it allows the clinicians to better time gestation - specific antenatal screening tests , reduces erroneous labelling of pregnancies as very preterm , preterm , and small - for - gestational - age , and decreases the risk of inappropriate induction of labour [ 15 ] . in the first trimester , there is a very little biologic variation in fetal size compared with later trimesters .
it is therefore a good time in pregnancy to determine gestational age by ultrasound where the crown - rump length ( crl ) is measured and compared to published reference charts .
in contrast , dates calculated from the first day of the last menstrual period ( menstrual age ) may have inaccuracies arising from imprecise recollection of dates , variation in the timing of ovulation , or time to conception .
a number of crl reference charts have been proposed and different versions are in common use ( table 1 ) . most of these charts are based on menstrual age to estimate gestational age at the day of the ultrasound examination , based on modest sample sizes , generated many years ago using ultrasound machines of poorer resolution , or used transabdominal measurements ( which gives poorer pictures relative to a transvaginal approach ) [ 68 ] . as such , there is considerable variability between current reference charts , and estimated gestational age can vary significantly depending on which chart is used .
it should be possible to generate very accurate reference charts by using a large in vitro fertilisation ( ivf ) cohort where crl measurements could be correlated with gestational age precisely calculated from date of fertilisation .
accuracy could be further enhanced by using measurements obtained from high - resolution transvaginal scans by sonologists specialising in women 's health .
we examined crl lengths at 69 weeks of gestation measured in a large ivf cohort where gestational age could be calculated from date of fertilisation .
we assessed the ability of six commonly used reference charts to accurately date these pregnancies .
given all these charts showed inaccuracies in their ability to date these ivf pregnancies , we developed a new reference chart based on ivf dates in a singleton cohort .
we retrospectively obtained clinical details on 1268 singleton ivf pregnancies conceived using a fresh embryo transfer cycle , had a transvaginal first trimester ultrasound done between 6 ( + 1d ) to 9 ( + 0d ) weeks of gestation where crl were measured , and progressed to viability ( > 24 weeks of gestation ) .
these were identified from a total cohort of 4971 first trimester ultrasound reports of ivf and naturally conceived pregnancy scans .
pregnancies that resulted from frozen embryos transfer , complicated by fetal structural anomalies , delivered before 24 weeks , or had missing data were all excluded . of our cohort of 1268 pregnancies ,
84 were scanned twice , and 2 pregnancies were scanned three times , giving a total population size of 1182 . in these pregnancies with multiple scans , all data were included in the analysis as discrete values .
we restricted our analysis to those who had fresh embryo transfer since we were concerned with the need to add the time from egg pickup to freezing , together with the time from subsequent thawing to transfer might introduce inaccuracies .
we determined gestational age on the day of the ultrasound ( ivf dates ) by nominating the day of egg pickup and fertilisation as day 14 of gestation .
we first compared ivf dates with estimated dates determined using six existing reference charts : australian society for ultrasound medicine ( asum ) , hadlock et al . ,
note that some charts did not have corresponding gestational ages for all measurements which accounts for the variability in sample size seen in the comparison of charts .
we then generated reference charts using the crl measurements and ivf dates by smoothing out the data ( see statistical analysis below ) , which we named the monash chart . to validate our chart
, we obtained crl measurements from an ivf twin cohort ( fresh embryo transfer ) at 6 ( + 1d ) to 9 ( + 0d ) weeks of gestation where gestational age was calculated ( fertilisation age + 14 days ) . taking each twin as a discrete measurement
, we determined the accuracy of all six existing reference charts and the monash chart in estimating gestational age .
we then applied all six existing reference charts and our chart to estimate gestational age of crl measurements obtained from 3052 consecutive first - trimester singleton ultrasound scans pregnancies at 69 weeks of gestation .
ethics approval was obtained before we commenced the study ( project 05063 , monash surgical private human research ethics committee , clayton , vic , australia ) . for this retrospective database study where we used de - identified data in aggregate ,
all examinations were performed at three ultrasound centres that exclusively perform women 's health ultrasounds .
all ultrasounds were transvaginal , done on advanced technology laboratories hdi 5000 ultrasound machines by experienced sonographers .
after confirmation of a live intrauterine pregnancy , the crl was measured in the midsagittal plane by the placement of ultrasound callipers at the outer edges of the head and rump of the fetus , excluding the limbs and yolk sac .
for comparison of data , an unpaired student 's t - test was used to compare two groups with continuous variables that were normally distributed and nonparametric data was compared using mann - whitney u test .
