Mode of birth and women's psychological and physical wellbeing in the postnatal period

Ingrid J Rowlands
Maggie Redshaw
Mode of birth and women's psychological and physical wellbeing in the postnatal period
R E S E A R C H A R T I C L E Open Access
Background: Physical and psychological problems after childbirth are common, and may have a significant negative and long-term impact on women's wellbeing and daily functioning. The method of birth may be a particularly important factor influencing women's health and wellbeing following birth, however, population-wide evidence is limited. This study uses data from 5,332 women who responded to a national survey of women's experiences of maternity care in England. We examined women's postnatal wellbeing in the first three months after birth, and whether these varied by mode of birth.Methods: This is a secondary analysis of survey data using a random sample of women selected from birth registration. We used multinomial logistic regression models to examine the association between women's self-reported psychological symptoms, health problems and mode of birth. Results: Women who had forceps-assisted vaginal births and unplanned caesarean section births reported the poorest health and wellbeing, while those of women who had unassisted vaginal births and planned caesarean section births were less affected by the birth process. Most women's physical and emotional health appeared to improve with time, however, those who had a forceps-assisted vaginal birth were more likely to report ongoing posttraumatic-type symptoms several months after the birth.Conclusions: Mode of birth was associated with differences in outcomes at three months. By comparison to women who had unassisted vaginal births, the risk of reduced postnatal health and wellbeing was higher amongst the women who had forceps-assisted vaginal births but not amongst women who had ventouse-assisted vaginal births. This would suggest that it is important to differentiate the different types of instrumental birth in outcome studies. Of concern was the higher rate of posttraumatic-type symptoms among women who had forceps-assisted vaginal births relative to the other modes of birth. Women who have forceps-assisted births should be monitored carefully by health professionals in the postnatal period, and in the months after childbirth, when they could be offered the opportunity to discuss their labour and birth.

Background

Physical and psychological problems after childbirth are common, and may have a significant negative and possibly long-term impact on women's wellbeing and daily functioning [1]. One factor that has been more consistently identified as influencing the duration and severity of women's physical and psychological symptoms following childbirth is the mode of birth. However, population-wide evidence to demonstrate this association is largely lacking; the quality of the research varies considerably and few studies have examined both women's psychological and physical outcomes and explored the possibility that these symptoms may influence one another. Although findings from earlier studies related to postnatal outcomes and mode of birth were based on relatively large samples [2,3], subsequent studies have relied on small samples, and there are few studies that have been able to separate the psychological and physical outcomes for women who have forceps-assisted births from ventouse-assisted births. A recent large, population-based study from Norway, focusing on antenatal and postnatal emotional health, did not differentiate between forceps and ventouse-assisted vaginal births and found no association with mode of birth [4].

Perineal problems, backache, pain during intercourse, and extreme tiredness are common problems for women during the first three months after birth [5,6]. Although caesarean section births have been identified as contributing to reductions in women's postnatal physical wellbeing, more recently there has been interest in exploring the impact of assisted vaginal births. In the first few months after birth, assisted vaginal births have been associated with a greater likelihood of perineal pain [7,8] urinary incontinence, haemorrhoids, and sexual problems [7,[9][10][11] than unassisted vaginal births, even when taking into account aspects related to the birth such as the length of labour and degree of perineal trauma [7,11]. Women who have assisted vaginal births, particularly forceps-assisted vaginal births may also be at risk for ongoing and continuing physical problems [12]. A recent longitudinal study from Birmingham found that faecal incontinence at 12 years postpartum was increased among women who had a birth history that included a forceps-assisted vaginal birth, but not with a birth history that included a ventouse-assisted vaginal birth [13].

Obstetric intervention, particularly emergency caesarean section, has also been associated with depressive symptoms after birth, however, results are inconsistent [14] and recent evidence from large cohort studies has found no association [4,15,16]. More consistently, caesarean section births have been associated with feelings of dissatisfaction related to the birth and with poor body image and lower self-esteem [14], possibly increasing the risk of depression in the longer term. Recently, there has been an emergence of studies focusing on anxiety disorders, particularly posttraumatic stress disorder (PTSD) after childbirth, which occurs in a small proportion of women (1-6%) [17]. Studies show that the risk of postnatal PTSD symptoms is higher for women who have had unplanned caesarean section births followed by women who have had assisted vaginal births [18][19][20]. However, other researchers have found that women who have had an assisted vaginal birth are more likely to perceive the birth as "extremely distressing" six weeks after birth than women who have had a caesarean section or unassisted vaginal birth [21]. This study, however, did not differentiate between forceps and ventouse-assisted births and this may be important for understanding the influence of mode of birth on postnatal PTSD-type symptoms.

The experience of childbirth can be complex due to a wide range of individual, medical and social factors that can interact to influence women's experiences and outcomes. This study examines the physical and psychological outcomes of women in the first three months after birth, and whether these varied by mode of birth. We specifically examined whether women who had assisted vaginal births and operative births experienced poorer psychological and physical health than women who had unassisted vaginal births. Because women's physical symptoms after birth may affect the severity and duration of their self-reported psychological outcomes and vice versa [22], we adjusted for concurrent psychological and physical symptoms in our analyses when examining women's postnatal outcomes. The results of this study will provide important information for health professionals about those women who may be in greater need of support during and after childbirth.

Methods

Participants

The study used data collected as part of the National Maternity Survey 2010, which aimed to document women's experiences of maternity care in England, to describe current practices, the care received and changes in these since the last survey conducted in 2006 [23,24]. Based on birth registration, surveys were sent by the Office for National Statistics to a random sample of 10,000 women aged 16 years and over, who had recently given birth in England over a two week period. Questionnaires were mailed out in January 2010 at three months postpartum, and women were asked about their experiences during pregnancy, birth and during the postnatal period. Two methods of responding to the survey were offered: a paper questionnaire returned by post or an online survey. Tailored reminders were sent out: a letter after two weeks, a further questionnaire after four weeks and a further reminder letter four weeks after that.

Measures

The National Maternity Survey was designed to document the care and support provided to women during pregnancy, birth and after birth. Women were also asked about their health and wellbeing over this time, about partner engagement and support for infant care. Demographic information including age, parity, ethnicity, age left full-time education, marital status, employment status, Index of Multiple Deprivation (IMD) (a small area based measure) and country of birth were also collected. Mode of birth was self-reported and categorised as five groups: unassisted vaginal birth, ventouse-assisted vaginal birth, forceps-assisted vaginal birth, unplanned caesarean-section and planned caesarean-section.

Women's psychological and physical wellbeing after birth were assessed using a checklist of common postnatal psychological and physical symptoms based on that used in earlier national maternity surveys [25]. Women were asked, "Did you experience any of the following. . ." and chose from a list of physical problems and psychological symptoms: "the blues", painful stitches, breastfeeding problems, depression, wound infection, stress incontinence, fatigue or severe tiredness, backache, anxiety, pain during intercourse, sleep problems not related to the baby, flashbacks to the labour and birth and difficulties in concentrating. At 10 days, 1 month and 3 months, women were asked to tick the box for each symptom if the symptom was present at that particular time point after the birth.

The study undertaken was carried out as a secondary analysis of survey data. The original survey on which the evaluation of maternity services in England was based was passed by the Trent Multi-Centre Research Ethics Committee.

