Effects of a midwife psycho-education intervention to reduce childbirth fear on women's birth outcomes and postpartum psychological wellbeing

Jennifer Fenwick
Jocelyn Toohill
Jenny Gamble
Debra K Creedy
Anne Buist
Erika Turkstra
Anne Sneddon
Paul A Scuffham
Elsa L Ryding
Effects of a midwife psycho-education intervention to reduce childbirth fear on women's birth outcomes and postpartum psychological wellbeing
10.1186/s12884-015-0721-yR E S E A R C H A R T I C L E Open Access
Background: High levels of childbirth fear impact birth preparation, obstetric outcomes and emotional wellbeing for around one in five women living in developed countries. Higher rates of obstetric intervention and caesarean section (CS) are experienced in fearful women. The efficacy of interventions to reduce childbirth fear is unclear, with no previous randomised controlled trials reporting birth outcomes or postnatal psychological wellbeing following a midwife led intervention.Method: Between May 2012 and June 2013 women in their second trimester of pregnancy were recruited. Women with a fear score ≥ 66 on the Wijma Delivery Expectancy / Experience Questionnaire (W-DEQ) were randomised to receive telephone psycho-education by a midwife, or usual maternity care. A two armed non-blinded parallel (1:1) multi-site randomised controlled trial with participants allocated in blocks of ten and stratified by hospital site and parity using an electronic centralised computer service. The outcomes of the RCT on obstetric outcomes, maternal psychological well-being, parenting confidence, birth satisfaction, and future birth preference were analysed by intention to treat and reported here.Results: 1410 women were screened for high childbirth fear (W-DEQ ≥66). Three hundred and thirty-nine (n = 339) women were randomised (intervention n = 170; controls n = 169). One hundred and eighty-four women (54 %) returned data for final analysis at 6 weeks postpartum (intervention n = 91; controls n = 93). Compared to controls the intervention group had a clinically meaningful but not statistically significant reduction in overall caesarean section (34 % vs 42 %, p = 0.27) and emergency CS rates (18 % vs 25 %, p = 0.23). Fewer women in the intervention group preferred caesarean section for a future pregnancy (18 % vs 30 %, p = 0.04). All other obstetric variables remained similar. There were no differences in postnatal depression symptoms scores, parenting confidence, or satisfaction with maternity care between groups, but a lower incidence of flashbacks about their birth in the intervention group compared to controls (14 % vs 26 %, p = 0.05). Postnatally women who received psycho-education reported that the 'decision aid' helped reduce their fear (53 % vs 37 %, p = 0.02).

(Continued from previous page) Conclusion: Following a brief antenatal midwife-led psycho-education intervention for childbirth fear women were less likely to experience distressing flashbacks of birth and preferred a normal birth in a future pregnancy. A reduction in overall CS rates was also found. Psycho-education for fearful women has clinical benefits for the current birth and expectations of future pregnancies.

Keywords: Childbirth fear, Midwife psycho-education, Caesarean section, BELIEF study Background Few randomised controlled trials (RCT) have tested interventions to reduce childbirth fear in pregnant women and improve birth outcomes and emotional wellbeing. The first of two previous Finnish RCTs reported improved vaginal birth rates in fearful women who had either six cognitive behavioural sessions or two intensive sessions with an obstetrician [1]. In the second study women who received six group psycho-education sessions with a psychologist had lower CS rates compared to women in the control group. [2]. Obstetrician counselling or group psycho-education with a psychologist enhanced preparedness for birth and positive parenting in women with intense fear [1,3]. However no published RCTs have used the skills of midwives.

The BELIEF (Birth Emotions and Looking to Improve Expectant Fear) trial investigated the efficacy of a midwife-led psychoeducational intervention for reducing women's fear during pregnancy. The protocol for the study has been published [4]. Antenatal outcomes of the RCT showed a reduction in pre-birth fear level (W-DEQ), improved childbirth self-efficacy (CBSEI), and a trend to reducing decisional conflict (DCS) and depressive symptoms (EPDS) [5]. This current paper reports secondary outcomes for the RCT at 6 weeks postpartum with respect to mental health and obstetric outcomes of women receiving the intervention compared to controls.

Childbirth fear

Childbirth fear has been recognised and investigated in Scandinavian countries for more than three decades [6]. In Sweden women are routinely treated for childbirth fear within multidisciplinary teams but managed predominantly by midwives [7,8]. In Australia, childbirth fear has only recently started to attract greater attention [9]. Toohill et al. [10] reported high fear, as measured by the W-DEQ (score ≥66), to affect approximately 20 % of Australian childbearing population. This figure is similar to others reported by Swedish, Canadian and United Kingdom (UK) researchers over the last 15 years [10]. However more recently there has been a focus on identifying severe levels of fear (W-DEQ ≥85). This level of fear appears to occur in about 10 % of women, with some indication that it may be slightly higher in European countries, and is said to impact women's daily functioning (such as attending work or mothering the baby) [11,12].

A woman's emotional and psychological wellbeing contributes significantly to her perceptions and experiences of pregnancy and birth. Poor emotional health is associated with increased childbirth fear and risk of depression [13], birth trauma [14][15][16][17], an inability to interact positively with the baby and meet infant developmental needs [18,19], and can be a stressor to the couple relationship [19,20]. In addition, pregnant women with childbirth fear more often prefer a caesarean section (CS) [12,21]. They are also at increased risk of obstetric interventions such as elective or emergency CS [22]. In the absence of routine screening and intervention, fearful Australian women may be at higher risk for CS than their northern European counterparts who receive education and support.