nonparametric data was expressed as median and interquartile range , while parametric data was expressed as mean ( standard deviation ) . to determine the relationship between true gestational age and crl
, we constructed a scattergram , plotting crl lengths against true gestational age in our singleton ivf cohort ( figure 1(a ) ) .
investigation using fractional polynomial regression analysis   revealed that a straight line best described the mean . the standard deviation ( sd )
varied very little at every week of gestational age , which was unsurprising given our significant cohort size .
the  goodness of fit  was determined by a plot of the standard deviation score or standardised residual which was normally distributed . to develop the monash chart ,
a linear prediction plot was fitted to the scattergram ( figure 1(a ) ) and appeared to be the best model to fit the data with a very narrow 95% confidence interval ( figure 1(b ) ) .
the final crl reference chart was derived from the equation describing the line of best fit .
when we compared the six existing crl reference charts to either ivf dates or to gestational ages derived for the monash chart , we calculated the mean differences of the gestational ages from the six charts from either the ivf true gestational age or the monash chart gestational age ( depending on the analysis being undertaken ) , and compared them with paired t - tests .
we identified 1268 first trimester ultrasound scans done at 6 ( + 1d ) to 9 ( + 0d ) weeks of gestation where gestational age at the day of the ultrasound could be precisely determined using the ivf dates .
we calculated ivf dates by noting the number of days from fertilisation until the date of the ultrasound assessment .
since day of egg pickup is day 14 of gestation by convention , an extra 14 days were added to this number in order to calculate the ivf dates .
we noted the raw crl measurement in millimetres and compared gestational age estimated by six existing crl reference charts with actual ivf dates .
we found the mean gestational age estimated by all these charts varied significantly to ivf dates ( p < 0.0001 for all charts ) , with mean dates varying from 1.2 to 2.1 days ( table 3 ) .
we plotted crls against gestational age determined by ivf dating on a scattergram ( figure 1(a ) ) . after using fractional polynomial regression analysis
we determined a line of best fit with a 95% confidence interval noted to be very narrow across the entire gestational age range ( figure 1 ) . using the equation that describes this line ( gestational age = 0.82 [ crl in mm ] + 42.1 ) , we derived a new crl reference chart based on ivf dates ( table 4 ) that we termed the monash chart . to ensure the generation of a line of best fit to create
the monash chart did not significantly distort prediction of gestational age , we compared estimated gestational age calculated from our chart with actual ivf dates .
we then compared gestational age derived from the monash chart with that predicted by the six existing reference charts and found significant differences for all charts ( mean difference in estimated gestational age varied from 1.3 days to 2.1 days ; p < 0.0001 for all charts ) .  
we next sought to validate the monash chart . to do this , we chose an independent ivf twin pregnancy ( n = 185 twin pairs , or 370 independent observations ) conceived after fresh embryo transfer where exact gestational age ( fertilisation age + 14 days ) was known , and the result was delivery of two babies > 24 weeks of gestation .
first , it would validate the use of our chart for twins , a situation where calculation of estimated delivery dates is especially important given obstetric risks .
secondly , the use of ivf pregnancies again allows accurate determination of gestational age by calculating ivf dates .
while there may be differences in growth between twins and singletons in late pregnancy , there is no evidence that differences in crl between singletons and twins exist .
biological differences in crl as large as millimetres at this early gestation would be very unlikely .
the mean ( sd ) maternal age of the ivf twin cohort was 33 ( 3.9 ) years , mean ( sd ) gestation at birth was 36 ( 2.5 ) weeks ' gestation .
mean birthweight for twin 1 was 2492 ( 548 )  gms and twin 2 was 2467 ( 576 )  gms .
median ( range ) treatment cycle number was 3 ( 115 ) and the median ( range ) number of embryos transferred was 2 ( 13 ) . in this cohort of ivf twins , predicted gestational age from the six existing reference charts was significantly different from ivf dates , with mean differences ranging from 1.1 days to 2.3 days ( p  0.0005 ) .
only the monash chart was not statistically significantly different to ivf dates in the twin cohort ( p = 0.6835 ) .
we next applied our chart to crl measurements obtained from 3052 consecutive first - trimester viability ultrasounds .
the purpose was to see whether the monash chart would pragmatically alter expected dates of delivery compared to existing charts .
we found dates derived from our chart were significantly different to the existing reference charts ( mean difference in estimated gestation ranged from 1.8 days to 1.8 days ; p  0.0047 for all charts , see table 5 ) .
ultrasounds performed at 69 weeks ' gestation are done very often , where gestational age is derived from the crl measurements .
we have developed a potentially highly accurate crl reference chart to date pregnancies at the viability ultrasound .