Exploratory factor analysis, namely principal components analysis, was conducted to determine the factor structure underlying the postnatal symptoms. We specified an oblique promax rotation to account for the correlations among the subscales. Initial eigen values for the first four factors were all above 1, which was reflected in the scree plot where values "levelled off" after first four factors, suggesting a four factor model. The first four factors explained 47% of the total variance, with each factor explaining 22%, 9%, 8% and 8% of the variance, respectively. The factor loadings showed that the psychological symptoms loaded highly on the first factor and the three PTSD items clustered with these items. The physical symptoms: "stress incontinence", "backache" and "difficulties/pain during intercourse" loaded highly together on the second factor, while "breastfeeding difficulties" loaded on the third factor and "painful stitches" and "wound infection" loaded together on the fourth factor. Severe tiredness/ fatigue did not appear to load highly with any of the other items, however, as this was an important and prevalent postnatal symptom, this variable was included in our analysis.

Based on the results of the factor analysis, psychological theory and literature [26][27][28], the items were grouped as follows: psychological symptoms (e.g. "the blues", depression, anxiety); posttraumatic stress-type symptoms (e.g. flash-backs, difficulties concentrating, sleep problems not related to the baby); bodily changes (e.g. stress incontinence, backache; difficulties/pain during intercourse); birth-related symptoms (e.g. painful stitches, wound infection); breastfeeding problems and severe fatigue. All items in each grouping were combined to form a single variable representing those items, except for the psychological symptoms, breastfeeding problems and severe fatigue, which were examined separately.

Statistical analysis

Univariate analyses (analysis of variance, chi-square tests) were used to test for associations between demographic and obstetric characteristics, psychological and physical symptoms and mode of birth at 10 days, 1 month and 3 months. We used multinomial logistic regression models to examine whether women's psychological and physical outcomes at the three time points varied according to the mode of birth. The models were adjusted for key sociodemographic variables including mother's age (in years), parity (primiparous, multiparous), age on leaving full-time education (<16, 17-18, ≥19), Index of Multiple Deprivation (IMD; quintiles), ethnicity (white, other) and all other symptoms. Although univariate and multivariate analyses were conducted to examine the differences between mode of birth and psychological and physical symptoms at all three time points, we have presented the results for 1 month and 3 months only, as these time points are most likely to be associated with longer-term problems. All statistical analyses were performed using SPSS version 19.

Results

Participant characteristics

Of the 5,332 women who responded to the survey, more than half (58%) were between the ages of 25 and 34 years. The median age of babies at the time of women responding was 3.1 months (mean 3.3 months, s.d. 0.65 and range 2.2-6.6 months). Half the women had given birth before, 12% were single parents at the time of the survey, 22% had left school by the age of 16 years, 14% were from Black and Minority Ethnic Groups and 21% had been born outside the UK. Data on women who did not respond to the survey were provided by the Office of National Statistics and comparisons showed that nonrespondents were more likely to be younger, living in a more disadvantaged area and from a Black and Minority Ethnic background [23]. Table 1 shows the characteristics of the women included in the analysis according to mode of birth. Most women (n = 3275, 61%) had unassisted vaginal births. Ventouse-assisted vaginal births and forcepsassisted vaginal births were reported less frequently by 6% (n = 302) and 7% (n = 359) of women, respectively. A total of 12% (n = 630) of women had a planned caesarean and for 13% (n = 675) their caesarean was unplanned. Women who had unassisted vaginal births were more likely to be younger, not married, to have previously given birth and less likely to have been in fulltime education after the age of 19 years. In contrast, women who reported having a ventouse-assisted vaginal birth were more likely to be married, to have recently given birth to their first baby, to have received further  (14) 20 (7) 34 (10) 129 (20) 96 (16) Age left full-time education (years) ≤ 16 751 (23) 51 (17) 63 (18) 115 (17)  education after the age of 19 years and to be from a white ethnic background. Women who had a forcepsassisted vaginal birth were similar to women having a ventouse-assisted vaginal birth on marital status, parity and ethnicity but they were less likely to have received any further education after the age of 19 years. Women reporting an unplanned caesarean were also similar to women who had had assisted vaginal births on marital status and parity but they were more likely to be from a black minority ethnic background and not born in the UK. Women who had planned caesareans were more likely to be older, married and to have previously given birth than women delivering by other methods.

In relation to obstetric variables, women who had assisted vaginal births were more likely to have had constant electronic monitoring during labour (ventouse: n =221, 73%; forceps: n =255, 71%); to have laboured for more than 18 hours (ventouse: n =109, 37%; forceps: n =165, 47%); to have had an episiotomy (ventouse: n =234, 79%; forceps: n =337, 94%) and perineal repair (ventouse: n =286, 96%; forceps: n =352, 99%) than women who had unassisted vaginal births (all p < 0.001). Women who had a forceps-assisted vaginal birth were also more likely to have had a third or fourth degree tear (n =41, 12%) than women who had a ventouse-assisted birth (n =13, 5%) or unassisted vaginal birth (n = 141, 4%) (p < 0.001).

Prevalence of symptoms

The most common psychological symptom reported during the first 10 days was "the blues" (34%) followed by "anxiety" and "difficulties concentrating" (both 16%). While all women reported a decline in symptoms over time, PTSD-type symptoms such as "flashbacks to the labour and birth" (11 %) and "difficulties concentrating" (15%) were more common at 3 months amongst women who had a forceps-assisted vaginal birth (see Figures 1   and 2). Amongst all women the most common physical symptoms reported at 10 days after the birth were fatigue or severe tiredness (37%), breastfeeding problems (35%), painful stitches (34%) and backache (28%). As with the psychological symptoms a similar pattern of reducing prevalence over time was observed for the physical symptoms, with backache, difficulties experienced during sexual intercourse and stress incontinence occurring more commonly in women who had assisted vaginal births at 3 months (see percentages for bodily changes in Tables 2 and 3).

Univariate analyses

In relation to mode of birth, women who had forcepsassisted vaginal births were more likely to report symptoms of depression (11%), anxiety (17%) and PTSD-type symptoms (1 symptom, 24%; 2 or 3 symptoms, 9%) at 1 month after birth than women who had unassisted vaginal births ( Table 2). There were no differences between the groups for "the blues". At 3 months after birth, women who had experienced a forceps-assisted vaginal birth were more likely to report two or more PTSD-type symptoms (7% compared with 2% or 3% for the other groups) but there were no differences between the groups on any of the other psychological outcomes (Table 3).

Physical symptoms including bodily changes were more prevalent at 1 month after birth among women who had ventouse (46%) and forceps-assisted vaginal births (43%) than women in the other groups. Birthrelated symptoms (30%), breastfeeding difficulties (20%) and fatigue (32%) were also more likely at 1 month after birth amongst women who had a forceps-assisted vaginal birth than women who had unassisted vaginal births ( Table 2). Although bodily changes and birth-related symptoms were higher at 3 months for those who had assisted vaginal births, there were no other differences    between the groups on any of the other physical outcomes (Table 3).