High CS rates are of concern across industrialised countries due to higher physical and psychological morbidity [23], the impact to women's reproductive life as a result of a scarred uterus [23] and the high likelihood of undergoing a repeat CS [24]. The cost of a CS is at least twice that of a non-operative birth in low risk women [25]. The influence of fear on women's birth decisions and how operative birth adds to the pervasiveness of fear in populations is of international interest [26][27][28][29][30][31][32][33][34].

Objectives

As part of our BELIEF RCT we hypothesised that women receiving midwife-led telephone psycho-education during pregnancy would report improved postnatal mental health six weeks after birth, experience higher levels of vaginal birth (reduced CS) and prefer a vaginal birth in a subsequent pregnancy compared to the control group.

Method

A two armed non-blinded parallel (1:1) multi-site randomised controlled trial was used. Details of the RCT have been published previously [4,5]. To summarise, women between 12 to 24 weeks gestation, aged 16 years and older, able to read, write and understand English and with capacity to consent were invited to participate. Women who required an interpreter, or had a fetal diagnosis of major abnormality or incompatibility with life were excluded. Women were recruited by research midwives in antenatal clinics of three metropolitan teaching hospitals in south-east Queensland, Australia between May 2012 and June 2013. Participants provided their written consent to the study. Human research ethics approval was obtained from Griffith University and Queensland Health multi-site hospital Human Research Ethics Committee for the three participating hospitals.

Data collection and measures

Immediately following consent to participate, women were asked to complete a questionnaire that sought data about demographic characteristics, obstetric history and psycho-social factors. The W-DEQ, which has been validated within the Australian context [9], was used to measure antenatal childbirth fear [35]. Women scoring high childbirth fear (≥66) were randomised to the BE-LIEF intervention or control group. An evidenced based birth decision aid booklet was provided to all randomised women. The booklet included information about common practices used around the time of birth, health outcome statistics associated with interventions and tools for decision-making and communicating consumer needs with care providers.

Further questionnaires were completed at 36 weeks gestation to ascertain course of pregnancy, and at six weeks postpartum to determine birth outcomes. At both time-points depression was measured using the Edinburgh Postnatal Depression Scale (EPDS) [36]. The ten item EPDS has shown both high sensitivity and specificity when used in the antenatal and postnatal periods [37]. At 6 weeks postpartum women completed the selfefficacy subscale of the Sense of Confidence and Satisfaction Scale which measures the extent to which a mother is confident in her abilities to effectively nurture her child [38]. In addition women were asked 'How often have you experienced distressing 'flash-backs' to your labour and birth since having your baby?' For more details see the study protocol [4].

Women who had not returned questionnaires at six weeks after birth were telephoned to prompt completion of questionnaires. These could be completed over the telephone or by hard copy returned by free post. After two reminders by telephone including calling alternative numbers or sms, and after an additional questionnaire was mailed with no response, women were considered lost to follow-up.

Intervention

Women in the BELIEF intervention group received psycho-education sessions at 24 and 34 weeks gestation by telephone at a scheduled time convenient to them. Psycho-education sessions were around 1 hour duration (First session range: 22-125 min; Second session range:

10-104 min) [5]. Women randomised to the control group received usual maternity care at their chosen facility.

The BELIEF study aimed to review women's current expectations and feelings around fear of childbirth, support the expression of feelings, and provide a framework for women to identify and work through distressing elements of childbirth [4,5]. A detailed description of the intervention has been published [4].

Outcomes

The secondary outcomes of the BELIEF study reported in this paper tested the efficacy of the intervention in reducing caesarean section, induction of labour (amniotomy, prostaglandin or syntocinon), epidural use in labour and neonatal admission to special care or intensive care nursery. Psycho-social outcomes included lower levels of depressive symptoms (EPDS), distressing flashbacks of the birth and improved parenting confidence (PSOC). Women's satisfaction with their ultimate birth mode and the decision aid assisting with decreasing feelings of fear are also reported.

Sample size

The sample was calculated after allowing for 30 % attrition, using a significance level of 5 %, power of 80 %, and a two tailed test. A sample of 150 women in each group was determined to detect a 10 point reduction in high fear scores between the intervention and control groups prebirth for the primary outcome.

Randomisation

A research assistant not involved in recruitment or provision of the intervention accessed the randomisation service following receipt of participant's written consent and completed baseline measures. Participants were allocated in blocks of ten and stratified by hospital site and parity using a centralised web-based service to either intervention or control group. A midwife providing the intervention was subsequently notified of women's details to initiate contact and the intervention. [39] was used for all analyses. Descriptive statistics were generated for all demographic variables and scale scores. Chi square tests were used to compare groups on categorical outcome variables and independent t-tests were conducted to compare continuous scale scores. An alpha level of 0.05 was used for all statistical tests. The Cronbach alpha values, which indicate internal consistency reliability, for the scales administered at Time 3 were .88 EPDS and .90 for the seven items of the self-efficacy and satisfaction sub-scale of the Sense of Confidence and Satisfaction Scale.

Statistical methods

SPSS Version 21

Results

Of 1410 women recruited into the study 339 (24 %) reported high childbirth fear and were randomised to their allocated study groups. One hundred and eighty-four (54.2 %) women provided data at completion of the study within the allocated time frame (10 weeks). Two women were incorrectly randomised and removed from analysis. (Refer Fig. 1 Study flow diagram).

