Antibiotic administration was associated with a reduction in the intensity of intra-amniotic inflammatory response in patients with preterm prelabor rupture of the membranes

Abstract

Background:

Preterm prelabor rupture of the membranes (PPROM) is frequently complicated by intra-amniotic inflammatory processes such as intra-amniotic infection and sterile intra-amniotic inflammation. Antibiotic therapy is recommended to patients with PPROM to prolong the interval between PPROM and delivery (latency period), to reduce the risk of clinical chorioamnionitis, and improve neonatal outcome. However, there is a lack of information regarding whether the administration of antibiotics can reduce the intensity of the intra-amniotic inflammatory response or eradicate microorganisms in patients with PPROM.

Objective:

The first aim of the study was to determine if antimicrobial agents can reduce the magnitude of the intra-amniotic inflammatory response in patients with PPROM by assessing the concentrations of interleukin (IL)-6 in amniotic fluid before and after antibiotic treatment. The second aim was to determine whether treatment with intravenous clarithromycin changes the microbial load of Ureaplasma spp. DNA in amniotic fluid.

Study design:

A retrospective cohort study included patients with a singleton gestation with PPROM between 24+0 and 33+6 weeks, who underwent transabdominal amniocentesis at the time of admission and received intravenous antibiotic treatment (clarithromycin for patients with intra-amniotic inflammation and benzylpenicillin/clindamycin in the cases of allergy in patients without intra-amniotic inflammation) for 7 days. Follow-up amniocenteses (every 7^th day after admission) were performed in the subset of patients with a latency period lasting longer than 7 days. Concentrations of IL-6 were measured in the samples of amniotic fluid with a bedside test, and the presence of microbial invasion of the amniotic cavity (MIAC) was assessed with culture and molecular microbiologic methods. Intra-amniotic inflammation was defined as a bedside IL-6 concentration ≥745 pg/mL in the samples of amniotic fluid. Intra-amniotic infection was defined as the presence of both MIAC and intra-amniotic inflammation; sterile intra-amniotic inflammation was defined as the presence of intra-amniotic inflammation without MIAC.

Results:

A total of 270 patients with PPROM were included in this study: 207 patients delivered within 7 days, and 63 patients delivered after 7 days of admission. Of the 63 patients who delivered after 7 days following the initial amniocentesis, 40 underwent a follow-up amniocentesis. Patients with intra-amniotic infection (n=7) and sterile intra-amniotic inflammation (n=7) were treated with intravenous clarithromycin. Patients without either MIAC or intra-amniotic inflammation (n=26) were treated with benzylpenicillin or clindamycin. Treatment with clarithromycin decreased the IL-6 concentration in amniotic fluid at the follow-up amniocentesis compared to the initial amniocentesis in patients with intra-amniotic infection (median: 295 pg/mL, IQR 72-673 vs. median: 2973 pg/mL, IQR 1750-6296; p=0.02) and those with sterile intra-amniotic inflammation (median 221 pg/mL, IQR 118-366 pg/mL vs. median 1446 pg/mL, IQR 1300-2941; p=0.02). Samples of amniotic fluid with Ureaplasma spp. DNA had a lower microbial load at the time of follow-up amniocentesis compared to the initial amniocentesis (median: 1.8x10⁴ copies DNA/mL 2.9x10⁴-6.7x10⁸ vs. median: 4.7x10⁷ copies DNA/mL IQR 2.9x10³-3.6x10⁷; p = 0.03).

Conclusion:

Intravenous therapy with clarithromycin was associated with a reduction in the intensity of the intra-amniotic inflammatory response in patients with PPROM with either intra-amniotic infection or sterile intra-amniotic inflammation. Moreover, treatment with clarithromycin was related to a reduction in the load of Ureaplasma spp. DNA in the amniotic fluid of patients with PPROM <34 weeks of gestation.

INTRODUCTION

Preterm prelabor rupture of the membranes (PPROM) is characterized by the rupture of fetal membranes and leakage of amniotic fluid before the onset of regular uterine activity <37 weeks of gestation.^1–10 PPROM complicates approximately 3%-4% of all deliveries and is responsible for one-third of all deliveries <37 weeks.^1–9 PPROM is one of the “great obstetrical syndromes”^11–15 because of its multiple etiologies,^16–19 a long pre-clinical phase,¹⁵ fetal involvement,^20–25 and a complex interaction between the maternal and fetal genome and the environment.^22,24–26

PPROM is often complicated by the presence of microorganisms in the amniotic fluid, i.e., microbial invasion of the amniotic cavity (MIAC)^27–39 with Ureaplasma spp. being the most frequent microorganisms,^{30–33,36,38–42} and intra-amniotic inflammation.^{29,31,33–35,38,40,43–58} The combination of these two conditions creates the following clinical subtypes of PPROM: (1) intra-amniotic infection (defined by the presence of both MIAC and intra-amniotic inflammation); (2) sterile intra-amniotic inflammation; (3) MIAC without intra-amniotic inflammation; or (4) without either MIAC or intra-amniotic inflammation.^39,59 There is evidence that the earlier the gestational age at PPROM, the higher the rates of MIAC,^39,59 intra-amniotic infection,^39,59 and sterile intra-amniotic inflammation.⁵⁹

The current standard management of patients with PPROM <34 weeks of gestation consists of an expectant approach, including the administration of corticosteroids and antibiotics.^60–62 Antibiotics are recommended for all patients with PPROM in order to prolong the latency period (interval between membrane rupture and delivery),^63–69 and reduce the rate of clinical chorioamnionitis,^63,70 early onset neonatal sepsis,⁶⁸ the need for surfactant therapy replacement, and oxygen therapy.⁶⁸ Antibiotics are thought to treat or prevent subclinical intra-amniotic infection from the ascension of bacteria from the vagina or cervix. ^61–72

Recently, antibiotic therapy has been shown to reduce the intensity of the intra-amniotic inflammatory response in a subset of patients with spontaneous preterm delivery with intact membranes⁷³ as well as in patients with cervical insufficiency.⁷⁴ However, the knowledge about the effect of antibiotics in the treatment of intra-amniotic inflammatory complications in PPROM is less clear.^53,75 In 2006, Gomez et al. reported that the antibiotic treatment of ceftriaxone, clindamycin, and erythromycin can rarely eradicate intra-amniotic infection.⁵³ Ten years later, Lee et al. showed that an intensive antibiotic treatment of ceftriaxone, clarithromycin, and metronidazole reduced intra-amniotic infection by about 33%.⁷⁵ These authors used clarithromycin—a semi-synthetic macrolide—instead of erythromycin.⁷⁵ The rationale for this choice was that Ureaplasma spp. are resistant to erythromycin in up to 80% of cases.^76,77

Clarithromycin, aside from its effective antibacterial activity against Ureaplasma spp.,^78–82 shows optimal placental passage.^83,84 In addition, this antibiotic is capable of inhibiting the production of pro-inflammatory cytokines.^85–92 Thus, clarithromycin might be effective not only in the treatment of intra-amniotic infection caused by Ureaplasma spp., but also in the treatment of sterile intra-amniotic inflammation in patients with PPROM. This antimicrobial agent also has anti-inflammatory properties through its actions in inhibiting the transcription factors of nuclear factor (NF) kappa B^93–95 and activator protein (AP)-1^92,96, which are major regulators in pro-inflammatory response.