Multivariate analyses Psychological symptoms by mode of birth

Unplanned caesarean section births, which may include both urgent and non-urgent procedures, were marginally associated with an increased risk of developing two or more PTSD-type symptoms (e.g. flashbacks, sleep problems unrelated to the baby) at 1 month after birth (OR = 1.52; 95% CI: 1.01-2.29) than women who had unassisted vaginal births, but there was no association at 3 months (OR = 1.16; 95% CI: 0.67-2.01). Although the associations were not significant, women who had a forceps-assisted vaginal birth had a somewhat greater risk of symptoms of anxiety at 1 month after birth (OR= 1.30; CI: 0.90-1.89) than women who had unassisted vaginal births. There was no association between a forceps-assisted vaginal birth and symptoms of anxiety at 3 months, however, the risk of PTSD-type symptoms remained significantly increased (

Physical symptoms by mode of birth

In comparison to women who had an unassisted vaginal birth, birth-related symptoms were increased at all three time points for the other birth groups, and were particularly high among the forceps-assisted vaginal births (

Discussion

Mode of birth may have a strong association with women's psychological and physical outcomes in the first Women who reported "the blues" were added to this group. few months after birth. As expected, women's symptoms were highest at 10 days after birth, and the health of most women improved emotionally and physically over the following 3 months. However, some outcomes appeared to be related to the type of delivery experienced, particularly when adjusting for sociodemographic variables and the other psychological and physical symptoms present at each time point. Specifically, women who had forceps-assisted vaginal births and unplanned caesareans appeared to have poor health and psychological wellbeing after birth. The health of women who had ventouse-assisted births appeared to be somewhat compromised, while the health of women who had unassisted vaginal births and planned caesareans did not appear to be influenced by the birth process. Women who had a forceps-assisted vaginal birth were most likely to have ongoing psychological problems after childbirth. These women were more likely to report two or more PTSD-type symptoms at 3 months, and this may be explained by the fact that their labours were more likely to have been longer, and there may have been concern about their labour or their baby as indicated by the higher levels of constant electronic monitoring reported. A lack of control, worry and an intense period of anxiety or uncertainty during labour and birth may explain the psychological symptoms reported by these women. This, in combination with other individual factors related to the mode of birth, such as a woman's labour and birth expectations may also predispose women to stress-related symptoms after birth, as well as factors occurring after the birth, including a lack of postnatal follow-up with a health professional when this might have been helpful [27]. In our study, more than half (58%) of women who had a forceps-assisted vaginal birth reported that they spoke about their labour with a health professional. However, of those women who didn't speak to a health professional, 43% would have liked to speak to someone. While health professionals Table 4 Results from logistic regression model: Association between psychological symptoms, bodily changes, birth-related symptoms, breastfeeding, severe fatigue and mode of birth at 3 months after birth may be more aware of the physical morbidity for women after childbirth, the ongoing psychological issues may be less clear or alternatively, perceived as more difficult to treat or beyond their area of expertise. Referral pathways need to be in place to address this point [29].

Although caesareans section births are often described as resulting in poorer postnatal psychological outcomes for women, it seems that the outcomes for women depend on whether the caesarean section is planned or not. Women having unplanned caesarean section births were marginally more likely to report PTSD-type symptoms, however, there was no association between PTSDtype symptoms and planned caesarean section births in our study. These findings are consistent with others [18][19][20] and are important for understanding the individual and social factors that may influence women's postnatal psychological wellbeing. For a proportion of women it appears that there may be benefits of a planned caesarean birth, especially for those with health problems, previous complications or adverse experiences [14,30]. However, a large study of 55,000 Norwegian women did not find an association between mode of birth and a measure of emotional distress at 6 months postpartum [4], which suggests that symptoms of anxiety in new mothers may attenuate significantly with time. On the other hand, in our study depression was not associated with mode of birth, and this may suggest the need to distinguish between symptoms of anxiety and depression rather than using general measures of distress. It is possible that because our study differentiated between assisted vaginal births (forceps vs. ventouse) when assessing women's psychological outcomes, this may have contributed to the difference in findings.

Birth related symptoms, such as painful stitches and wound infections after birth, were problematic for women during the first 3 months after birth. Those who had forceps-assisted vaginal births were more likely to report problems, and this may be explained by the high rates of episiotomy and third and fourth degree tears requiring perineal repair in this group. Consistent with previous research [7,[9][10][11], the risk of bodily changes such as stress incontinence and backache were higher amongst women who had assisted vaginal births whereas these symptoms were less likely to affect women who had operative births. This is one of the few studies to use population-based data to examine women's physical and psychological outcomes after childbirth whilst taking into account cooccurring conditions and other key sociodemographic variables. As has been reported previously, women's physical and psychological outcomes are important to take into account when examining their overall postnatal health and wellbeing [22]. In our study, we also found that women's risk of postnatal physical symptoms was reduced when adjusting for co-occurring psychological symptoms. Our results suggest that both the psychological and physical domains need to be assessed in terms of understanding the factors influencing the duration and severity of problems affecting women's postnatal health and wellbeing.

Although our findings are based on data from a large, national sample of women, a limitation of the study was the participation rate of 55%, a rate which is similar to that increasingly found in epidemiological studies [31]. Because women who did not participate in this study were more likely to be younger, living in a more disadvantaged area and from a BME background, and because these groups tend to have higher rates of poor health and wellbeing generally, our study may underestimate the magnitude of physical and psychological outcomes. However, the similarities between our results and those of previous studies strengthen our conclusions. Another limitation of this study was the reliance on women's retrospective self-reports of their health and wellbeing after birth. However, the timing of survey completion and return was close enough to the birth for accuracy as far as mode of delivery and other aspects of care were concerned, and for symptom reporting at three months post-partum. Nevertheless, pre-existing experiences and those occurring after birth may be significant in increasing women's vulnerability to distress and maintaining the distress [32] and we cannot be certain that women's responses were not influenced by prior events or those occurring at the time of survey completion. It is also not clear how well the symptoms recorded in our study could relate to an actual diagnosis of PTSD, and thus we cannot comment on rates of clinical levels of PTSD in our sample. However, given that our findings do reflect those of previous research carried out using smaller samples and different study designs, it seems that the items used in our survey to assess PTSD-type symptoms appear to detect disturbances in new mothers.

Conclusions

This study provides additional evidence about the importance of mode of birth in understanding women's postnatal health and wellbeing. Our results appear to suggest that there are unique issues for women according to the mode of birth, and thus future analyses should be aware of the potential differences in wellbeing among women who have planned and unplanned caesarean births as well as ventouse and forceps-assisted vaginal births relative to unassisted vaginal births. It is concerning that women who have a forceps-assisted vaginal birth are more likely to report ongoing psychological difficulties, but are not being offered the appropriate level of healthcare support. Because these women were mostly likely to report poorer psychological and physical health following childbirth, at the same time as adjusting to life with a new baby, it may be important for health professionals to initiate conversations with women about their birth to give women the opportunity to discuss distressing symptoms. Consistent with NICE guidelines, women who describe PTSD-type symptoms at 1 month after birth should be formally tested for PTSD using a standard instrument (e.g. Traumatic Event Scale-B (TES-B); PTSD Symptom Scale-Self Report (PSS-SR) [33]. Women at risk should be monitored carefully by health professionals and appropriate referral pathways should be used in an effort to prevent chronic symptoms. Ongoing support, however, may be necessary, particularly in relation to further pregnancies. The development of preventative and supportive strategies during pregnancy, birth and afterwards would be an initial step towards improving the long-term reproductive health and wellbeing of these women.

Figure 1
1Proportion of women reporting 'flashbacks to the labour and birth'. Proportion of women reporting 'flashbacks to the labour and birth' during the first 3 months after birth according to mode of birth.

