.9) 1 (7.7) 6.88 (1.43-40) 0.02
ǂ
PPROM <34 weeks 11 (64.7) 5 (38.5) 1.68 (0.83-3.86) 0.26
Antenatal steroids 13 (76.5) 4 (30.8) 2.51 (1.12-5.84) 0.02
Tocolysis 6 (35.3) 3 (23.1) 1.50 (0.47-4.94) 0.7
GA at PPROM (weeks) 28.4±5.6 19.3±0.5 9.0 (3.4-14.6) † 0.02
GA at delivery (weeks) 29.1±1.7 22.5±3.9 6.9 (2.9-10.9) † <0.01

Diagnosis to delivery 8.3±5.8 2.9±3.0 5.6 (2.0-9.3) †

0.02
interval (weeks)

Mode of delivery
• Vaginal delivery
4 (23.5) 10 (76.9) 0.30 (0.12-0.76) 0.008
both
13 (76.5) 3 (23.1) 3.30 (1.19-9.3)
• CS both
Clinical
2 (11.8) 3 (23.1) 0.51 (0.1-2.6) 0.62
chorioamnionitis
Histological
4 (23.5) 8 (61.5) 0.38 (0.14-1.0) 0.06
chorioamnionitis
Abruption 3 (17.6) 1 (7.7) 2.29 (0.26-19.6) 0.6
Birth weight Twin A 1331±850 529±479 801 (1306 to 297) † 0.003
Birth weight Twin B 1349±950 502±432 847 (1384 to 309) † 0.003
Birth weight (g)* 1349±874 544±434 823 (446 to 1200) † <0.0001
24/26 3.3
Birth weight <1500g* 21/34 (61.7) 0.67 (0.50-0.89) 0.007
(92.3)
Apgar <7 at 5 min Twin 4/17 11/13 0.27 (0.11-0.68) 0.003
 30

A
Apgar < 7 at 5 min
5/17 11/13 0.34 (0.16-0.75) 0.004
Twin B
22/26 1.7
Apgar < 7 at 5 min* 9/34 (26.5) 0.26 (0.16-0.50) <0.0001
(84.6)
10/13
Both twins died 2/17 (11.7) 0.15 (0.04-0.58) 0.005
(76.9)
One twin died 2/17 (11.7) 0/13 (0) NA 0.49
Both twins alive 13/17 (76.4) 3/17 (17.6) 3.31 (1.18-9.25) 0.009
At least 1 newborn
15/17 (88.2) 3/13 (0.23) 3.8 (1.4-10.4) 0.0005
home
Perinatal mortality Twin
3/17 (17.6) 10/13 (76.9 0.22 (0.1-0.67) 0.002
A
Perinatal mortality Twin 3/17 (17.6) 10/13
B (76.9) 0.22 (0.1-0.67) 0.002

Overall perinatal 6/34 (17.6) 20/26 1.7

mortality* (76.9) 0.23 (0.1-0.49) <0.0001

650
651 Variables described as mean ± standard deviation or frequencies (percentage).
652 GA: Gestational age
653 PTB: Preterm birth
654 PPROM: Preterm premature rupture of membranes
655 CS: Cesarean section
656 MD: mean difference
657 RR: relative risk
658 CI: Confidence interval
659 NNT: number needed to treat
660 * Data including both twins A and B
661 † Data correspond to mean difference
662 ǂ Antenatal admission refers to admission for expectant management but not admission
663 for imminent delivery
664

665

666

667

668 Table 3 Outcome of neonates born alive

 31

Physical
exam No
VARIABLE Indicated RR (Cl, 95%) or
Cerclage Cerclage p-value
MD (Cl, 95%)
n=30 n=6

Admission to NICU 22 (73.3) 6 (100) 0.73 (0.6-1.0) 0.3

GA admitted to NICU 0.15 (-5.1 to 5.4) † 0.9

27.9±3.6 28.1±3.4
(weeks)

Neonatal mortality 2 (6.6) 0 -1.3 (0.1 to -21.01) 1.0

Composite neonatal 14 (46.6) 3 (50) 0.93 (0.4-2.2) 1.0

outcome

NICU LOS (days) 68.2±65.3 92.3±53.4 29.8 (-14.6 to -74.2) † 0.3

RDS (intubation or 14 (46.6) 2 (33.3) 1.41 (0.42-4.6) 0.67

CPAP)

IVH stage 3,4 4 (13.3) 1 (16.6) 0.80 (0.1-5.9) 1.0

NEC stage 3,4 0 0 NA --

Sepsis 2 (6.6) 1 (16.6) 0.40 (0.04-3.74) 0.43

ROP/Laser 5 (16.6) 1 (16.6) 1.0 (0.14-7.1) 1.0

669
670 Variables described as mean ± standard deviation or frequencies (percentage).
671 RR: relative ratio
672 MD: Mean difference
673 CI: Confidence interval
674 GA: Gestational age
675 LOS: Length of stay in the hospital
676 NICU: Neonatal intensive care unit
677 RDS: respiratory distress syndrome
678 CPAP: continuous positive airway pressure
679 IVH: Intraventricular hemorrhage
680 NEC: Necrotizing enterocolitis
681 ROP: Retinopathy of prematurity
682 NA: not applicable
683 † Data correspond to mean difference
684 Table 4. Neonatal outcomes treating twin pair as cluster unit
685
 32

686
ICC (95% CI)
VARIABLE
OR (CI 95%)* p-value *

Admission to NICU** 0.37 (0.02 to 8.58) 0.53 1

Perinatal mortality 0.09 (0.02 to 0.51) 0.006 0.86 (0.73 to 0.93)

Neonatal mortality** 1.30 (0.05 to 33.63) 0.88 -0.34 (-0.38 to 0.32)

687
688 Variables described as mean ± standard deviation or frequencies (percentage).
689 Boldface data, statistically significant
690 OR: odds ratio
691 CI: Confidence interval
692 NICU: Neonatal intensive care unit
693 ICC: intracluster correlation coefficient
694 *Generalized mixed-model analysis, treating twin pair as cluster unit
695 **Outcomes of only neonates born alive
696

697

698

699

700

701

702

703

704

705

706

707

708 FIGURE LEGENDS

 33

709 Figure 1. Randomization and follow up of participants

710

711

712 Figure 2. Survival curves of twin pregnancies that remained undelivered across
713 gestation
714

715 Kaplan-Meier survival curves indicating the proportions of women with twin pregnancy

716 in the physical exam indicated cerclage and control groups. The log-rank test showed

717 significant difference Hazard Ratio 0.33 (95%CI 0.13-0.8) p=0.0006

718

719