This study had three objectives: (1) to determine the effect of intravenous clarithromycin therapy on the intensity of the intra-amniotic inflammatory response, by assessing the concentrations of interleukin (IL)-6 in amniotic fluid, in a subset of pregnancies with PPROM <34 weeks complicated by intra-amniotic infection and sterile intra-amniotic inflammation before and after antibiotic treatment; (2) to evaluate the effect of intravenous clarithromycin therapy on the microbial load of Ureaplasma spp. DNA in amniotic fluid; and (3) to assess the effect of intravenous therapy with non-macrolide antibiotics (benzylpenicillin/clindamycin) on the amniotic fluid IL-6 concentrations of patients without MIAC and intra-amniotic inflammation.

MATERIAL AND METHODS

A retrospective cohort study was conducted in pregnant patients who were admitted to the Department of Obstetrics and Gynecology, University Hospital Hradec Kralove, Czech Republic, between January 2014 and May 2019. Eligible patients who met the following criteria were selected for participation in this study: (1) age ≥18 years; (2) singleton pregnancy; (3) PPROM between gestational ages 24+0 and 33+6 weeks; and (4) written informed consent was obtained for transabdominal amniocentesis at the time of admission.

Patients were excluded if: (1) the planned amniocentesis was not performed; (2) they had pre-gestational or gestational diabetes mellitus and/or chronic hypertension or gestational hypertension, or preeclampsia; (3) the fetus had signs of fetal growth restriction, a congenital structural abnormality, or a chromosomal abnormality; or (4) active vaginal bleeding was present.

Gestational age was established by first-trimester fetal biometry. PPROM was diagnosed by examination with a sterile speculum to verify the pooling of amniotic fluid in the vagina. If uncertainty remained, leakage of amniotic fluid was confirmed by the presence of insulin-like growth factor binding proteins in the vaginal fluid (ACTIM PROM test; MedixBiochemica, Kauniainen, Finland). This test has been previously documented to be valuable for the diagnosis of PPROM,⁹⁷ and patients with a positive test and intact membranes are at risk for impending term or preterm delivery.^98–100 The performance of transabdominal amniocentesis to assess the intra-amniotic environment (bedside IL-6 test¹⁰¹ and MIAC) was offered to each patient admitted with a singleton pregnancy complicated by PPROM as a part of the clinical management of singleton pregnancies with PPROM.

Ultrasound-guided transabdominal amniocentesis was performed at the time of admission before administration of corticosteroids and antibiotics, and approximately 3 mL of amniotic fluid was aspirated. A total of 100 μL of non-centrifuged amniotic fluid was used for the bedside assessment of IL-6 concentrations. The remaining amniotic fluid was immediately dispensed into three polypropylene tubes. The first and second tubes, containing uncentrifuged samples of amniotic fluid, were immediately transported to the molecular biology and microbiology laboratories for polymerase chain reaction (PCR) testing for Ureaplasma spp., Mycoplasma hominis, and Chlamydia trachomatis to evaluate 16S rRNA, and for cultivation of amniotic fluid, respectively. The last tube was centrifuged for 15 minutes at 2000 g to remove cells and debris, divided into aliquots, and stored at −70°C.

Initial antibiotic treatment began once the results of amniotic fluid IL-6 were available. Patients with intra-amniotic inflammation (a concentration of bedside IL-6 ≥745 pg/mL) received clarithromycin intravenously, 500 mg every 12 hours, for 7 days, unless delivery occurred. Patients without intra-amniotic inflammation (a concentration of bedside IL-6 <745 pg/mL^101,102) were treated with benzylpenicillin—initially, 5.0 million IU intravenously and, further, 2.5 million IU every 6 hours intravenously for 7 days, unless delivery occurred. In case of a penicillin allergy, patients were treated with clindamycin, 900 mg intravenously every 8 hours for 7 days, unless delivery occurred. Once the final results from cultivation or PCR were known, the attending clinician made individualized treatment to determine the optimal antibiotic therapy.

Patients received a course of corticosteroid treatment—betamethasone 14 mg intramuscularly, 24 hours apart. Tocolytic therapy was not routinely used at admission; however, if steroids were administered to induce fetal lung maturity, tocolysis with nifedipine or atosiban was administered to patients without intra-amniotic inflammation if they experienced regular increased uterine contractility. The decision was based on the discretion of the attending clinician.

Patients with intra-amniotic infection (proven MIAC and intra-amniotic inflammation) >28+0 weeks of gestation were managed actively: labor was induced or an elective caesarean delivery was performed after completing corticosteroid treatment but no later than 72 hours after the rupture of the membranes. Other patients were managed expectantly.

A follow-up amniocentesis was offered to patients with PPROM when antibiotic treatment ended.

