Early implantation leads to a larger CRL and late implantation to a smaller CRL at 10–14 weeks, independent of CRL growth rate. Implantation timing is a major determinant of fetal size at 10–14 weeks and largely explains the variation in estimates of GA in the first trimester derived from embryonic or fetal CRL .

The median ovulation and implantation days were 16 and 27, respectively, with an O–I interval of 11 days. GA estimated from CRL at 10–14 weeks was on average 1.3 days greater than that derived from ovulation timing. CRL Z‐score was inversely related to O–I interval (ρ = −0.431, P = 0.0009). There was no significant relationship between CRL growth rate and the difference between observed CRL and expected CRL based on GA from last menstrual period (ρ = 0.224, P = 0.08) .

One hundred and forty‐three women who were trying to conceive were recruited prospectively. The timing of ovulation and implantation and the ovulation to implantation (O–I) interval were established in 101 pregnancies using home urinary tests for luteinizing hormone and human chorionic gonadotropin. In 71 ongoing pregnancies, GA determined by measurement of fetal CRL at 10–14 weeks' gestation was compared with GA based on ovulation and implantation day. First‐trimester growth was determined by serial ultrasound scans at 6–7, 8–9 and 10–14 weeks .

Introduction Assessment of gestational age (GA) forms the basis for interpreting early pregnancy ultrasound scans, diagnosing fetal growth restriction and making critical decisions regarding pregnancy management; GA at delivery is itself a determinant of perinatal outcome1. Early pregnancy ultrasound appearances can vary even with certain menstrual or in‐vitro fertilization (IVF) dates: a healthy ongoing pregnancy might appear to be of uncertain viability, leading to a false diagnosis of miscarriage and even termination of a potentially viable early pregnancy2. The Royal College of Obstetricians and Gynaecologists' guidance in relation to the diagnosis of miscarriage has recently been updated to reflect these uncertainties3. GA is routinely determined by measuring the fetal crown–rump length (CRL) at 10–14 weeks using ultrasound4, 5. The CRL charts in general use were constructed from observed first‐trimester CRL measurements in relation to GA calculated from the last menstrual period (LMP) in women with regular menstrual cycles, assuming that ovulation occurs mid‐cycle6-8. However, only 10% of women with a regular 28‐day menstrual cycle ovulate on day 149, 10, and the median ovulation day in women with regular cycles is day 1611, 12. The ovulation to implantation (O–I) interval in a natural conception varies by up to 6 days13. The impact of ovulation and implantation timing on apparent embryonic or fetal size and GA assessment has never been determined. A relationship between a small first‐trimester embryonic measurement and low birth weight has been shown in IVF pregnancies and spontaneous conception14-16. In these studies a single CRL measurement was used to define fetal growth restriction and no account was taken of variation in implantation timing and first‐trimester growth, as opposed to size14-16. The development of a urinary luteinizing hormone (LH) testing kit has enabled reliable prediction of ovulation17. Similarly, a highly sensitive urinary human chorionic gonadotropin (hCG) testing kit detects implantation13, 18, 19. It is now possible to establish the timing of both ovulation and implantation to a high degree of accuracy using home urinary testing kits in women trying to conceive. We prospectively observed ovulation timing, implantation timing and O–I interval in women trying to conceive naturally and investigated the effect of these observations on embryonic and fetal growth and size, and the estimation of GA derived from first‐trimester CRL measurement.