Participants characteristics were representative of Australian women giving birth [10]. Psycho-social characteristics of the baseline population have been reported elsewhere [40].

Fig. 1 Study Flow Diagram

Preliminary analyses were conducted to compare those women who completed both the baseline and postpartum questionnaires, and those that were lost to follow-up. There was no difference in the proportion of women in the intervention group and the control groups that dropped out of the study (46.5 % and 45 % respectively, p = .78). There was also no difference between the completed and lost to follow up groups on parity (p = .94), marital status (p = .50), preferred mode of birth (p = .29), baseline WDEQ scores (p = .35) or baseline EPDS scores (p = .79). The women in the lost to follow-up group were however younger (p = .006), less educated (p < .001), with lower household incomes (p < .001).

Mode of birth and obstetric events

No statistical difference in birth mode was found between the intervention and control groups. However fewer women in the intervention group had a CS (34 % vs. 42 %, p = 0.27). In particular, there was a lower emergency CS rate in the intervention group (18 % vs. 25 %, p = 0.23) ( Table 1). Women having their first baby in the intervention group had higher rates of vaginal birth (65 % vs. 55 %) (See Table 2). At 6 weeks postpartum fewer women who received psycho-education preferred a CS for their next pregnancy (18 % vs 30 %, p = 0.04). Refer to Table 1. There were no differences for induction of labour, epidural use, neonatal admission to a nursery or satisfaction with birth mode.

Psychological factors

There were no differences between the groups for depressive symptoms (EPDS: Mean 6.2 vs. 5.5 p = 0.30) or parenting confidence (Mean 32.4 vs. 33.3 p = 0.32). Fewer women in the intervention group, however, reported having flashbacks (p = 0.05). Women in the intervention group reported they gained more from the decision aid booklet compared to women in the control group (p = 0.02).

Discussion

The 8 % lower rate of CS in women who received midwife psycho-education is a noteworthy finding. While not statistically significant, this result is in line with the findings of Saisto and colleagues [1] who similarly demonstrated that CS rates could be reduced through antenatal counselling with fearful women. However while Saisto et al. [1] included women seeking elective CS of any parity, they did exclude women not eligible for a vaginal birth. Consistent with the work of Rouhe et al. [2] we also found vaginal birth rates were improved for women having their first baby. Once again, however, eligible criteria differed with Rouhe et al. [2] excluding women with significant psychological problems and only randomising women with severe levels of fear (W-DEQ >100). As our primary outcome was to reduce women's antenatal fear levels regardless of birth mode our sample included women who had previously experienced a CS, women carrying a multiple pregnancy and women with other mental health disorders. Consequently our study sample included women at higher obstetric and possibly higher psychological risk than the previous two trials, and included women with lower fear levels (WDEQ-A ≥ 66). While we were optimistic, it was unlikely that we would reduce elective CS rates given it is difficult for women in Australia to secure vaginal birth following a previous CS. The lower overall CS rate in the intervention group (34 % vs. 42 %) aligns with the Queensland [41] state average, indicating that CS rates in fearful women can be reduced to at least those of the general population. In our study this can be attributed most particularly to reducing emergency CS, however both emergency and elective CS was lower for primiparous women who received the intervention. We noted CS rates could have been further improved had clinical practice been similar at each study site given women in the intervention group at one site had half the CS rate to that of women in the control group. Similar to previous studies [1,2] there were no differences between groups for use of pharmacological analgesia, induction of labour or neonatal outcomes. Comparisons to work undertaken in Finland [2] shows that our Australian cohort of fearful women reported using epidural anesthetic during labour around half as often as women in Finland. This may be due to lower vaginal birth rates in our RCT and is also consistent with higher CS trends in Australia [41]. However induction of labour was more frequent in our Australian study population (33 %) compared to Finnish women (20 %) [2].

Randomised controlled trials to date indicate that women's anxiety [1] and/or fear levels [2,5] are improved following antenatal counseling and this translates to higher satisfaction with vaginal birth. We did not specifically determine women's satisfaction with their birth experience, but women were satisfied with the type of birth they eventually had. The statistically significant difference in women's lower preference for caesarean section in their next pregnancy could perhaps indicate that the midwife intervention assisted women to feel positive about normal childbirth in both the short and longer term. Perhaps this also explains why women in the intervention group were less likely to experience distressing flashbacks of their birth despite no differences found in postpartum depressive symptoms or parenting confidence.

Flashbacks may be a symptom of trauma. Women who received the intervention may be in a more positive psychological frame of mind about childbirth. We have previously reported that women receiving the intervention had higher levels of confidence and a trend to lower decisional conflict during pregnancy [5]. Postnatally, women in the intervention group also assessed the decision aid more positively than the control group. Women in the intervention group were encouraged to challenge care when they were uncomfortable or uncertain. This may increase women's sense of being active within the decision making process and thus emotionally protective. This is important given the strong link between birth trauma symptoms, fear, and requests for CS; and could have future health care savings in relation to potentially reducing CS rates.