The collection of amniotic fluid samples for research purposes was approved by the Institutional Review Board of University Hospital Hradec Kralove (March 19, 2008: No. 200804 SO1P; renewed in July, 2014: No 201407 S14P), and written informed consent was received from all participants prior to sampling. The use of amniocentesis for the management of PPROM in our department has been the subject of several reports.^103,104 This also applies to the use of serial amniocenteses to assess the response to anti-microbial therapy.^28,53,75

Bedside testing for the concentration of interleukin-6 in amniotic fluid

The IL-6 concentration in fresh uncentrifuged amniotic fluid was assessed with a Milenia® QuickLine IL-6 lateral flow immunoassay (Milenia® POCScan Reader, Milenia Biotec, GmbH, Giessen, Germany). The measurement range was 50 pg/mL to 10000 pg/mL. The intra- and inter-assay coefficients of variation were 12.1% and 15.5%, respectively. This point-of-care test is available for patient care in Europe and has been approved by regulatory agencies.^103–106

Detection of Ureaplasma spp., Mycoplasma hominis, and Chlamydia trachomatis

DNA was isolated from the amniotic fluid with a QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions (using the protocol for the isolation of bacterial DNA from biological fluids). Real-time PCR was performed on a Rotor-Gene 6000 instrument (QIAGEN) with the commercial kit AmpliSens® C. trachomatis/Ureaplasma/M. hominis- FRT (Federal State Institution of Science, Central Research Institute of Epidemiology, Moscow, Russia) to detect the DNA from Ureaplasma spp., Mycoplasma hominis, and Chlamydia trachomatis in a common PCR tube. As a control, a PCR run for beta-actin, a housekeeping gene, was included to assess the presence of inhibitors of the polymerase chain reaction. The amount of Ureaplasma spp. DNA in copies/mL was determined by absolute quantification employing an external calibration curve. Plasmid DNA (pCR4, Invitrogen, Waltham, MA) was used for the preparation of the calibration curve. The concentration of Ureaplasma spp. DNA in copies/μL was converted into copies/mL using the following formula: concentration of Ureaplasma spp. DNA (copies/μL) x elution volume (μL) / input volume (mL).

Detection of other bacteria in amniotic fluid

Bacterial DNA was identified by PCR targeting the 16S rRNA gene with the following primers: 5‘-CCAGACTCCTACGGGAGGCAG-3‘ (V3 region) and 5‘-ACATTTCACAACACGAGCTGACGA-3‘ (V6 region).^107,108 Each individual reaction contained 3 μL of target DNA, 500 nM of forward and reverse primers, and Q5® High-Fidelity DNA Polymerase (New England Biolabs Inc., Ipswich, MA, USA) in a total volume of 25 μL. The amplification was performed in a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The products were visualized on an agarose gel. Positive reactions yielded products of 950 bp, which were subsequently analyzed by sequencing. The 16S PCR products were cleaned and used in sequencing PCR reactions, utilizing the above-named primers and the BigDye® Terminator Cycle Sequencing Kit, version 3.1 (ThermoFisher Scientific, Waltham, MA, USA). Bacteria were then typed using the sequences obtained in BLAST® and SepsiTest™ BLAST. These tests are routinely offered clinically in our medical center and do not represent research tests.^39,109–114

Aerobic and anaerobic cultures of amniotic fluid

The amniotic fluid samples were cultured on Columbia agar with sheep’s blood, a Gardnerella vaginalis selective medium, MacConkey agar, a Neisseria-selective medium (modified Thayer–Martin medium), Sabouraud agar, or Schaedler anaerobe agar. The plates were cultured for 6 days and checked daily. The species were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with the MALDI Biotyper software (Bruker Daltonics, Bremen, Germany).

Diagnosis of microbial invasion of the amniotic cavity

MIAC was defined as the presence of microorganisms detected by culture and/or the detection of microbial nucleic acids in amniotic fluid.

Definitions of intra-amniotic inflammation, intra-amniotic infection, sterile intra-amniotic inflammation

The diagnosis of intra-amniotic inflammation in pregnancies with PPROM was defined as a concentration of bedside interleukin IL-6 in amniotic fluid ≥745 pg/mL.^115,116 We are aware of other studies in which a cutoff concentration of IL-6 of 2.6 ng/mL was used to define intra-amniotic inflammation¹¹⁷; however, our study employed a point-of-care immunoassay in which the cutoff for the detection of intra-amniotic inflammation was ≥745 pg/mL.

Intra-amniotic infection was defined as the presence of MIAC with intra-amniotic inflammation. Sterile intra-amniotic inflammation was defined as the presence of intra-amniotic inflammation without MIAC.

Diagnosis of histologic chorioamnionitis and funisitis

The degree of polymorphonuclear leukocyte infiltration was assessed separately in the free membranes (amnion and choriodecidua), chorionic plate, and umbilical cord according to the criteria provided by Kim et al.⁵⁹ The diagnosis of histologic chorioamnionitis was based on the presence of inflammatory changes in the choriodecidua (grades 3-4), chorionic plate (grades 3-4), umbilical cord (grades 1-4), and/or amnion (grades 1-4). The diagnosis of funisitis was based on the presence of inflammatory changes in the umbilical cord (grades 1-4)¹¹⁸.

Diagnosis of short-term neonatal morbidity

Maternal and perinatal medical records were reviewed by five investigators (MK, MS, JS, JM, and IM). “Composite neonatal morbidity” was defined in this study as: the need for intubation during hospitalization and/or respiratory distress syndrome (defined by the presence of two or more of the following criteria: evidence of respiratory compromise, persistent oxygen requirement for more than 24 hours, administration of exogenous surfactant, and radiographic evidence of hyaline membrane disease); and/or bronchopulmonary dysplasia (defined as oxygen requirement at 28 days of age); and/or pneumonia (diagnosed by abnormal findings on chest X-rays); and/or retinopathy of prematurity (identified using retinoscopy); and/or intraventricular hemorrhage (diagnosis made using cranial ultrasound examinations according to the procedure of Papile et al.¹¹⁹); and/or necrotizing enterocolitis (defined as radiologic findings of either intramural gas or portal venous gas with or without free intra-abdominal gas); and/or early- (during the first 72 hours of life) or late-onset (between the ages of 4 and 120 days) sepsis (either proven by bacterial culture or strong clinical suspicion of sepsis); and/or neonatal death before hospital discharge.

Statistical analysis

Demographic characteristics were compared by the non-parametric Kruskal-Wallis and Mann-Whitney U tests for continuous variables and presented as the median (interquartile range [IQR]). Categorical variables were compared with the chi-square or Fisher’s exact test and presented as numbers (%). Normality of the data was tested using the Anderson-Darling and D’Agostino-Pearson omnibus normality tests. Concentrations of IL-6 and microbial loads of Ureaplasma spp. in amniotic fluid between the initial and follow-up amniocenteses were compared with the Wilcoxon matched-pairs signed rank test. Differences were considered statistically significant at p<0.05. All p-values were obtained from two-sided tests, and all statistical analyses were performed using GraphPad Prism 8.1.2 for Mac OS X (GraphPad Software, San Diego, CA, USA) or the SPSS 19.0 statistical package for Mac OS X (SPSS Inc., Chicago, IL, USA).