while others have proposed crl charts based on ivf dates before , they have been based on small numbers ( 36160 participants ) [ 6 , 1518 ] .
in contrast , ours was generated from a more sizable population ( n = 1268 ) . given a possible association between shorter than expected crl and miscarriage ,
we validated the performance of our chart using an independent twin cohort , and showed in a further cohort of 3052 consecutive ultrasounds it would materially alter dates if it used instead of any of six preexisting charts .
hence we believe our chart may possibly be the most accurate of any published chart to date pregnancies between 69 weeks of gestation .
in addition , we found inaccuracies in the ability of commonly used charts to estimate gestational age among ivf singleton and twin cohorts where exact dates are known .
while some showed only very slight differences in the estimation of dates compared to ivf dates ( e.g. , 0.57 days mean difference for daya chart ) and others showed larger differences ( 2.1 days for asum chart ) , all were highly statistically different ( table 3 ) .
of further concern is the fact that there appears to be significant disagreement between existing charts where some given crl lengths , predicted gestational age can vary by many days depending on which chart is referenced . in order to determine precisely gestational age on the day of the ultrasound scan
, we have necessarily derived our reference chart from an ivf population . while there may be some differences in final birthweight among those conceived by ivf compared to spontaneous conceptions
given variations of even a millimetre or two would represent significant proportional differences in length at these early gestations , we consider it unlikely the monash chart would not be valid for spontaneously conceived pregnancies . in this study , we were unable to generate a reference chart that encompassed crl reference ranges across the whole first trimester .
the reason is that crl measurements were rarely performed across 911 weeks of gestation among our ivf cohort .
the likely reason is that being ivf pregnancies , clinicians had exact dates with which to time the late first trimester ultrasound to 12 + weeks of gestation , when nuchal translucency is best assessed .
we attempted modelling a chart incorporating these late first trimester crl lengths , but we could not be confident that the integrity and high accuracy of the 69 week chart we report was maintained . also , we did not include gestations under 6 weeks given crl at ( 6 + 1 ) is already just 1  mm , and it is not possible for ultrasound to accurately measure differential crl lengths present at earlier gestations .
nevertheless , we believe our chart is still clinically useful since many spontaneous pregnancies will have the first ultrasound between 69 weeks of gestation . strengths of our study includes the fact we only used measurements obtained from high - resolution transvaginal ultrasounds of crls at centres that exclusively perform obstetrics and gynecological ultrasounds .
also , we utilized a large cohort , reflected by the fact that the 95% confidence intervals are very narrow ( figure 1 ) .
accurate dating is important since obstetric management throughout pregnancy is strongly based on gestational age .
for instance , the first trimester nuchal translucency measurements are most accurate if performed during the 12th week of gestation .
many units offer an induction of labour at exactly ten to fourteen days after the expected date of delivery , and no later given concerns that stillbirth rates may increase more steeply beyond two weeks after the expected date of delivery .
a cochrane systematic review concluded that accurate dating indeed reduces the rates of induction of labour for postdates . at the thresholds of viability ,
many would offer conservative management if a delivered baby is judged to be around 23 weeks + 3 days of gestation , but may consider actively resuscitating a baby estimated to have reached 24 weeks + 2 days of gestation .
therefore , it is important to be as accurate as possible in determining gestational age . in conclusion
, we have generated the monash chart that we believe may be the most accurate crl chart reference chart yet proposed to date pregnancies at 69 weeks ' gestation .
 accurate determination of gestational age underpins good obstetric care . we assessed the performance of six existing ultrasound reference charts to determine gestational age in 1268 singleton ivf pregnancies , where  
 true  
 gestational age could be precisely calculated from date of fertilisation . 
 all charts generated dates significantly different to ivf dates ( p < 0.0001 all comparisons ) . 
 thus we generated a new reference chart , the monash chart , based on a line of best fit describing crown - rump length across 6 + 1 to 9 + 0 weeks of gestation ( true gestational age ) in the ivf singleton cohort . 
 the monash chart , but none of the existing charts , accurately determined gestational age among an independent ivf twin cohort ( 185 twin pairs ) . 
 when applied to 3052 naturally - conceived singletons scans , the monash chart generated estimated due dates that were different to all existing charts ( p  0.004 all comparisons ) . 
 we conclude that commonly used ultrasound reference charts have inaccuracies . 
 we have generated a crl reference chart based on true gestational age in an ivf cohort that can accurately determine gestational age at 69 weeks of gestation . 
1. Introduction
2. Methods
3. Results
4. Discussion