Limitations

There are a number of limitations to consider when interpreting the results of this secondary analysis. While sample size was powered to detect a difference in the primary outcome of childbirth fear, this difference should have also been sufficient to show statistical differences in overall CS rates based on a previous study [1]. Inclusion in the current study of women who were not eligible for vaginal birth may have impacted our findings for birth mode. In addition, despite using a number of engagement strategies our postnatal attrition rate was much higher than expected. Younger, less educated women with lower household incomes were harder to engage. Future studies that include all risk pregnancies need to recruit larger samples of women and incorporate additional strategies to minimise attrition in vulnerable childbearing women. The sample included women attending publicly-funded antenatal clinics. The proportion of women who give birth in private hospitals in Australia is 29 % [41]. Consequently, findings cannot be generalized to women with childbirth fear who receive private care. Additionally study sites adopted different practices in respect of women's labour and birth care, for example; considerably higher CS rates were shown at two sites compared to the third site. The reasons for this were not investigated in this study and therefore unable to be extrapolated to wider birthing communities.

Conclusion

This is the first RCT to report midwife counseling for women with childbirth fear. Women who received the psycho-education had lower rates of CS compared to controls, and this was associated to lower rates of emergency CS. Additionally women reported fewer flashbacks of their birth indicating that midwife counseling for fear may also reduce the development of trauma symptoms. These are important findings in relation to improving normal birth rates and women's emotional wellbeing.

The study supports the role of midwives in assisting women with high levels of childbirth fear.

Table 1
1Outcomes six weeks post-partum CS caesarean section, SVD spontaneous vaginal delivery, B/fed breastfed, EPDS Edinburgh Postnatal Depression Scale, CI confidence interval 95 %Variable
Intervention group (n = 91)
Control group (n = 93 )
P
C I

Prefer CS next birth, n (%)
16 (17.6)
28 (30.1)
0.04
0.24 -0.99

SVD, n (%)
44 (48.4)
39 (41.9)
0.38
0.72 -2.31

Forceps/ vacuum, n (%)
16 (17.6)
15 (16.1)
0.79
0.51 -2.40

CS, n (%)
31 (34.1)
39 (41.9)
0.27
0.39 -1.30

Elective CS, n (%)
15 (16.5)
16 (17.2)
0.88
0.43 -2.05

Emergency CS, n (%)
16 (17.6)
23 (24.7)
0.23
0.31 -1.32

Induction of labour, n (%)
34 (37.4)
27 (29.1)
0.25
0.76 -2.75

Missing
11 (12.1)
13 (14)

Narcotic in labour, n (%)
26 (28.6)
29 (31.2)
0.65
0.44 -1.66

Missing
11 (12.1)
12 (12.9)

Epidural analgesia, n (%)
33 (36.3)
33 (35.5)
1.00
0.53 -1.94

Missing
13 (14.3)
13 (14)

Preterm birth, n (%), range weeks
7 (7.7), 32-36
3 (3.2), 28-35
-
-

Admit to nursery, n (%)
16 (17.6)
18 (19.4)
0.75
0.42 -1.87

Mother readmission, n (%)
3 (3.3)
5 (5.4)
-
-

Baby readmission, n (%)
8 (8.8)
6 (6.5)
-
-

B/fed 6weeks P/N, n (%)
76 (83.5)
73 (78.5)
0.38
0.66 -2.91

Flashbacks of birth, n (%)
13 (14.3)
24 (25.8)
0.05
0.22 -1.01

EPDS Mean (SD), range
6.2 (5), 0-22
5.5 (4.7), 0-23
0.30
-.67 -2.14

Satisfaction birth mode
53 (58.2)
61 (65.6)
0.30
.40 -1.32

Satisfaction of Decision Aid in Decreasing fear, n (%)
48 (53.3)
34 (37)
0.02
-.31 --.02

Parenting confidence Mean (SD), range
32.4 (6.2), 10-42
33.3 (6.6), 15-42
0.32
−2.81 -0.94

Table 2
2Birth mode by parityVaginal birth
Emergency caesarean
Elective caesarean

Study Group
Nullip n (%)
Multip n (%)
Nullip n (%)
Multip n (%)
Nullip n (%)
Multip n (%)

Intervention
33/51 (64.7)
27/40 (67.5)
15/51 (29.4)
1/40 (2.5)
3/51 (5.9)
12/40 (30.0)

Control
29/53 (54.7)
25/40 (62.5)
19/53 (35.9)
4 /40 (10.0)
5 /53 (9.4)
11/40 (27.5)

AcknowledgementsThe study was funded by the National Health & Medical Research Council, NHMRC grant number APP1025099. We are grateful for statistical analysis support from Associate Professor Julie Pallant, the research midwives who recruited women to the study, midwives who provided the psychoeducation, the research assistants who maintained contact with women and collected data. Importantly we thank the women who participated.Human research ethics committee approval number HREC/11/QGC/162 (Queensland Health); NRS/45/11/HREC (Griffith University).Competing interestsThe authors declare that they have no competing interests.Authors' contributions JT drafted the manuscript. JF, JT and DKC interpreted the data following analysis by a statistician. JF, JT, JG and DKC contributed equally to refining the manuscript. JF is CIA of the BELIEF study and co-ordinated with all listed authors (JT, JG, DKC, AB, EK, AS, PAS, ELR) revision of the manuscript. All authors have read and approved the final manuscript.
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Perinatal statistics series no. 28. Cat. no. PER 59. Canberra: AIHW; 2013. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Z Li, R Zeki, L Hilder, E Sullivan, Australia's mothers and babies. Submit your manuscript at www.biomedcentral.com/submitLi Z, Zeki R, Hilder L, Sullivan E. Australia's mothers and babies 2011, Perinatal statistics series no. 28. Cat. no. PER 59. Canberra: AIHW; 2013. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