RESULTS

Three hundred seventeen patients with PPROM met the entry criteria. An initial amniocentesis was performed in 98% (312/317) of patients (amniocentesis could not be performed due to anhydramnios and delivery occurred before the procedure in three and two patients, respectively). Subsequently, 42 patients were excluded from the study because of fetal growth restriction (n=18), gestational diabetes mellitus (n=9), chronic hypertension (n=7), pregestational diabetes mellitus (n=5), preeclampsia (n=1), gestational hypertension (n=1), and active severe vaginal bleeding (n=1). Therefore, the remaining 270 patients are included in this report. The overall rate of MIAC and intra-amniotic inflammation was 29% (79/270) and 28% (75/270), respectively. The presence of intra-amniotic infection was diagnosed in 20% (55/270), sterile intra-amniotic inflammation in 7% (20/270), and MIAC without intra-amniotic inflammation in 9% (24/270) of patients.

Twenty-three percent (63/270) of patients delivered >7 days after admission and 37% (23/63) of these did not have a follow-up amniocentesis for the following reasons: 1) the follow-up amniocentesis was declined by the patient (n=12); 2) the managing clinicians decided not to perform a follow-up amniocentesis (n=4); 3) labor began on the day of the follow-up amniocentesis (n=4); or 4) the follow-up amniocentesis could not be performed due to anhydramnios (n=3). The remaining 63% (40/63) of patients underwent a follow-up amniocentesis (Figure 1). The flow and antibiotic management of the patients who underwent a follow-up amniocentesis are shown in Figure 2.

Figure 1.

Flow of patients with a follow-up amniocentesis.

Figure 2.

Flow of patients throughout the study.

Abbreviations: MIAC, microbial invasion of the amniotic cavity.

The demographical and clinical data of patients who delivered ≤7 days and >7 days after admission without or with follow-up amniocentesis are shown in Table 1. Bacterial species identified in amniotic fluid from the initial amniocentesis are listed in Table 2. Short-term outcomes of the newborns from PPROM pregnancies are listed in Table 3.

Table 1.

Maternal and clinical characteristics of patients with preterm prelabor rupture of the membranes between gestational ages 24+0 and 33+6 weeks

Characteristic	Delivery ≤ 7 days after admission (n=207)	Delivery > 7 days after admission without a follow-up amniocentesis (n=23)	Delivery > 7 days after admission with a follow-up amniocentesis (n=40)	p-value
Maternal age [years, median (IQR)]	31 (27-36)	33 (29-37)	31 (29-34)	0.47
Primiparous [number (%)]	98 (47%)	10 (44%)	12 (30%)	0.13
Pre-pregnancy body mass index [kg/m2, median (IQR)]	23.6 (21.1-26.7)	24.1 (21.6-28.3)	23.4 (20.4-25.9)	0.52
Smoking [number (%)]	36 (17%)	3 (13%)	5 (13%)	0.67
History of preterm prelabor rupture of the membranes [number (%)]	13 (6%)	2 (9%)	2 (5%)	0.84
Time interval between rupture of the membranes and amniocentesis [hours, median (IQR)]	5 (3-10)	5 (2-10)	7 (3-14)	0.25
Gestational age at admission [weeks, median (IQR)]	32+1 (30+5-33+2)	30+0 (27+5-31+2)	28+0 (25+3-31+1)	<0.0001
Gestational age at delivery [weeks, median (IQR)]	32+4 (30+6-33+4)	31+4 (29+2-33+1)	32+0 (29+4-33+2)	0.12
Latency period [days, median (IQR)]	2 (1-3)	12 (9-14)	17 (12-28)	<0.0001
Amniotic fluid interleukin-6 concentrations at admission [pg/mL, median (IQR)]	250 (134-887)	188 (50-327)	383 (158-1424)	0.12
Maternal serum C-reactive protein concentrations at admission [mg/L, median (IQR)]	6.1 (2.7-11.3)	5.8 (3.1-8.6)	4.1 (1.6-8.1)	0.07
Maternal white blood cells count at admission [x109 L, median (IQR)]	13.0 (10.5-16.0)	12.2 (9.1-14.5)	12.1 (10.2-14.2)	0.05
Vaginal-rectal presence of Streptococcus agalactiae	25 (12%)	3 (13%)	3 (8%)	0.69
Microbial invasion of the amniotic cavity [number (%)]	67 (32%)	5 (22%)	7 (18%)	0.06
Intra-amniotic inflammation [number (%)]	58 (28%)	3 (13%)	14 (35%)	0.17
Intra-amniotic infection [number (%)]	46 (22%)	2 (9%)	7 (18%)	0.28
Sterile intra-amniotic inflammation [number (%)]	12 (6%)	1 (4%)	7 (18%)	0.03
Microbial invasion of the amniotic cavity without intra-amniotic inflammation [number (%)]	23 (11%)	3 (13%)	0 (0%)	0.08
Administration of antibiotics [number (%)]	207 (100%)	23 (100%)	40 (100%)	-
Administration of corticosteroids [number (%)]	201 (97%)	22 (96%)	40 (100%)	0.49
Spontaneous vaginal delivery [number (%)]	138 (67%)	7 (30%)	21 (53%)	0.001
Cesarean delivery [number (%)]	68 (33%)	16 (70%)	19 (47%)	0.02
Forceps delivery [number (%)]	1 (1%)	0 (0%)	0 (0%)	0.86
Birth weight [grams, median (IQR)]	1880 (1550-2130)	1700 (1190-1960)	1630 (1310-2060)	0.03
Apgar score <7; 5 minutes [number (%)]	6 (3%)	2 (9%)	2 (5%)	0.34
Apgar score <7; 10 minutes [number (%)]	5 (2%)	0 (0%)	0 (0%)	0.46

Abbreviations:

IQR: interquartile range

Continuous variables were compared using a nonparametric Kruskal-Wallis test. Categorical variables were compared using the chi-square test. Continuous variables are presented as median (IQR) and categorical as number (%).

Statistically significant results are marked in bold.

IQR: interquartile range

Table 2.

Bacterial species identified in the amniotic fluid obtained during the initial amniocentesis from patients with preterm prelabor rupture of the membranes.