Methods One hundred and forty‐three women who were trying to conceive were recruited prospectively via open advertisement in the hospital, GP surgeries, newspapers, pre‐school groups or by invitation letter. This was part of a larger study on cardiovascular changes in pregnancy. All women were healthy non‐smokers and not known to have diabetes, thrombophilia or fertility problems. The study received ethical approval from the local research ethics committee, and written consent was obtained at the time of recruitment. Age, ethnicity, LMP, detailed menstrual history, obstetric and cardiovascular history, height, weight and body mass index were recorded at recruitment. The women started using digital urinary home ovulation and pregnancy test kits at least a month after stopping contraception. They were asked to perform daily ovulation tests from the 6th day of their menstrual period until the urinary LH surge was detected and then carry out daily pregnancy tests from 8 days after the LH surge until their next period or until they had three consecutive positive pregnancy tests. They continued testing in every menstrual cycle until they became pregnant or for up to 6 to 12 months if no clinical pregnancy occurred. The test kits were provided free of charge by Swiss Precision Diagnostics, GmbH (Bedford, UK). A rise in urinary LH predicts ovulation at a mean of 20 h from the initial rise17. We therefore calculated the ‘LH surge + 1 day’ to define the day of ovulation, as previously described11, 17. The day of the first positive pregnancy test using sensitive digital urine pregnancy test kits was reported as the ‘implantation day’ in a similar way to previously described13. The O–I interval was calculated as the time interval between the presumed day of ovulation as indicated by the urinary LH test kits and the first positive pregnancy test. The urinary tests had been previously validated against urinary assays for detection of LH surge and hCG by Swiss Precision Diagnostics, GmbH20. Ultrasound scans were performed at 6–7, 8–9 and 10–14 weeks' gestation from LMP in all women with a regular 28‐day cycle, and where this was not the case they were scheduled from the date of the LH surge. CRL was measured transvaginally at 6–7 and 8–9 weeks by placing the calipers at the outer side of the crown and rump of the embryo or fetus in a longitudinal, midsagittal section21, 22. Of three CRL measurements taken, the one that most closely conformed to the standard described above was used for analysis. Fetal CRL at 10–14 weeks was imaged transabdominally in a midsagittal plane with the genital tubercle and the fetal spine longitudinally in view and the maximum CRL was measured16, 22. GA for pregnancies viable at 10–14 weeks was assigned according to the observed CRL measurement, and only women with ongoing pregnancies at 10–14 weeks were included in the study. GA adjusted for ovulation timing (GAOV) was derived by subtracting 14 days from the predicted ovulation date (LH + 1) to derive the effective LMP, as is the convention for pregnancy dating. GA adjusted for implantation timing (GAIMP) was derived by adding the difference between the observed implantation day and the median implantation day (which in this study was day 27) if the observed implantation day was earlier than day 27. Similarly, we subtracted the difference if the observed implantation day was later than day 27. Statistical analysis was performed using the MedCalc software for windows (MedCalc Software bvba, Version 11.6, Mariakerke, Belgium) and Statistical Package for Social Sciences (SPSS Version 18.0.0, 2009, SPSS Inc., Chicago, IL, USA). The data were checked for normality of distribution and are expressed as mean ± SD and median (interquartile range (IQR)) or median (range), as appropriate. The timing of ovulation and implantation and the O–I interval in ongoing pregnancies at 10 weeks, pregnancies that miscarried at less than 6 weeks and pregnancies that miscarried after 6 weeks were compared using the Kruskal–Wallis test. Bland–Altman plots were constructed to compare the differences between GA predicted by CRL measurement (GACRL) and that based on LMP (GALMP), adjusted for ovulation (GAOV) and adjusted for implantation (GAIMP). Pairwise differences were plotted against the mean GA in each case23. 95% limits of agreement (LoA) were calculated for the differences between the GA estimated from a single CRL measurement and that estimated from LMP (GALMP), GAOV and GAIMP. CRL growth rate (mm/day) was calculated by dividing change in CRL by change in GA. An average was taken of the growth rate between scans 1 and 2 and that between scans 2 and 324. CRL Z‐score at the 10–14‐week scan was calculated as: (measured CRL at the 10–14‐week scan − expected CRL based on GAOV)/SD, with respect to the Robinson and Fleming curve6. The relationship between implantation day, O–I interval and first‐trimester growth was investigated. Associations between pairs of continuous variables were evaluated using Spearman's correlation coefficient (ρ).