Comparison of human chordoma cell-kill for 290 MeV/n carbon ions versus 70 MeV protons in vitro

Hiroshi Fujisawa
Paula C Genik
Hisashi Kitamura
Akira Fujimori
Mitsuru Uesaka
Takamitsu A Kato
Comparison of human chordoma cell-kill for 290 MeV/n carbon ions versus 70 MeV protons in vitro
R E S E A R C H Open Access
Background: While the pace of commissioning of new charged particle radiation therapy facilities is accelerating worldwide, biological data pertaining to chordomas, theoretically and clinically optimally suited targets for particle radiotherapy, are still lacking. In spite of the numerous clinical reports of successful treatment of these malignancies with this modality, the characterization of this malignancy remains hampered by its characteristic slow cell growth, particularly in vitro. Methods: Cellular lethality of U-CH1-N cells in response to different qualities of radiation was compared with immediate plating after radiation or as previously reported using the multilayered OptiCell™ system. The OptiCell™ system was used to evaluate cellular lethality over a broad dose-depth deposition range of particle radiation to anatomically mimic the clinical setting. Cells were irradiated with either 290 MeV/n accelerated carbon ions or 70 MeV accelerated protons and photons and evaluated through colony formation assays at a single position or at each depth, depending on the system. Results: There was a cell killing of approximately 20-40% for all radiation qualities in the OptiCell™ system in which chordoma cells are herein described as more radiation sensitive than regular colony formation assay. The relative biological effectiveness values were, however, similar in both in vitro systems for any given radiation quality. Relative biological effectiveness values of proton was 0.89, of 13-20 keV/μm carbon ions was 0.85, of 20-30 keV/μm carbon ions was 1.27, and >30 keV/μm carbon ions was 1.69. Carbon-ions killed cells depending on both the dose and the LET, while protons depended on the dose alone in the condition of our study. This is the first report and characterization of a direct comparison between the effects of charged particle carbon ions versus protons for a chordoma cell line in vitro. Our results support a potentially superior therapeutic value of carbon particle irradiation in chordoma patients. Conclusion: Carbon ion therapy may have an advantage for chordoma radiotherapy because of higher cell-killing effect with high LET doses from biological observation in this study.

Introduction

In cancer therapy, the main treatment modalities are, or include, surgery, chemotherapy and radiation therapy. Radiation therapy has been evolving since X-rays were discovered. More recently, intensifying efforts have been invested in devising improved and novel radiation therapy treatments based on innovative technologies and devices [1]. While intensity modulated radiation therapy (IMRT) and image guided radiation therapy (IGRT) using photons are two outstanding examples of such recent developments in radiation therapy [1], the most notable instance in the field of a vastly improved therapeutic gain would be that derived from charged particle radiation therapies with accelerated carbon ions or protons [1]. Accelerated carbon ions and protons have been very successfully used for treating solid cancers primarily due to their advantageous property of excellent physical dose-distribution and deposition around the Bragg peak [2,3]. Several recent clinical studies have reported that outcomes from charged particle beam radiation therapy yield equivalent or superior outcomes to surgical modalities in a number of cases such as prostate, lung, head and neck, bone and soft tissue tumors [4,5]. In bone tumors, namely chordomas, malignancies appear to be highly responsive to charged particle radiation therapy which also maximally preserves adjacent organs and their function, whereas the latter are difficult to preserve through unavoidably highly invasive surgery [5][6][7][8][9].

Chordomas are primary malignant bone tumors that arise from vestigial notochordal remnants and appear more commonly in the clivus, the basilar process of the occipital bone and the sacrum or coccyx, vertebrae at the caudal region of the spine [10,11]. Chordomas represent only 1 to 4% of all primary malignant tumors and among their hallmark characteristics are containment and slower growth kinetics together with less frequent metastases [12]. While the first-line therapy of choice for chordoma treatment remains surgery, radiation therapy is also used [4,13,14] because of the deeply ensconced anatomical location of these tumors and the severe negative quality of life issues resulting from surgical resections [6]. Radiation therapy, especially in the case of charged particle beams involving carbon ions or protons, has been commanding much attention of late particularly in the treatment of bone and soft tissue sarcomas [6,15]. Both carbon ions and protons have thus been successfully used in the treatment of chordomas, and their superior therapeutic outcomes have been reported in clinical practice [4,8,15]. Notwithstanding these remarkable advances with charged particle beam therapies of chordoma, there remains a severe paucity of basic biological data on this tumor cell type. Basic scientific investigation into the biology of these tumors has been hampered due to several limiting characteristics of their primary cell type, namely their long mean doubling time and the lack of types corresponding cell lines [16,17]. Nonetheless, a few noteworthy cytogenetic studies describing the karyotypes and chromosomal aberrations through multicolor fluorescence in situ hybridization and array have been reported [18,19]. We also have previously reported that U-CH1-N, a newly established chordoma cell subline from U-CH1, is very sensitive to high Linear Energy Transfer (LET) radiation involving charged particle beams such as those of carbon, neon, silicon, argon and iron ions [16]. To date, however, the sensitivity of chordoma tumors to carbon and protons remains illdefined.