Intra-amniotic infection (n=55)			Microbial invasion of the amniotic cavity without intra-amniotic inflammation (n=24)
Delivery ≤7 days after admission (n=46)	Delivery > 7 days after admission without a follow-up amniocentesis (n=2)	Delivery > 7 days after admission with a follow-up amniocentesis (n=7)	Delivery ≤7 days after admission (n=21)	Delivery > 7 days after admission without a follow-up amniocentesis (n=3)	Delivery > 7 days after admission with a follow-up amniocentesis (n=0)
Ureaplasma species 21x	Ureaplasma species 1x	Ureaplasma species 6x	Ureaplasma species 14x	Ureaplasma species 2x
Ureaplasma species + Chlamydia trachomatis 5x	Ureaplasma species + Chlamydia trachomatis 1x	Anaerococcus tetradius 1x	Mycoplasma hominis 2x	Lactobacillus iners 1x
Haemophilus influenzae 4x			Chlamydia trachomatis 1x
Fusobacterium nucleatum 2x			Haemophilus influenzae 1x
Streptococcus agalactiae 2x			Gardnerella vaginalis 1x
Streptococcus anginosus 2x			Ureaplasma species + Chlamydia trachomatis 1x
Streptococcus pneumonia 2x			Ureaplasma species + Leptotrichia amnionii 1x
Chlamydia trachomatis 1x
Parvominas micra 1x
Peptoniphilus species 1x
Sneathia sanguinegens 1x
Streptococcus intermedius 1x
Leptotrichia amnionii + Chlamydia trachomatis 1x
Ureaplasma species + Sneathia sanguinegens 1x
Ureaplasma species + Veilonella species 1x

Table 3.

Selected aspects of short-term morbidity of the newborns from pregnancies complicated by preterm prelabor rupture of the membranes between gestational ages 24+0 and 33+6 weeks.

	Delivery ≤ 7 days after admission (n = 207)	Delivery > 7 days after admission without a follow-up amniocentesis (n = 23)	Delivery > 7 days after admission with a follow-up amniocentesis (n = 40)	p-value
Respiratory distress syndrome	99 (48%)	12 (52%)	19 (48%)	0.92
Need for intubation	13 (6%)	1 (4%)	6 (15%)	0.13
Intraventricular hemorrhage	53 (26%)	4 (17%)	7 (18%)	0.41
Intraventricular hemorrhage (I-II)	51 (25%)	3 (13%)	7 (18%)	0.32
Intraventricular hemorrhage (III-IV)	2 (1%)	1 (4%)	0 (0%)	0.26
Necrotizing enterocolitis	6 (3%)	0 (0%)	0 (0%)	0.39
Early-onset sepsis	14 (7%)	1 (4%)	1 (3%)	0.55
Late-onset sepsis	4 (2%)	0 (0%)	0 (0%)	0.54
Bronchopulmonary dysplasia	15 (7%)	3 (13%)	5 (13%)	0.40
Retinopathy of prematurity	3 (2%)	0 (0%)	1 (3%)	0.73
Pneumonia	4 (2%)	1 (4%)	0 (0%)	0.46
Neonatal death	4 (2%)	1 (4%)	0 (0%)	0.46
Composite neonatal morbidity	126 (61%)	15 (65%)	24 (60%)	0.91

Composite neonatal morbidity was defined as a need for intubation and/or respiratory distress syndrome and/or pneumonia and/or bronchopulmonary dysplasia and/or retinopathy of prematurity and/or intraventricular hemorrhage and/or necrotizing enterocolitis and/or early-onset sepsis and/or late-onset sepsis and/or neonatal death before hospital discharge.

Categorical variables were compared using the chi-square test.

The effect of clarithromycin therapy on intra-amniotic inflammation in a subset of patients with intra-amniotic infection

In the subset of patients with intra-amniotic infection who had a follow-up amniocentesis and delivered >7 days after admission, the concentrations of IL-6 in amniotic fluid were lower in the samples from the follow-up amniocentesis than in the initial amniocentesis (follow-up: 295 pg/mL, IQR 72-673 versus initial: 2973 pg/mL, IQR 1750-6296; p=0.02; Figure 3a). Importantly, 86% (6/7) of the patients did not have intra-amniotic inflammation at the time of the follow-up amniocentesis.

Figure 3.

Concentrations of interleukin-6 in amniotic fluid from the subset of the patients with preterm prelabor rupture of the membranes with intra-amniotic infection (a), with sterile intra-amniotic inflammation (b), and without either microbial invasion of the amniotic cavity or intra-amniotic inflammation (c) who underwent a follow-up amniocentesis.

Description: full circle = intra-amniotic infection; empty circle = sterile intra-amniotic inflammation; full triangle = microbial invasion of the amniotic cavity without intra-amniotic inflammation; empty triangle = without either microbial invasion of the amniotic cavity or intra-amniotic inflammation; dotted line = cut-off value of 745 pg/mL Patients who received clindamycin are marked in red. Abbreviations: ATB, antibiotic; MIAC, microbial invasion of the amniotic cavity; PPROM, preterm prelabor rupture of membranes.

In the subset of patients with intra-amniotic infection who had the follow-up amniocentesis, 86% (6/7) had proven Ureaplasma spp. DNA in the amniotic fluid samples from both the initial and follow-up amniocenteses. The microbial load for Ureaplasma spp. DNA was lower in the amniotic fluid from the follow-up amniocenteses than from the initial amniocentesis (follow-up: 1.8x10⁴ copies DNA/mL, IQR 2.9x10³-3.6x10⁷ versus initial: 4.7x10⁷ copies DNA/mL, IQR 2.9x10⁴-6.7x10⁸; p = 0.03; Figure 4). One patient had Anaerococcus tetradius in the amniotic fluid collected from the initial amniocentesis (proven by both PCR and culture methods); however, the amniotic fluid from the follow-up amniocentesis after antibiotic therapy was negative (by both PCR and culture studies).

Figure 4.

Microbial load of Ureaplasma spp. DNA in amniotic fluid from the subset of patients with preterm prelabor rupture of the membranes with intra-amniotic inflammation who underwent a follow-up amniocentesis.

Description: full circle = intra-amniotic infection; full triangle = microbial invasion of the amniotic cavity without intra-amniotic inflammation.

Abbreviations: ATB, antibiotic; PPROM, preterm prelabor rupture of the membranes.

The effect of clarithromycin therapy on intra-amniotic inflammation in a subset of patients with sterile intra-amniotic inflammation

In the subset of the patients with sterile intra-amniotic inflammation who delivered >7 days after admission and underwent a follow-up amniocentesis, the concentrations of IL-6 in amniotic fluid were lower in the samples from the follow-up amniocenteses than the initial procedures (follow-up: median 221 pg/mL, IQR 118-366 pg/mL versus initial: median 1446, IQR 1300-2941 pg/mL; p=0.02; Figure 3b). At the time of the follow-up amniocentesis, 86% (6/7) no longer had intra-amniotic inflammation.