Figure 2
2Proportion of women reporting 'difficulties concentrating'. Proportion of women reporting 'difficulties concentrating' during the first 3 months after birth according to mode of birth.

Table 1
1Characteristics of women in the national maternity survey by mode of deliveryNon-operative births
Operative births

Unassisted
Vaginal a

Ventouse-
assisted a

Forceps-
assisted a

Unplanned
Caesarean a

Planned
Caesarean a

p

n = 3275
n = 302
n = 359
n = 675
n = 630

n (%)
n (%)
n (%)
n (%)
n (%)

DEMOGRAPHIC

Mother's age (years)

M (SD)
30.11 (5.75)
30.27 (5.26)
30.30 (5.49)
31.07 (5.55)
32.88 (5.31)
<0.001

Parity

Primiparous
1342 (42)
246 (83)
299 (83)
492 (75)
193 (32)
<0.001

Multiparous
1880 (58)
51 (17)
51 (15)
164 (25)
420 (69)

Ethnicity

White
2780 (86)
279 (93)
322 (90)
534 (81)
521 (84)
<0.001

Other b
445

Table 2
2Association between psychological symptoms, bodily changes, birth-related symptoms, breastfeeding, severe fatigue and mode of birth at 1 month after birthUnassisted
Vaginal a

Ventouse-
assisted a

Forceps-
assisted a

Unplanned
Caesarean a

Planned
Caesarean a

p

n = 3275
n = 302
n = 359
n = 675
n = 630

n (%)
n (%)
n (%)
n (%)
n (%)

The blues

No
2850 (87)
266 (88)
295 (82)
574 (85)
544 (86)
0.08

Yes
425 (13)
36 (12)
64 (18)
101 (15)
86 (14)

Depression

No
3055 (93)
289 (96)
320 (89)
627 (93)
589 (94)
0.02

Yes
220 (7)
13 (4)
39 (11)
48 (7)
41 (7)

Anxiety

No
2961 (90)
272 (90)
298 (83)
596 (88)
568 (90)
<0.001

Yes
314 (10)
30 (10)
61 (17)
79 (12)
62 (10)

PTSD symptoms

No
2646 (81)
229 (76)
243 (68)
515 (76)
529 (84)
<0.001

1
494 (15)
56 (19)
85 (24)
110 (16)
76 (12)

≥2
135 (4)
17 (6)
31 (9)
50 (7)
25 (4)

Bodily changes

No
2156 (66)
164 (54)
202 (56)
444 (66)
453 (72)
<0.001

Yes
1119 (34)
138 (46)
157 (43)
231 (34)
177 (28)

Birth -related symptoms

No
3017 (92)
242 (80)
250 (70)
546 (81)
534 (84)
<0.001

Yes
258 (8)
60 (20)
109 (30)
129 (19)
96 (15)

Breastfeeding difficulties

No
2828 (86)
246 (82)
286 (80)
551 (82)
555 (88)
<0.001

Yes
447 (14)
56 (19)
73 (20)
124 (18)
75 (12)

Severe tiredness/ Fatigue

No
2487 (76)
222 (74)
246 (69)
488 (72)
469 (74)
0.02

Yes
788 (24)
80 (27)
113 (32)
187 (28)
161 (26)

a

Numbers may not sum to total because some data missing.

Table 4 :
4OR = 1.86;

Table 4 :
43 month OR = 4.89; 95% CI: 2.68-8.94). At 3
months, women who had a forceps-or ventouse-assisted
vaginal birth had a greater risk of bodily changes and
women who had caesarean section births were less likely
to report bodily changes (e.g. stress incontinence, back-
ache, painful intercourse) at 10 days, 1 month and 3
months. No association was found between breastfeed-
ing and mode of birth at any time point.

Table 3
3Association between psychological symptoms, bodily changes, birth-related symptoms, breastfeeding, severe fatigue and mode of birth at 3 months after birthUnassisted
Vaginal a

Ventouse-
assisted a

Forceps-
assisted a

Unplanned
Caesarean a

Planned
Caesarean a

p

n = 3275
n = 302
n = 359
n = 675
n = 630

n (%)
n (%)
n (%)
n (%)
n (%)

Depression b

No
3050 (93)
285 (94)
328 (91)
632 (94)
591 (94)
0.53

Yes
225 (7)
17 (6)
31 (9)
43 (6)
39 (6)

Anxiety

No
3123 (95)
287 (95)
335 (93)
636 (94)
597 (95)
0.41

Yes
152 (5)
15 (5)
24 (7)
39 (6)
33 (5)

PTSD symptoms

No
2842 (87)
254 (84)
283 (79)
567 (84)
557 (88)
<0.001

1
340 (10)
43 (14)
51 (14)
87 (13)
56 (9)

≥2
93 (3)
5 (2)
25 (7)
21 (3)
17 (3)

Bodily changes

No
2445 (75)
188 (62)
212 (59)
506 (75)
499 (79)
<0.001

Yes
830 (25)
114 (38)
147 (41)
169 (25)
131 (21)

Birth-related symptoms

No
3238 (99)
288 (95)
337 (94)
645 (96)
607 (96)
<0.001

Yes
37 (1)
14 (5)
22 (6)
30 (4)
23 (4)

Breastfeeding difficulties

No
3147 (96)
291 (96)
334 (93)
643 (95)
605 (96)
0.08

Yes
128 (4)
11 (4)
25 (7)
32 (5)
25 (4)

Severe tiredness/ Fatigue

No
2942 (90)
273 (90)
316 (88)
615 (91)
558 (89)
0.46

Yes
333 (10)
29 (10)
43 (12)
60 (9)
72 (11)

a

Numbers may not sum to total because some data missing.

b

Note. The reference group is unassisted vaginal births.Ventouse-assisted
Adjusted

Forceps-assisted
Adjusted

Unplanned
Caesarean
Adjusted

Planned
Caesarean
Adjusted

OR a (95% CI)
OR a (95% CI)
OR a (95% CI)
OR a (95% CI)

Depression

No
1
1
1
1

Yes
0.85 (0.49-1.48)
0.99 (0.62-1.58)
0.93 (0.62-1.37)
0.89 (0.59-1.34)

Anxiety

No
1
1
1
1

Yes
1.17 (0.63-2.16)
1.05 (0.62-1.79)
1.40 (0.90-2.17)
1.32 (0.83-2.09)

PTSD symptoms

No
1
1
1
1

1
1.18 (0.80-1.74)
1.25 (0.87-1.79)
1.36 (1.02-1.82)
0.85 (0.61-1.17)

≥2
0.46 (0.17-1.20)
1.86 (1.06-3.24)
1.16 (0.67-2.01)
0.88 (0.48-1.63)

Bodily changes

No
1
1
1
1

Yes
1.46 (1.12-1.92)
1.56 (1.21-2.01)
0.76 (0.61-0.95)
0.71 (0.56-0.89)

Birth -related symptoms

No
1
1
1
1

Yes
4.31 (2.16-8.62)
4.89 (2.68-8.94)
4.21 (2.44-7.27)
4.36 (2.50-7.62)

Breastfeeding difficulties

No
1
1
1
1

Yes
0.63 (0.33-1.21)
1.07 (0.65-1.77)
0.87 (0.56-1.35)
0.97 (0.61-1.53)