In this study, we compare the relative biological effectiveness (RBE) of carbon ion particles to that of proton particles in a human chordoma cell line, UCH1-N, cultured in the OptiCell ™ system. We previously established and characterized this cell line de novo [16] with a notably abridged doubling time of approximately 3 days compared to that of the original 7 days in the originating cell line. The OptiCell ™ system is a commercially available cell culture technology that was designed as a unique cell culture format with respect to growing, monitoring, and transporting of cells in culture [20]. These vessels consist of an enclosed sterile cell culture chamber with two parallel gas-permeable cell culture-treated polystyrene membranes. The thin profiles of these chambers (2 mm in width) offer very specific advantages. More detailed acquisition of biological data at each position (LET and dose) is possible with respect to experimental particle beam pathways.

The present study is the first to undertake and report a direct comparison between the cell killing effects of 290 MeV/n carbon ions and 70 MeV protons in a chordoma cell line in a clinically relevant in vitro system. Our data lead to a description of chordomas as an even more radiation resistant solid tumor than previously understood, which responds significantly to carbon particle beams in an LET and dose dependent manner rather than to protons, with an enhanced RBE greater than one. Further work will be needed to determine whether this characterization applies to other tumor types and beam qualities and doses. Finally, our data suggest that estimates of cell killing in a classical cell culture system overestimate the responsiveness of tumor cells to all radiation qualities, particularly in the case of protons and strongly support the heightened relevance of carbon beam particle therapy as the optimal treatment of chordoma tumors in the clinic.

Methods and materials

Cells and culture

The human chordoma cell line U-CH1 was kindly supplied by the Chordoma Foundation (Greensboro, NC, USA). The U-CH1-N cells used in this study are a subpopulation derived from the original U-CH1 chordoma cells at National Institute of Radiological Sciences (NIRS) [16]. Cells were grown in α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (Sigma, Japan) and 1% (v/v) antibiotics and antimycotic (Invitrogen, Carlsbad, CA, USA) and they were maintained in a tissue culture incubator at 37°C in a 100% humidified atmosphere of 5% CO 2 in air.

Irradiations

Hadron irradiations were conducted at the NIRS in Chiba, Japan. Carbon ions were accelerated to 290 MeV/n using the Heavy Ion Medical Accelerator in Chiba (HIMAC) and protons were accelerated to 70 MeV using the NIRS-930 cyclotron delivery port in C-8. Dose rates for carbon ions and protons were set at 1 Gy/min. Monoenergetic Cell survival assays using OptiCell ™ culture chambers Cultured cells were trypsinized and re-suspended into complete growth medium. Once re-suspended, 10 ml of medium containing 8,000 cells was placed into each individual OptiCell ™ cell culture vessel (Thermo Fisher Scientific, Pittsburgh, PA, USA) within 3 hours prior to irradiation. The chamber dimensions were 2 × 65 × 150 mm with a maximum 10 ml volume capacity. All samples were stacked in layers and arranged perpendicular to the beam paths. Immediately following radiation at doses of 1, 2 and 3 Gy (of exposure for the first chamber) with carbon and proton particle beams and of 2, 4, 6 and 8 Gy with gamma-rays, cells were incubated at 37°C in 100% humidified atmosphere of 5% CO 2 in air for 3 weeks. After this culture period, cells were washed with 0.9% NaCl, fixed in 100% ethanol and stained with 0.1% crystal violet. Colonies containing more than 50 cells were recorded as reproductively viable surviving cells. Cell survival assays were carried out in as many as four independent replicates.

Cell survival assays using standard culture dishes

Exponentially growing U-CH1-N cells cultured in T12.5 flasks (Becton Dickinson, Franklin Lakes, NJ, USA) were irradiated with 70 keV/μm of near Bragg peak carbon ions, entrance region protons or X-rays at room temperature, then trypsinized and plated onto 100 mm cell culture dishes. They were kept in an incubator for three weeks without medium change. Up to 4 replicate experiments were conducted and after colonies were formed, cells were fixed and stained as described above.

Statistical analysis

Data were analyzed using Prism 5 ™ (GraphPad, La Jolla, CA, USA). Standard errors of the means for data were calculated and were depicted in each figure.

Results

Cell survival versus depth in water

Cell survival assays were conducted with our stacked OptiCell ™ cell culture system, and cells irradiated with carbon ion and proton particles. Positions of OptiCell ™ chambers at each depth were altered to the depths in water using the physical parameters determined in our previous report [21]. Bragg peaks of each carbon and proton beams were delivered at depths of 14 and 4 cm, respectively ( Figure 1). Survival fractions were gradually decreased and minimized at the Bragg peaks. Survival fraction near the Bragg peaks for carbon showed lower survival fractions compared to those for protons (Figure 2A-F).

Cell survival at low LET and at high LET

Cell survival in the OptiCell ™ system for U-CH1-N cells was evaluated in response to three groups of LET values for carbon particles. LET values were assigned to 13-20, 20-30, and >30 keV/μm value ranges. Higher LETs killed more cells. Proton particles were considered to be of low LET value only. Depths in water were translated to radiation doses by referring to physical parameters as our previous report (Figure 1) [21]. The survival fractions that correspond to the doses were then plotted ( Figure 3A-C). Cellular lethality caused by carbon ions was found to depend on both the dose and the LET, while in contrast, that caused by protons depended exclusively on the dose in the range of LETs in our study (1 to 10 keV/μm).