The effect of non-macrolide antibiotics (benzylpenicillin/clindamycin) therapy on intra-amniotic inflammation in a subset of the patients without either microbial invasion of the amniotic cavity or intra-amniotic inflammation

In the subset of patients without either MIAC or intra-amniotic inflammation, the concentrations of IL-6 were lower in the amniotic fluid from the follow-up amniocenteses than from the initial amniocenteses (follow-up: median 79 pg/mL, IQR 50-301 versus initial: median 175 pg/mL, IQR 118-369; p=0.045, Figure 3c). After excluding the patients treated with clindamycin (n=5), the concentrations of IL-6 in amniotic fluid from the follow-up amniocenteses were lower than those from the initial amniocenteses (follow-up: median 77 pg/mL, IQR 50-195 versus initial: median 159 pg/mL, IQR 106-341; p = 0.02).Two cases of intra-amniotic infection, caused by Candida albicans (benzylpenicillin group) and Ureaplasma spp. (clindamycin group) developed during the latency period.

Outcome of patients with microbial invasion of the amniotic cavity without intra-amniotic inflammation

Among patients with MIAC without intra-amniotic inflammation, 88% (21/24) delivered ≤7 days after admission and 12% (3/24) delivered >7 days after admission without the performance of a follow-up amniocentesis.

Outcomes of patients with preterm prelabor rupture of the membranes who underwent a follow-up amniocentesis

Table 4 shows the outcomes of 14 patients with PPROM with intra-amniotic infection and sterile intra-amniotic inflammation. Table 5 describes the outcomes of 26 patients with PPROM without either MIAC or intra-amniotic inflammation who had a follow-up amniocentesis.

Table 4.

Details of the presentations from the initial and follow-up amniocenteses and the outcomes of patients who were treated with clarithromycin and underwent a follow-up amniocentesis.

	Gestational age at admission (week+days)	Initial amniocentesis at the time of admission			Follow-up amniocentesis after 7 days			Latency (days)	HCA (Yes/No)	Funisitis (Yes/No)	Short-term neonatal morbidity
		Culture	PCR	IL-6 (pg/mL)	Culture	PCR	IL-6 (pg/mL)
1.	24+0	Negative	Ureaplasma spp. (2.0x109 cp/mL)	10000	Negative	Ureaplasma spp. (1.3x108 cp/mL)	655	14	Yes	Yes	RDS, ROP, BPD
2	24+1	Anaerococcus tetradius	Anaerococcus spp.	5125	Negative	Negative	295	40	Yes	No	RDS
3.	26+1	Negative	Ureaplasma spp. (3.0x104 cp/mL)	6296	Negative	Ureaplasma spp. (5.3x103 cp/mL)	10000	38	Yes	No	RDS
4.	26+5	Negative	Ureaplasma spp. (2.8x107 cp/mL)	2973	Negative	Ureaplasma spp. (5.1x106 cp/mL)	215	14	Yes	Yes	RDS, BPD
5.	27+0	Negative	Ureaplasma spp. (2.3x108 cp/mL)	2282	Negative	Ureaplasma spp. (2.2x103 cp/mL)	943	21	Yes	Yes	RDS, IVH I
6.	27+6	Negative	Ureaplasma spp. (2.6x104 cp/mL)	828	Negative	Ureaplasma spp. (2.8x103 cp/mL)	72	16	Yes	No	RDS
7.	30+4	Negative	Ureaplasma spp. (6.7x107 cp/mL)	1755	Negative	Ureaplasma spp. (2.3x104 cp/mL)	50	8	Yes	No	-
8.	24+0	Negative	Negative	1359	Negative	Negative	842	58	No	No	-
9.	24+0	Negative	Negative	1246	Negative	Negative	221	74	No	No	RDS
10.	24+1	Negative	Negative	10000	Negative	Negative	295	26	Yes	Yes	-
11.	25+0	Negative	Negative	1800	Negative	Negative	50	26	Yes	No	RDS, BPD
12.	25+2	Negative	Negative	1251	Negative	Negative	118	29	Yes	No	RDS
13.	25+5	Negative	Negative	2941	Negative	Negative	152	16	No	No	RDS
14.	29+5	Negative	Negative	1446	Negative	Negative	366	16	Yes	No	RDS

Abbreviations:

BPD: bronchopulmonary dysplasia

HCA: histological chorioamnionitis

IL-6: interleukin-6

IVH: intraventricular hemorrhage

PCR: polymerase chain reaction

RDS: respiratory distress syndrome

ROP: retinopathy of prematurity

Spp.: species

Table 5.

Details of the presentations from the initial and follow-up amniocentesis and the outcomes of patients who were treated with benzylpenicillin / clindamycin and underwent a follow-up amniocentesis

	Gestational age at admission (week+days)	Initial amniocentesis at the time of admission			Follow-up amniocentesis after 7 days			Latency (days)	HCA (Yes/No)	Funisitis (Yes/No)	Short-term neonatal morbidity
		Culture	PCR	IL-6 (pg/mL)	Culture	PCR	IL-6 (pg/mL)
1.*	24+5	Negative	Negative	441	Negative	Negative	538	23	Yes	Yes	RDS, EOS, BPD
2.	24+6	Negative	Negative	158	Negative	Negative	136	33	No	No	RDS
3.	25+1	Negative	Negative	73	Negative	Negative	50	13	Yes	Yes	RDS, BPD
4.	26+5	Negative	Negative	50	Negative	Negative	50	17	Yes	No	RDS
5.	27+0	Negative	Negative	454	Negative	Negative	177	17	Yes	Yes	RDS
6.	27+4	Negative	Negative	225	Negative	Negative	189	38	No	No	IVH 1-2
7.	28+0	Negative	Negative	599	Negative	Negative	59	31	Yes	Yes	-
8.	28+0	Negative	Negative	166	Negative	Negative	50	42	Yes	No	IVH 1-2
9.	28+0	Negative	Negative	65	Negative	Negative	50	23	Yes	Yes	-
10.	29+3	Negative	Negative	356	Negative	Negative	128	12	Yes	No	-
11.*	29+4	Negative	Negative	335	Negative	Negative	137	17	No	No	RDS, IVH 1-2
12.	29+6	Negative	Negative	660	Negative	Negative	317	9	Yes	Yes	RDS
13.*	30+0	Negative	Negative	262	Negative	Negative	52	17	No	No	IVH 1-2
14.	30+3	Negative	Negative	326	Negative	Negative	296	18	No	No	-
15.	30+6	Negative	Negative	182	Negative	Negative	50	15	Yes	No	-
16.	31+1	Negative	Negative	50	Negative	Negative	79	18	No	No	-
17.*	31+1	Negative	Negative	409	Negative	Negative	369	12	Yes	No	-
18.	31+4	Negative	Negative	107	Candida albicans	Negative	10000	8	Yes	Yes	-
19.	31+5	Negative	Negative	167	Negative	Negative	50	11	No	No	RDS
20.	31+5	Negative	Negative	105	Negative	Negative	50	10	Yes	Yes	-
21.	32+1	Negative	Negative	159	Negative	Negative	200	10	Yes	No	-
22.	32+3	Negative	Negative	507	Negative	Negative	592	8	Yes	Yes	IVH 1-2
23.	33+0	Negative	Negative	122	Negative	Negative	50	8	No	No	-
24.	33+0	Negative	Negative	123	Negative	Negative	78	14	Yes	Yes	-
25.	33+0	Negative	Negative	132	Negative	Negative	73	9	No	No	RDS, IVH 1-2
26.*	33+6	Negative	Negative	193	Negative	Ureaplasma spp.(1.3x106 cp/mL)	7173	9	Yes	Yes	-