Severe tiredness/ Fatigue

No
1
1
1
1

Yes
0.86 (0.55-1.34)
0.88 (0.59-1.31)
0.79 (0.56-1.10)
1.10 (0.81-1.51)

a

Adjusted for age (in years), parity (primiparous, multiparous), age on leaving full-time education (<16, 17-18, ≥19), IMD (quintiles), ethnicity (white, other) and all
other birth-related outcomes presented in the table.

http://www.biomedcentral.com/1471-2393/12/138
AcknowledgementsWe are most grateful to the many women who participated in the survey. This paper reports on an independent study which is part-funded by the Policy Research Programme in the Department of Health. The views expressed are not necessarily those of the Department. The European Health Psychology Society provided funding to support the collaboration which enabled this study.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsThe authors listed have contributed to this paper: IJR performed the statistical analysis and drafted the manuscript. MR was responsible for the concept and conduct of the UK 2010 National Maternity Survey and provided assistance in the interpretation of results and drafting the manuscript. IJR and MR have read and approved the final manuscript.
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Cite this article as: Rowlands and Redshaw: Mode of birth and women's psychological and physical wellbeing in the postnatal period. BMC Pregnancy and Childbirth. 12138Cite this article as: Rowlands and Redshaw: Mode of birth and women's psychological and physical wellbeing in the postnatal period. BMC Pregnancy and Childbirth 2012 12:138.


Open Access RESEARCH Phylogenetic assessment of alignments reveals neglected tree signal in gaps Alignments for phylogenetics Tree-based tests of alignment methods enable the evaluation of the effect of gap placement on the inference of phylogenetic relationships
2010

Gil Genome Dessimoz
Biology
Open Access RESEARCH Phylogenetic assessment of alignments reveals neglected tree signal in gaps Alignments for phylogenetics Tree-based tests of alignment methods enable the evaluation of the effect of gap placement on the inference of phylogenetic relationships
11372010BioMed Central Research
Background: The alignment of biological sequences is of chief importance to most evolutionary and comparative genomics studies, yet the two main approaches used to assess alignment accuracy have flaws: reference alignments are derived from the biased sample of proteins with known structure, and simulated data lack realism.Results: Here, we introduce tree-based tests of alignment accuracy, which not only use large and representative samples of real biological data, but also enable the evaluation of the effect of gap placement on phylogenetic inference. We show that (i) the current belief that consistency-based alignments outperform scoring matrix-based alignments is misguided; (ii) gaps carry substantial phylogenetic signal, but are poorly exploited by most alignment and tree building programs; (iii) even so, excluding gaps and variable regions is detrimental; (iv) disagreement among alignment programs says little about the accuracy of resulting trees.Conclusions:This study provides the broad community relying on sequence alignment with important practical recommendations, sets superior standards for assessing alignment accuracy, and paves the way for the development of phylogenetic inference methods of significantly higher resolution.

Background

The study of biological sequences almost inevitably begins with the process of alignment. The goal of this process is usually to match homologous characters, that is, characters that have a common ancestry [1]. In turn, these sets of homologs, the columns of the alignment, can be used for a variety of applications, such as identifying residues with analogous structural or functional role, or inferring the phylogenetic tree of the underlying sequences. The accuracy of multiple sequence alignment programs has been the object of numerous comparative studies [2][3][4], which evaluate alignments either by using trusted reference alignments obtained from structural data, or by using simulation. Unfortunately, both approaches have flaws. Trusted benchmark alignments such as Balibase, Prefab, Homstrad, or Sabmark [5][6][7][8] are all derived from protein structure information, exploiting the tendency of structure to evolve more slowly than sequence [9].

However, proteins with resolved structure remain a small and highly biased sample of all proteins [10,11]. In addition, homology inferred from structural information is inherently restricted to conserved regions, thereby providing little guidance for correct gap placement. The other approach to validating alignments is simulation [12][13][14][15][16][17][18]. Yet, results obtained from simulated data strongly depend on the choice of model used to generate the data, and most biological processes are difficult to model realistically. For instance, current insertion-deletion models are known to be insufficient [19]. Even if a good model can be formulated, it will never fully capture the complexity of real biological data. Consequently, the results observed on simulated data differ significantly from those measured on empirical data [1].

Results and discussion

There is, therefore, a need for alternative evaluation procedures that do not rely on structural information while applicable to a large and representative sample of real biological data. In this work, we propose two such tests.

We then show how they offer answers to three of the most important open questions regarding sequence alignment for phylogenetic inference: (i) Which alignment approach leads to the most accurate trees? (ii) Are gap regions informative for phylogenetic inference or should they be ignored? (iii) What is the impact of alignment uncertainty on tree inference?

Phylogeny-based tests of alignment accuracy

The principle of the phylogeny-based tests of alignment accuracy is simple: the more accurate the resulting trees, the more accurate the alignments (in terms of homology matching) are assumed to be. Therefore, we can use tree accuracy as surrogate for alignment accuracy. The first phylogeny-based test we propose ('species-tree discordance') compares alignments of orthologous genes from species whose phylogeny is resolved and undisputed (Figure 1). By Fitch's definition of orthology [20], trees inferred from orthologs are expected to have the same topology as the underlying species. Thus, holding all else constant, if a particular method produces alignments that result more frequently in trees congruent with the phylogeny of the species, it is likely to be more accurate. A similar idea was previously used in the context of model comparison [21], and verification of orthology [22]. The second test ('minimum duplication') takes homologous sequences as input and uses a parsimony argument rather than knowledge about the phylogeny of the species: holding all else constant, the gene tree with the least number of duplication nodes is the most likely ( Figure 1, [23][24][25]). Hence, if a sequence alignment method results in tree topologies with consistently fewer duplications, it is likely to produce better alignments. Given a tree, a conservative estimate of the number of duplication events can be obtained using the concept of species overlap [26]. By accepting practically any gene family as input, the two tests can be performed on sequences relevant to a given biological study. Moreover, note that by design, the tests are robust to sources of errors that affect all alignment methods equally on average, such as stochastic errors in tree inference, lateral gene transfers, or the choice of evolutionary model. For instance, although the parsimony assumption may occasionally underestimate the true number of duplicated genes (for example, in gene families with many duplications/losses), as long as this underestimation does not favor a particular alignment method, the ranking of the methods is unaffected.