RBE values for U-CH1-N

RBE values were calculated based on the D 10 values, at doses resulting in one decade of cell killing. RBEs were obtained from D 10 values for gamma-rays divided by the D 10 for each ion particle. Carbon ions displayed the highest RBEs in this charged particle study: LET >30 keV/ μm 290 MeV/n carbon ions had an RBE of 1.69 and lower LET (13-20 and 20-30 keV/μm) 290 MeV/n carbon ions showed an RBE of 0.86 and 1.27 respectively. The proton's RBE value was 0.89 and very similar to LET 13-20 keV/ μm carbon ions.

Cell survival and RBE in standard cell culture

Survival fractions following irradiation of U-CH1-N were also obtained using classic cell culture dishes. Both gamma-rays and x-rays were used for the standard photon radiation. U-CH1-N showed very similar sensitivity to both kinds of photon radiation. Survival fractions were shown to depend on radiation dose, decreasing as doses increased (Figure 3). The entrance region of 70 MeV proton cell survival curves showed similar profiles to those for gamma-rays and x-rays, while in contrast, carbon ions survival curves revealed a markedly more sensitive response to carbon ions than to photon radiation. The calculated RBEs based on D 10 for carbon ions and protons against gamma-rays (D 10 = 4.11) and xrays (D 10 = 4.12) in this second in vitro system were higher than those determined in the OptiCell ™ system and were of 2.27 and 0.90, respectively.

Discussion

Chordomas grow relatively slowly in vitro and the doubling time of human chordoma U-CH1 cells in vitro is reported as 7 days [16]. Because of its characteristic kinetics as slow-growing, radiobiological characterization of chordoma cells is still lacking, though many clinical trials have been conducted and their outcomes reported [6,16]. Recently, however, an established cell line with a shortened doubling time of 3 days was developed with cell cycle kinetics thereby of particular usefulness towards acquiring invaluable biological information on chordoma cell growth and sensitivity to radiation or anti-tumor drugs in vitro [16] and ultimately, in vivo.

In the present study we chose to focus on the cell survival of chordoma cells in vitro irradiated with two very different clinically relevant hadron beams in terms of quality, namely carbon ions and protons, currently used for charged particle radiation therapy in a limited number of highly specialized centers throughout the world  [5, [22][23][24]. Qualitatively and quantitatively, our understanding of chordomas and data accrual for carbon treatments of this tumor lag far behind those for protons in light of the much smaller number of carbon treatment facilities worldwide. In this study, we show that exposure to carbon ions results in cell survival fractions lower than those for protons near the Bragg Peak. In the OptiCell ™ system, RBE values against gamma-rays were 1.69 for higher LET (>30 keV/μm) carbon ions, 0.85 for low LET (13-20 keV/μm) carbon ions, 1.27 for low LET (20-30 keV/μm) carbon ions, and 0.89 for the entire range of protons (1-10 keV/μm) ( Figure 3) and thereby demonstrate a marked superiority of carbon to proton charged particles in killing chordoma cells, explaining at least in part carbon's superior ability to treat tumors in the clinic. Dose-response curves also show these differences between carbon ions and protons. While the latter in vitro cellular determinations reflect the clinical course of these actual chordoma tumors following carbon versus proton therapy in vivo, they do define a larger quantitative therapeutic differential between the two particle beams in question.

The cell killing effectiveness of carbon ions in our study reveals for the first time a dependency of the chordoma cell lethality of these hadrons on both the radiation dose and the particles' LET values in a threedimensional in vitro setting [25]. The present study extends and refines the qualitatively similar effects of carbon ions in a standard tissue culture environment as we have previously shown [16]. A marked novelty and advantage of our irradiating U-CH1-N cells in stacked OptiCell ™ chambers with accelerated carbon beams is that we concurrently include both high LET particles with values near the Bragg peak and low LET particles with values outside of the Bragg peak area. In this study, we show that high LET carbon beams which are at present used in clinical treatment [26] do indeed kill more U-CH1-N cells than do low LET carbon particle beams. Moreover, we show that while proton particle beams which are considered to be of low LET also result in a cell-kill profile which consists of both a plateau and a Bragg peak areas, the dose response curve for chordoma cell-kill as a function of exposure to protons presents no significant differences when the survival data are segregated into outside-and near-Bragg peak areas. Thus, low LET protons and carbon are noticeably similar effective in killing chordoma cells (proton D 10 = 0.89 and 13-20 keV/μm carbon ions D 10 = 0.85). Cell kill data following proton beam exposure in our study, moreover, suggest that the effect of protons on cell kill would be far less therapeutically controllable or modifiable than that of a carbon beam as the former depends on the dose of alone. From the results above, the use of carbon ions in particle therapy of chordoma offers a distinct advantage with respect to both effectiveness and efficiency in that its therapeutic gain depends on LET and a demonstrated higher cell killing. These tendencies confirm chordoma cells patterns of superior cell kill by carbon ions recently shown in other cell lines, namely Chinese Hamster Ovary (CHO) cells [21] and human salivary gland cancer HSG cells [27].