Abbreviations:

BPD: bronchopulmonary dysplasia

HCA: histological chorioamnionitis

IL-6: interleukin-6

IVH: intraventricular hemorrhage

PCR: polymerase chain reaction

RDS: respiratory distress syndrome

Spp.: species

Five patients (*) received clindamycin instead of benzylpenicillin because of allergy

DISCUSSION

Principal findings of this study

(1) Treatment with clarithromycin was associated with a reduction in the intensity of the intra-amniotic inflammatory response in patients with PPROM and intra-amniotic infection who underwent a follow-up amniocentesis; (2) treatment with clarithromycin was related to the reduction of a microbial load of Ureaplasma spp. measured by the gene copy numbers in amniotic fluid; (3) treatment with clarithromycin was associated with an attenuation of the intensity of the intra-amniotic inflammatory response in the patients with PPROM and sterile intra-amniotic inflammation who underwent a follow-up amniocentesis; (4) treatment with non-macrolide antibiotics was also associated with a reduction in the intensity of the intra-amniotic inflammatory response in the subset of patients with PPROM without either MIAC or intra-amniotic inflammation who had a follow-up amniocentesis. Collectively, these observations provide objective evidence that antimicrobial agents can down-regulate and/or eradicate the intra-amniotic inflammatory response, as well as the presence of microorganisms in a subset of patients with PPROM.

The results of the study in the context of what is known

Although there is no clear consensus about the specific type of antibiotics or the optimal combination for patients with PPROM to improve maternal and neonatal outcomes, the administration of erythromycin alone or in a combination is broadly recommended and used.^60,62,71 Nevertheless, some clinicians advocate for the use of the other macrolide antibiotics instead of erythromycin, given that many Ureaplasma spp. are resistant to this antibiotic.^76,77 One alternative is azithromycin, favored for its higher efficacy against Ureaplasma spp.^78,120 In addition, a recent multicenter retrospective cohort study has shown that azithromycin could be an alternative to erythromycin in the management of patients with PPROM because there were no differences in the latency period, the rate of chorioamnionitis, or neonatal outcomes between the patients treated with azithromycin or erythromycin.¹²¹

However, clarithromycin, the other member of the macrolide family, may be an even better choice in PPROM, since the resistance of Ureaplasma spp. to this antimicrobial agent is approximately 7.5%⁷⁸ compared to azithromycin, whose resistance is approximately 22%.⁷⁸ In addition, an ex vivo study has shown that clarithromycin also has a higher transplacental transfer rate (6%) than erythromycin (3%) or azithromycin (2%).⁸⁴ Another study identified an even better transplacental transfer of clarithromycin (8%) in placentas from PPROM pregnancies <34 weeks of gestation.⁸³ The next most important factor supporting the use of clarithromycin is that this antibiotic, along with its active metabolite (14-OH-R-clarithromycin), is mostly eliminated by the liver and kidneys.^122,123 The latter mechanism of elimination is important given, that this would result in an even higher concentration in amniotic fluid to eradicate bacteria in this location. The urinary excretions of clarithromycin and 14-OH-R-clarithromycin are approximately 18% and 14%, respectively.^122,123

In this study, all patients with intra-amniotic infection who underwent a follow-up amniocentesis were treated with intravenous clarithromycin. In this subset of patients, attenuation of the intensity and resolution of intra-amniotic inflammation was identified in 100% (7/7) and in 86% (6/7) of patients, respectively.

However, the patient who still met criteria for intra-amniotic inflammation after the treatment with clarithromycin showed clear improvement as demonstrated by a latency period of 21 days and the absence of serious neonatal morbidity.

It is well known that Ureaplasma spp. are the most common microorganisms found in amniotic fluid from PPROM pregnancies.^{30–33,36,38–42,53,103,124–130} Although the first report documented the successful eradication of Ureaplasma urealyticum from the amniotic fluid in one patient with PPROM treated with erythromycin, ampicillin, gentamycin, and clindamycin in 1992²⁸, however, Gomez et al. reported only very limited eradication (14% [1/7]) of genital mycoplasmas (Ureaplasma spp. and Mycoplasma hominis) in patients with PPROM treated with a combination of ceftriaxone, clindamycin, and erythromycin 14 years later.⁵³ Recently, Tanaka et al. have shown eradication of Ureaplasma spp. in 57% of patients with PPROM treated with a combination of ampicillin-sulbactam and azithromycin.¹³¹ In the current study, we employed sensitive and specific PCR-based assays to assess the presence and burden of Ureaplasma spp. in amniotic fluid. We are aware that this method may represent a double-edged sword. On one hand, PCR can amplify DNA from dead Ureaplasma spp. as DNA remains stable after the death of bacteria. Consequently, the use of specific PCR assays could prevent identification of the successful eradication of Ureaplasma spp. from amniotic fluid. On the other hand, real-time PCR enabled measurement of the microbial load of Ureaplasma spp. DNA and monitoring of the effect of therapy against Ureaplasma spp. in a quantitative manner. In this study, we found that the use of intravenous clarithromycin therapy in intra-amniotic infection, caused by Ureaplasma spp., reduced the load of Ureaplasma spp. DNA in amniotic fluid. As expected, after clarithromycin treatment, evidence of Ureaplasma spp. DNA was still present in all amniotic fluid samples.