Assessment of alignment methods

To address the question of alignment accuracy, we used the tests to evaluate 13 MSA software packages, which can be classified into roughly three alignment scoring strategies: scoring matrix-based Mafft FFT-NS-2, Muscle, Clustal W2, DiAlign/-T/-TX, Kalign [6,[27][28][29][30][31][32][33]; consistency-based Mafft L-INS-i, T-Coffee, Mummals, Prob-Cons, ProbAlign [27,28,[34][35][36][37]; and tree-aware-gapplacing Prank [38]. We tested the alignment software both on amino-acid and on nucleotide data, with the Figure 1 Schematic of the phylogeny-based tests of alignment accuracy. Both tests are based on large-scale genomic data: (a) The species-tree discordance test samples sets of orthologs inferred by OMA among species with a well-accepted phylogeny (Additional file 1, Figure S1). Each sample is aligned by the different packages. The resulting alignments are evaluated by reconstructing trees from them, and comparing with the reference topology. All else being equal, trees from better alignment packages show higher average congruence with the reference topology. (b) The minimum duplication test follows a similar idea, but differs from the first test in two ways. First, it samples sets of homologs rather than the more specific orthologs. Second, the evaluation is based on a parsimony argument rather than knowledge about the phylogeny of the species: all else being equal, alignments yielding trees with fewer duplication nodes on average are more accurate.  exception of Mummals and ProbCons, which only run on amino-acid data. For the species-tree discordance test, we sampled sets of 6 orthologs as inferred by OMA [39] among 57 eukaryotic, 11 fungal, and 418 bacterial genomes, under the constraint that the branching order of the species represented in each set be well-accepted (Additional file 1, Figure S1). For the minimum duplication test, we retrieved groups of up to 60 homologs from 18 metazoan and 18 fungal genomes. Trees were reconstructed by maximum likelihood (ML) from both aminoacid and nucleotide alignments. In addition, to compare the two types of alignments under the same evolutionary model, ML trees were also reconstructed from backtranslated amino-acid alignments, using the actual codons from the corresponding nucleotide sequences. In total, the tests required computing over 100,000 alignments of up to 60 sequences, at a cost of over 20,000 CPU hours.

In general, we observed fewer differences among programs aligning amino-acids than aligning nucleotides ( Figure 2). Trees from nucleotide alignments fared significantly worse than those from back-translated aminoacid alignments in practically all cases. Since the only difference between the two types of trees resides in the alignment process, we conclude that current alignment packages align amino-acids more accurately than nucleotides (Additional file 1, Figure S7), as previously observed in simulation by [13]. In terms of alignment strategy, and contrary to current beliefs [3,4], consistency-based alignment methods as a class did not outperform their scoring matrix-based counterparts, yet they were up to 300 times slower ( Figure 2, Additional file 1, Figure S6). Thus, the additional time spent by consistency-based programs did not necessarily translate into more accurate trees. In addition, the consistency-based methods surveyed here tended to perform unevenly across different datasets, which suggests that their underlying models and/or parameters are relatively sensitive to input data characteristics. The potential misguidance of current benchmarks is exemplified in the results obtained from the different versions of DiAlign: although both simulated and structure-based reference alignments indicated that DiAlign had significantly improved over the course of the three releases investigated here [32], the present tests do not support this conclusion. While significant differences among the versions can be observed in particular datasets, no DiAlign variant demonstrated superior performance. In terms of individual programs, only small differences could be observed with amino-acid sequences. It nonetheless appears that DiAlign TX and Prank were consistently among the best programs (Additional file 1, Figure S6). With nucleotide sequences, the differences were greater. Mafft L-INS-i was the only package consistently among the best on nucleotide data. At the other end of the spectrum, T-Coffee, KAlign and DiAlign T exhibited subpar nucleotide alignment performance. Overall, as we have seen that alignments are almost invariably more accurate on amino-acid data, the best nucleotide alignments are obtained by back-translating amino-acid alignments.

To limit the risk of systematic biases or unrecognized factors, these observations were confirmed by two kinds Figure 2 Comparison of alignment methods. Assessment of various alignment methods under default parameters using (a) the species-tree discordance and (b) the minimum duplication tests, on eukaryotic data. Consistency-based alignment methods do not improve over scoring matrixbased methods. The relative performance between alignment programs is more variable for nucleotide data than for amino-acid data. On amino-acid data, Mafft-FFT-NS-2, DiAlign TX and Prank were never outperformed; on nucleotide data, Mafft L-INS-i (right column) was never outperformed (see also Additional file 1, Figure S6). Average compute times (per alignment) are plotted as triangles (amino-acids) and circles (nucleotides). Error bars correspond to ± 1 s.d. Significant difference from best alignment program is denoted with a minus symbol at the basis of relevant bars (Wilcoxon double-sided test, P < 0.01).  50
30.00 M a f f t F F T - N S - 2 M u s c le C lu s t a l W 2 D iA li g n D iA li g n T D iA li g n T X K a li g n M a f f t L - I N S - i T - C o f f e e M u m m a ls P r o b C o n s P r o b A li g nM a f f t F F T - N S - 2 M u s c le C lu s t a l W 2 D iA li g n D iA li g n T D iA li g n T X K a li g n M a f f t L - I N S - i T - C o f f e e M u- - - - - ---- Consistency Consistency
of controls. First, we considered the effect of the tree building method used in the test procedure. We ran the tests under a different model of evolution and using least squares distance trees instead of ML. The results were highly consistent (Additional file 1, Figures S8 and S9, relative accuracy of the two methods correlates with 0.90, P < 10 -10 , t-test). Second, we tested the dependence of the results on characteristics of the input data. We re-evaluated the tests on partitioned data and estimated the correlations between the relative accuracy of each partition with its full datasets. The data was segmented according to sequence length (Additional file 1, Figure S10, r = 0.62, P < 10 -10 ), sequence divergence (Additional file 1, Figure  S11, r = 0.67, P < 10 -10 ) and number of sequences (Additional file 1, Figure S12, r = 0.89, P < 10 -10 ). Furthermore, we contrasted the results of different pairs of lineages (Additional file 1, Figure S6, 0.68 <r ≤ 0.94, all P < 10 -3 ). In all cases, our conclusions above stand.

Guide trees make or break progressive alignments

Since sequence insertion and deletion events are generally assumed to take place along a tree, most aligners rely on guide trees to construct and score alignments. Some of them -in our case Mafft, Muscle, Clustal W2, T-Coffee and Prank -allow specification of the guide tree by the user. To investigate their sensitivity to tree specification, we ran the species-tree discordance test on two extreme cases: we provided either a random guide tree, or the reference species tree as guide (Additional file 1, Figure  S13). Unsurprisingly, the input trees hardly affected methods refining their guide trees iteratively (Muscle) or relying strongly on consistency (T-Coffee), a mostly treeindependent objective function. In contrast, strictly progressive methods (Mafft-FFT, Clustal W2, Prank) were highly sensitive to the provided guide tree. With such methods, guide tree specification is a double-edged sword: prior knowledge of the underlying sequence phylogeny, depending on its accuracy, can either improve the resulting alignments, or worsen them. Consequently, if the tree is known with high confidence, we recommend using it in conjunction with Prank or Mafft. If not, one might wonder which program infers the best guide trees, and whether feeding them to the other aligners could improve results overall. Our results suggest that on average, the best guide trees are inferred by Prank on aminoacid data, and Mafft on nucleotide data (Additional file 1, Figure S14). The difference is however not sufficiently large that the other alignment methods consistently profit from these improved guide trees (Additional file 1, Figure S15).