OptiCell ™ tissue culture chambers are cell culture systems optimized for cell growth, culture monitoring, and transportation. They are designed for maximizing tissue culture incubator space in light of their narrow crosssectional profile and their highly specific enclosed-system which is readily applied to edifying three-dimensional stacks of monolayer culture systems as in vitro models for cell [20,28]. In the present study, we devised stacked three-dimensional in vitro OptiCel ™ arrays in configurations modeling in vivo clinically relevant anatomical constructs which we then used to determine cell survival fractions at each depth of beam energy deposition. Thus, development and alignment of these live-cell biological phantoms in three dimensions along a beam path more relevantly mimic a desired intravital geometry. To study relative in vitro survivals at varying depths, several other physical culture methods have been used, including Petri dishes fixed in a particular device [29], and a "cell stack chamber" [30]. Relative to these other specialized cell culture methods, the most advantageous feature of the stacked OptiCell ™ chamber system is the acquisition of many more -and greater detailed -data points, with an optimal resolution of 2 mm. Since the maximum dose of protons accelerated to 70 MeV was delivered at a depth of 4 cm in this study, a detailed analysis was necessary to acquire data near the proton Bragg peak. One significant difference between our present study protocol and previously used methods is the relative timing of cell plating and that of irradiations. There are essentially two different approaches to performing studies involving colony formation assays. A first strategy involves the plating cells before exposure to drugs or radiation, while the other relies on treating cells prior to subsequent passaging them to assess their resulting clonogenic ability [31]. Survival fractions are known to depend on whether plating of cells is performed before or after irradiation [32,33]. In the present study, cells were diluted and plated prior to irradiation and maintained undisturbed for the subsequent three weeks, which contrasts with the methodology employed in our previously reported study [16]. In this study, cell survival fractions following carbon or proton irradiation within OptiCell ™ chambers demonstrate a marked radioresistance when compared with cells cultured in standard tissue culture dishes (Figure 3). The survival difference in both culturing conditions can also be explained in part by the respective sequences of plating and irradiation.

In summary, this is the first report of a direct and therapeutically relevant in vitro comparison between the cell killing effects of carbon ions and protons on human chordoma tumor derived cell lines. Our data describe an experimental setting in which we have begun to investigate the superior therapeutic index of high LET carbon charged particle beams over all other charged particle and photon radiation modalities. We contend that use of the OptiCell ™ system provides an overall more reliable understanding of the biology of cells within a native in vivo chordoma tumor environment and should form the basis of future chordoma studies.

Conclusion

Our chordoma model describes a more biologically representative assessment of the true radiation responsiveness or resistance to varying radiation qualities (high versus low LET carbon relative to protons and photons) and doses with respect to charged particle beams at energies and at doses currently used in the clinic. Our data for a rare and difficult to treat chordoma tumor type indeed confirm that chordomas are exquisitely more responsive targets to carbon ion than to proton radiotherapy from an in vitro biological standpoint, principally in light of a unique dependency of carbon ion effects on both LET and dose on cellular lethality.

290 MeV/n carbon ions have a LET value of 13 keV/μm on entrance. Monoenergetic 70 MeV protons have a LET value of 1 keV/μm on entrance. Maximum doses of carbon ions and protons were delivered at depths of 14 and 4 cm in water, respectively (Figure 1) [21]. X-ray irradiations were performed using a TITAN irradiator at 200 kVp, 20 mA and 0.5 cm aluminum and copper filters (Shimadzu, Japan), at a dose rate of 1 Gy/min. Gammaray irradiations were carried out at a dose rate of 2.5 Gy/ min at Colorado State University (Fort Collins, CO, USA) using a 6,000 Ci (nominal activity) 137 Cesium sealed source [Model Mark I-68A (SS0056) J.L. Shepherd, San Fernando, CA, USA]. Irradiations were carried out at room temperature.

Figure 1 Figure 2
12Depth in Water (cm) LET (keV/µm)Relative Dose (Gy) Dose and LET at each depth in water. Open circles represent relative dose of proton (70 MeV) compared to entrance, and closed circles represent LET spectrum of proton. Open triangles represent relative dose of carbon ions (290 MeV/n) compared to entrance, and triangles represent LET spectrum of carbon ions. Cell survival curves at each depth in water. Cell survival assays were conducted with stacked OptiCell™ cell culture systems, irradiated with 290 MeV/n carbon ions: A-C and 70 MeV protons: D-F. Error bars indicate standard errors of the means from as many as four independent experiments.

Figure 3
3Dose response curves for proton and carbon ions. Survival fraction per dose obtained by OptiCell™ was calculated from Figure 1 and 2. Cell survival curves in standard cell culture dishes are also plotted. A. photon radiation, gamma-ray survival obtained by OptiCell™ was described by closed circles. Standard survival assay for x-ray was described by closed triangles and gamma-rays was described by open circles. B. proton, closed triangles represent OptiCell™ survival and open triangles represent standard survival. C. carbon ions, closed squares represent LET 13-20 keV/μm, open circles represent LET 20-30 keV/μm, closed circles represent LET >30 keV/μm obtained by the OptiCell™ system. Open triangles represent monoenergetic carbon ions LET 70 keV/μm. Error bars indicate standard errors of the means from as many as four independent experiments.
http://www.ro-journal.com/content/8/1/91
AcknowledgementsThis work was conducted as part of the Research Project with Heavy Ions at the NIRS (National Institute of Radiological Sciences)-HIMAC and with the NIRS-Cyclotron. This research was partially supported by the International Open Laboratory at the NIRS and by the Graduate Program for Leaders in Life Innovation (GPLLI) from the Japan Society for the Promotion of Science (JSPS).Competing interestsThe authors declare that they have no competing interests.
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