Macrolide antibiotics are not only a potent tool to treat bacterial infection: they also have other biologic properties, including anti-inflammatory activity. Macrolide antibiotics have been shown to: (1) diminish production of various cytokines^{85,86,89,91,92} and adhesion molecules;^88,94 (2) reduce superoxide production by neutrophils in a dose-dependent manner;^132,133 (3) accelerate apoptosis of neutrophils;^132,133 (4) reduce expression of NF kappa B, a key pro-inflammatory nuclear transcription factor^93–95; (5) suppress expression of the transcription factor AP-1^92,96; and (6) reduce the production of matrix metalloproteinase^93,134;In addition, clarithromycin has been shown to: (1) block toll-like receptor 4 signaling by the inhibition of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and anti-MAPK kinase;¹³⁵ (2) have a positive effect on the attenuation of systemic and local inflammatory responses after surgical trauma¹³⁶ and during sepsis;¹³⁷ and (3) inhibit contractions of human myometrium in vitro.¹³⁸ Given the anti-inflammatory effects of clarithromycin, the administration of this antibiotic could be effective in diminishing intra-amniotic inflammation, even in patients with sterile intra-amniotic inflammation.

Complications arising from sterile intra-amniotic inflammation affect 5%-29% of PPROM pregnancies.^39,59,128 The development of sterile intra-amniotic inflammation in pregnancies with PPROM might be caused by the following conditions: i) damage of fetal membranes leading to the release of endogenous molecules (alarmins) into the amniotic fluid, resulting in a subsequent inflammatory response through pattern recognition receptors;^{18,39,52,56,59,139} ii) infection in the choriodecidual space, leading to the release of inflammatory mediators from the fetal membranes into the amniotic fluid;¹⁴⁰ or iii) a combination of those two processes.

In this study, all patients with sterile intra-amniotic inflammation who underwent a follow-up amniocentesis were treated with intravenous clarithromycin. In this subset of the study participants, attenuation of the intensity and resolution of intra-amniotic inflammation was identified in 100% (7/7) and in 86% (6/7) of patients, respectively. Although clarithromycin therapy was not associated with the resolution of intra-amniotic inflammation in one patient, this case can also be considered as a partial success for two reasons: the latency period between admission and delivery was 58 days and the newborn did not have any morbidity.

The final aim of this study was to assess whether the treatment with non-macrolide antibiotics—benzylpenicillin, or clindamycin in case of a penicillin allergy—could affect the concentration of IL-6 in amniotic fluid from PPROM pregnancies without either MIAC or intra-amniotic inflammation. Despite low concentrations of IL-6 in the amniotic fluid of the subset of patients without MIAC and/or intra-amniotic inflammation, the median concentration of IL-6 in amniotic fluid from the follow-up amniocentesis was even lower when compared to the initial amniocentesis. The results of this study corroborate with the findings of previous studies showing that ampicillin and penicillin can inhibit the release of IL-6 by amnion¹⁴¹ and decidual cells,¹⁴² respectively, independent of their antibacterial properties.

Clinical implications

In this study, we describe for first time that antibiotic treatment with clarithromycin was associated with an attenuation of the intensity of the intra-amniotic inflammatory response in a subset of PPROM pregnancies complicated with intra-amniotic infection and sterile intra-amniotic inflammation. This information is clinically relevant: the presence of intra-amniotic inflammation in PPROM pregnancies may affect short-term outcomes that may include the length of the latency period,^{35,59,128,143} gestational age at delivery,^{35,59,128,143} and neonatal morbidity,^35,59 as well as long-term neurodevelopmental and neurological outcomes.^144,145

Strengths and limitations

The main strength of this study is the utilization of paired amniotic fluid samples obtained before and after intravenous antibiotic treatment in pregnancies complicated by a well-defined phenotype of spontaneous preterm delivery (PPROM) <34 weeks of gestation. The second important strength is the combination of non-cultivation (specific PCR for Ureaplasma spp., Mycoplasma hominis, and Chlamydia trachomatis and non-specific PCR for detection of 16S rRNA gene) and cultivation approaches utilized to assess MIAC.

This study also has some limitations. First, the follow-up amniocenteses were not performed in all patients with intra-amniotic infection and sterile intra-amniotic inflammation, but only in the subset of those who delivered >7 days from admission. Therefore, the results should be interpreted with caution and need to be validated. Second, all patients who underwent follow-up amniocenteses received a course of corticosteroids after the initial amniocentesis. Glucocorticoids, particularly dexamethasone, have been shown to inhibit the production of IL-6 by amnion cells on the cell culture model^146,147 but not on the explant tissue model from term fetal membranes.¹⁴⁸ In addition, betamethasone administration did not alter concentrations of IL-6 in amniotic fluid in the sheep model.¹⁴⁹ Regardless of this conflicting evidence, one cannot fully exclude an effect of corticosteroids on IL-6 concentration in amniotic fluid samples collected from the follow-up amniocenteses. On the other hand, we have shown not only a diminishing concentration of IL-6 in amniotic fluid but also a reduction in the microbial load of Ureaplasma spp. DNA, measured by the gene copy numbers. This effect could not be achieved by the administration of corticosteroids.

Third, the changes in the intensity of intra-amniotic inflammation associated with antibiotic treatment were characterized in this study just by the assessment of the single amniotic fluid protein IL-6. To obtain a more comprehensive view, as well as a more precise determination of the inflammatory changes related to antibiotics therapy, the employment of modern high-throughput “–omics” technologies would be desirable. Last, implementation of the PCR method to identify bacteria in amniotic fluid prevented an answer to the question of whether intravenous clarithromycin therapy eradicated Ureaplasma spp. from amniotic fluid since DNA in amniotic fluid can come from live or dead bacteria. To address this point, some approaches can potentially be used: i) a combination of specific PCR and cultivation for Ureaplasma spp.; ii) detection of bacterial mRNA in amniotic fluid; or iii) use of selective quantitative real-time PCR with propidium monoazide to identify only viable bacteria¹⁵⁰.

Conclusion

Intravenous clarithromycin therapy was associated with a reduction in the intensity of the intra-amniotic inflammatory response associated with intra-amniotic infection and sterile intra-amniotic inflammation, and a reduction in the microbial load of Ureaplasma spp. DNA in the amniotic fluid of patients with PPROM <34 weeks of gestation.