Gaps carry substantial unexploited tree signal

A notable advantage of our evaluation approach lies in its capacity to assess the accuracy and phylogenetic informa-tion content of gap regions. Given that structural alignments are inherently limited to regions of conserved structure, previous assessment of gap region accuracy were typically performed on simulated data only (for example, [40]). Using simulation, Löytynoja and Goldman, the authors of Prank, have recently argued that other alignment programs infer less phylogenetically plausible alignments [41]. However, though competitive, Prank did not show a clear advantage over the other alignment strategies in the tests described above, especially considering its much higher computational cost ( Figure 2). As it turns out, this is mainly a consequence of gap treatment in current ML tree building methods: by modeling each gap position as unknown character, they ignore much of the phylogenetic signal from gaps. To assess the phylogenetic signal of gaps, we repeated our tests using a tree inference method that only uses gap signals: maximum parsimony on binary gap/no-gap characters. On amino-acid data, the results using gap parsimony trees clearly show that Prank outperforms the other programs regarding gap placement on real biological sequences, at times quite dramatically (Figure 3a). On nucleotide data, Prank was occasionally surpassed by one of the DiAlign variants, but showed solid performance overall (Additional file 1, Figure S16). More importantly, although parsimony trees obtained from gaps are on average much less accurate than ML trees from substitutions, with Prank, the difference between the two considerably diminishes, especially at high levels of sequence divergence (Figure 3b). In one extreme case (fungal nucleotide data, species-tree discordance test), the gap parsimony trees from alignments by Prank largely surpassed the ML trees from alignments by several other methods (Additional file 1, Figures S6 and S16). The broader implication of these results is that gaps carry significant phylogenetic signal, information that is currently ignored by most alignment and tree reconstruction programs (and certainly not completely exploited in the simplistic parsimony approach employed here). We stress that this unexpected result could only be observed by combining the recent improvements in alignment afforded by Prank, our alignment evaluation methods, and a tree inference procedure that exploits gap patterns.

Excluding gaps and variable regions harms

It has been argued that even if gap regions carry potential phylogenetic signal, inclusion of these regions, which are usually more difficult to align than conserved ones, results in an overall decrease in the signal-to-noise ratio of alignments [42]. And indeed, the common recommendation of excluding 'gaps and ambiguous sites' in phylogenetic analyses tends to support this view as well. Even so, in some cases, studies on particular gene families [43,44], or using simulation [18,45], have supported the opposite view. We investigated this issue by comparing trees reconstructed from full alignments versus from alignments without gap columns (that is, without columns containing gaps), and full alignments versus alignments curated by Gblocks [42]. By default, Gblocks identifies and removes both gap columns and variable regions. For amino-acid alignments, excluding gap columns never improved tree accuracy, and often worsened it (Figure 4, Additional file 1, Figures S18 and S19). Removing variable regions in addition to gaps, as performed by Gblocks, had a strong negative impact on the accuracy of trees. For nucleotide alignments, the effects were not nearly as detrimental; in some cases, the filtering helped (Additional file 1, Figure S19). But remember that alignment programs often have difficulty with nucleotide sequences; almost invariably, the best trees were obtained from unfiltered, amino-acid sequence alignments. Most striking about these findings is that, as pointed out above, the standard tree building methods used here do not exploit gap patterns; it appears that character substitution patterns inside gap and variable regions carry enough phylogenetic signal to warrant inclusion of those segments under current methods.

Alignment variability poorly predicts tree accuracy

We have seen that different alignment programs can give rise to trees of varying accuracy. But in the broader context of tree inference, sequence alignment is not the only source of tree uncertainty. By 'uncertainty', we mean the expected addition of systematic and random error, that is, the expected inaccuracy. For instance, the amount of input data (that is, sequence lengths), the divergence between sequences, the model of evolution, or the tree searching algorithm all affect the accuracy of reconstructed trees, and one's confidence therein. This raises the question of the relative contribution of alignment uncertainty to tree uncertainty. Wong et al. recently quantified the observation that different alignment programs often lead to different tree topologies [46]. They found a correlation (Spearman-rank correlation r s = 0.53) between alignment variability (average distance between alignments from different methods) and tree variability (average topological distance among trees estimated from different alignment methods). But constrained by a lack of measure of total tree error, their analysis only focused on the random component of tree uncertainty. We exploited the tree accuracy measure from the speciestree discordance test to estimate the correlation between alignment variability and tree accuracy. Interestingly, accounting for both random and systematic errors suggests a weaker connection between alignment and tree quality: the negative correlation between alignment variability and tree accuracy was low for amino-acid and back-translated data (Additional file 1, Figure S20, -r s < 0.16, P < 0.01, t-test). Thus, alignment variability says little about overall tree uncertainty for amino-acid alignments. To put the results into perspective, we also estimated the correlation between bootstrap tree support and tree accuracy. Surprisingly, even though bootstrap Figure 3 Phylogenetic signal of gaps. (a) Assessment of gap accuracy under default parameters using the species-tree discordance test with parsimony trees on presence/absence patterns of gap characters in aminoacid alignments. By taking into account gap information, this test demonstrates that the gap placement of Prank is significantly better than other alignment methods. This cannot be observed either using standard tree building methods (Figure 2), or using structure-based benchmarks. Error bars correspond to ± 1 s.d. Significant difference from Prank is denoted with a minus symbol at the basis of relevant bars (Wilcoxon double-sided test, P < 0.01). (b) Accuracy of maximum likelihood (ML) trees on amino-acid substitution patterns versus parsimony on binary gap presence/absence characters, on fungal data. The phylogenetic signal of gaps inferred by Prank increases with divergence. For distant sequences, the proportion of correctly inferred splits from gaps alone is close to that from amino-acids substitutions by ML. Thus, tree building methods could capture up to twice as much phylogenetic signal from the same data. Moreover, note that the crude approach used here to infer the gap trees likely understates the potential of gap patterns. assumes correct alignments, it was a consistently better predictor of tree accuracy than alignment variability (Additional file 1, Figure S20, r s, Bootstrap > -r s, AlignmentVar , P < 0.006, see methods). For nucleotide alignments, shown above to be often worse than amino-acid alignments, we found a higher correlation between alignment variability and tree accuracy than for the amino-acid counterparts. Still, alignment variability was never a better predictor of tree accuracy than tree support (Additional file 1, Figure  S20). Since tree support is usually computed anyway, this casts doubt on the usefulness of trying more than one alignment method for the purpose of phylogenetic inference [47]. Rather, we recommend that practitioners stick with an accurate alignment method, as identified by tests such as the ones presented here.

Conclusions

In summary, the use of trees rather than protein structure to assess alignments is advantageous in that it more closely fits a common application of alignments, it is not restricted to the relatively small and biased sample of proteins with known structure, and it also allows the evaluation of gap regions. Indeed, our results show that consistency-based alignment methods, which score best in structural benchmarks, do not yield significantly better trees than their scoring matrix-based counterparts. Our tests also demonstrate that gaps often carry a strong phylogenetic signal, which at present is not well exploited, either by most alignment methods, or by standard tree building methods; but even with such methods, excluding gaps and variable regions worsen the resulting trees. Finally, the low correlation we observed between alignment variability and tree accuracy suggests that there is little to gain from the common practice of trying more than one alignment program on a given dataset. This latter result, as well as the analysis on the impact of guide tree specification, rely exclusively on the species-tree discordance test, because they require knowledge of a refer-
M a ff t F F T - N S - 2 M u s c l e C l u s t a l W 2 D i A l i g n D i A l i g n T D i A l i g n T X K a l i g n M a ff t L - I N S - i
T -C o ff e e M u m m a l s P r o b C o n s P r o b A l i g n P r a n k + F Mean number of duplication

Scoring matrix Consistency

Not curated Gap removed Gblocks ------------------ence topology. As such, the conclusions are based on sixtaxa trees only. How well they generalize to larger trees is yet to be investigated. Besides, further interesting questions remain: how do alignment methods perform on data not represented in this study, such as promoter regions or other non-coding sequences? How can we best extend our current models of sequence evolution to take into account the phylogenetic signal of gap patterns? How do the methods investigated here compare with the statistical approach of joint alignment and tree inference?

The methodology introduced here gives us the means to investigate these issues. Beyond alignments, the ability to measure tree accuracy under realistic conditions allows assessment of further important aspects of phylogeny inference, such as evolutionary models, tree building algorithms, or tree confidence measures.

Materials and methods

Sets of orthologous protein sequences

The Species Tree Discordance Test was performed on three sets of species: eukaryotes, fungi, and bacteria (detailed list in Supplementary Information Sect. 1.1). For all three sources of data, we retrieved sets of orthologs as inferred by OMA (Release of September 2008) [48].

Although cases of misclassification cannot be excluded, it has been shown in a previous study that the false-positive rate of OMA's predictions is low compared with other similar projects [22]. More importantly, though the presence of non-orthologs reduces the power of our test, it does not bias the results toward a particular alignment program. Sequences were sampled according to reference trees with a comb topology (Additional file 1, Figure S1). This topology ensures that all sequences in a sample are orthologous to each other [22]. In each trial, a starting sequence from a random species in the innermost leaf was randomly chosen. Then, for each remaining leaf, a random orthologous sequence was sampled.

Sets of homologous protein sequences

We performed the Minimum Duplication test on two sets of organisms: metazoa and fungi (detailed list in Supplementary Information Sect. 1.2). Sets of homologs were constructed by taking the transitive closure of pairs of sequences with high alignment scores (E-value below 10 -10 ). The sets were restricted to a maximal size of 60 sequences by removing sequences randomly from sets of excessive cardinality.

Definition: absolute minimum number of duplications

For any set of homologous genes, consider partitions of the sequences according to their genome of origin: each resulting partition consists of same-species paralogs. Let m be the maximum cardinality of these partitions. For m paralogs to be observed in the same genome, at least m-1 duplications had to take place. We denote m-1 as absolute minimum number of duplications for the set of homologs.

Species-tree discordance test

The species-tree discordance test evaluates a sequence alignment program in terms of the average accuracy of the trees reconstructed from its alignments. The test requires a large number of sequence sets whose phylogeny is known. Given that orthologous genes (by definition) follow the species tree, we sampled orthologs provided by OMA [48] from species with known and undisputed branching order (Additional file 1, Figure S1). Agreement between obtained and reference topologies was quantified by the proportion of wrong splits [49].

Minimum duplication test

In a gene tree, the split of two same-species paralogs is necessarily a duplication event. By a parsimonious argument, the tree with the least duplication splits represents the most likely evolutionary history. The minimum duplication test evaluates a sequence alignment program in terms of the average minimum number of gene duplication events implied in the trees reconstructed from its alignments of homologous sequences. Given a rooted tree, a lower bound on the number of duplications can be obtained by counting nodes that have subtrees with overlapping sets of species [26]. Since the placement of the root of the tree is usually unknown, we considered all possible rootings and retained the minimum number of duplications. This measure was normalized by subtracting the absolute minimum number of duplications from it (see above). An example computation can be found in Additional file 1, Figure S2.

Tree reconstruction

Gene trees were reconstructed by maximum likelihood using PhyML v. 2.4.4 [50] from the sequences aligned with the different programs under JTT+I+Γ for aminoacids and HKY+I+Γ for nucleotides. To investigate the accuracy of gap placement, the two tests were also performed using Wagner parsimony on the presence/ absence patterns of gaps (for a given alignment, each column containing at least one gap was considered a character and the presence/absence of a gap its state). To avoid over-counting, neighboring columns with identical gappatterns were combined into single characters.

Alternative tree building methods

As control, we recomputed the trees using a least-square distance approach instead of maximum likelihood: we reconstructed variance weighted least-squares distance trees using the MinSquareTree function in Darwin [51]. The pairwise input-distances were computed by maximum likelihood using the GCB matrices [52] for aminoacid data. For nucleotide data we used an unpublished, empirical nucleotide substitution matrix estimated from mammalian orthologs in OMA [48]. Likewise, as an alternative (and control), we recomputed the Gap Parsimony Trees without combining repeated columns. Furthermore, for a subset of the tests we repeated the computation of the ML trees using the software RAxML v. 7.0.4 [53].

Filtering of gaps and variable regions

We define a gap column as a column of the multiple sequence alignment in which at least one sequence has a gap character. To filter both gaps and variable regions, we used Gblocks version 0.91b [42] with default settings. In addition and as control, we also relaxed the settings according to Talavera et al. [42]. At times, any of the three filtering variants (no gap, Gblocks default, Gblocks relaxed) could yield alignments with no column left, that is, of null length. Such samples were excluded.

Measures to relate alignment uncertainty to tree inference

The measures used in the section Alignment Variability Poorly Predicts Tree Accuracy and Additional file 1, Figure S18 are defined as follows: Tree accuracy was measured by one minus the normalized Robinson-Foulds distance [49] between the inferred and the accepted topology. Tree support was measured by the proportion of bootstrap replicates agreeing with the inferred topology. Tree variability was measured by the average Robinson Foulds distance among trees estimated from different alignment methods. Alignment variability was measured by the average distance between alignments [54] from different alignment methods. This measure has been shown [46] to strongly correlate (Spearman's rank correlation r s = 0.92, P < 0.0001) with Bayesian-inferred alignment variability.

Comparing two correlation coefficients

We have stated in the main text (see also Additional file 1, Figure S18) that tree support (BS) is a better predictor for tree accuracy (TA) than alignment variability (AV). This can be assessed by the following test: As a null hypothesis, equal predictive power of the two measures is assumed, that is r s (BS, TA) = -r s (AV, TA). The observation (Additional file 1, Figure S18) that for all datasets r s (BS, TA) > -r s (AV, TA) is formulated as an alternative hypothesis. We assume that the pair samples are normal bivariate distributed.

is approximately standard normal distributed, where z(·) denotes the Fisher Z-transform.

Additional material

Authors' contributions CD and MG contributed equally to this work.

Figure 4
4Effect of excluding gaps and variable regions. The plot shows the effect of filtering on the minimum duplication test with back-translated, fungal amino-acid alignments. Removing gapped sites tends to worsen the accuracy of the induced maximum likelihood trees. Removing variable regions in addition to gapped sites (Gblocks, default settings) drastically reduces the accuracy of reconstructed trees. Error bars correspond to ± 1 s.d. Significant difference between results from original and curated alignments is denoted with a minus symbol at the basis of relevant bars (Wilcoxon double-sided test, P < 0.01).
This article is available from: http://genomebiology.com/content/11/4/R37 © 2010 Dessimoz and Gil; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Genome Biology 2010, 11:R37
AcknowledgementsWe thank Olivier Gascuel for early ideas leading to the design of the minimum duplication test, and Adrian Altenhoff, Maria Anisimova, Gina Cannarozzi, Gaston Gonnet, Heather Murray, Adrian Schneider, Jörg Stelling, Hervé Vanderschuren, as well as two anonymous reviewers for helpful remarks on the manuscript.Author Details
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Cite this article as: Dessimoz and Gil, Phylogenetic assessment of alignments reveals neglected tree signal in gaps. Genome Biology. 1137Cite this article as: Dessimoz and Gil, Phylogenetic assessment of align- ments reveals neglected tree signal in gaps Genome Biology 2010, 11:R37