Results One hundred and one women became pregnant while enrolled in the study (Figure 1). The median time taken to conceive from when they started trying was 5 (IQR, 2–7) months and this constituted a median of two cycles (IQR, 1–5) after entry to the study. One woman was lost to follow‐up and 29 suffered early pregnancy loss, thus there were 71 women with an ongoing pregnancy after 10 weeks (Figure 1); their characteristics are given in Table 1. The median ovulation day, implantation day and O–I interval in all pregnancies are given in Table 2. The median O–I interval and median implantation days were 14 and 31 days, respectively, in pregnancies that miscarried at < 6 weeks (n = 13), compared with 11 and 27 days in ongoing pregnancies and 11 and 25 days in those that miscarried at > 6 weeks (n = 12) (P < 0.001 for O–I interval; P = 0.004 for implantation day). Figure 1 Open in figure viewer PowerPoint Categories are not mutually exclusive. LH, luteinizing hormone; O–I, ovulation to implantation. Recruitment flowchart for the study.Categories are not mutually exclusive. LH, luteinizing hormone; O–I, ovulation to implantation. Table 1. Characteristics of study participants with ongoing pregnancy after 10 weeks Characteristic Participants (n = 71) Age (years) 32 (29–35) Ethnicity White 65 (91.5) Black 2 (2.8) Asian 2 (2.8) Other 2 (2.8) Parity Nulliparous 37 (52) Parous 34 (48) Table 2. Summary of menstrual cycle data in all pregnancies Pregnancy ongoing at > 10 weeks (n = 71) Miscarriage at < 6 weeks (n = 13) Miscarriage at > 6 weeks (n = 12) P Ovulation day 16 (11–39) (n = 59)* 15 (14–23) 14 (12–20) 0.2 Menstrual cycle length 28 (21–60) (n = 69)* 30 (27–47) 28 (25–35) 0.2 Implantation day 27 (23–44) (n = 58)* 31 (26–37) 25 (23–32) 0.004† Ovulation to implantation interval 11 (9–20) (n = 56)* 14 (11–17) 11 (9–13) < 0.001† We compared the differences between GACRL and GALMP, GACRL and GAOV and that between GACRL and GA adjusted for median implantation of 27 days (GAIMP) using Bland–Altman plots. The mean difference between GACRL and GALMP was −0.8 days (95% LoA, –11.8 to 10.1 days) (Figure 2a), that between GACRL and GAOV was 1.3 days (95% LoA, –3.8 to 6.4 days) (Figure 2b) and that between GACRL and GAIMP was 0.4 days (95% LoA, –4.0 to 4.9 days) (Figure 2c). Figure 2 Open in figure viewer PowerPoint CRL) with: (a) GA based on last menstrual period (GALMP), (b) GA adjusted by ovulation day (GAOV) and (c) GA adjusted in each pregnancy to a median implantation day of 27 days (GAIMP). The bold line represents the mean difference with 95% CI ( ) and dashed lines ( ) represent 95% limits of agreement (± 1.96 SD). Bland–Altman plots showing comparison of gestational age (GA) estimated from crown–rump length measurement at 10–14 weeks' gestation (GA) with: (a) GA based on last menstrual period (GA), (b) GA adjusted by ovulation day (GA) and (c) GA adjusted in each pregnancy to a median implantation day of 27 days (GA). The bold line represents the mean difference with 95% CI () and dashed lines () represent 95% limits of agreement (± 1.96 SD). CRL Z‐score at the 10–14‐week scan (with expected CRL based on GAOV and with respect to the Robinson and Fleming curve) showed a negative linear relationship with the O–I interval (ρ = −0.431, P = 0.0009; Figure 3a). The difference between GACRL and GAOV showed a similar negative linear relationship with O–I interval (ρ = −0.430, P = 0.0009; Figure 3b). Figure 3 Open in figure viewer PowerPoint Scatterplots of crown–rump length (CRL) Z‐score at 10–14 weeks (a) and difference between gestational age (GA) according to CRL (GACRL) and GA derived from ovulation timing (GAOV) (b) against the ovulation–implantation (O–I) interval. The smaller the O–I interval, the greater the CRL Z‐score (ρ = −0.431, P = 0.0009) and the greater the difference between GACRL and GAOV (ρ = −0.430, P = 0.0009). Regression lines are shown. There was no significant relationship between CRL growth rate and the difference between observed and expected CRL based on GALMP (ρ = 0.224, P = 0.08) or between CRL growth rate and O–I interval (ρ = 0.122, P = 0.371). However, there was a significant relationship between CRL growth rate and the difference between observed and expected CRL based on GAOV (ρ = 0.308, P = 0.017) and that between observed and expected CRL based on GAIMP (ρ = 0.459, P = 0.001).

Discussion The major novel finding of this study was that fetal size at 10–14 weeks' gestation is mainly a composite of ovulation and implantation timing. There was an 11‐day range in O–I interval that translated directly to fetal size for a given gestation. In other words, at 10–14 weeks an early implanting embryo (short O–I interval) was larger than expected and a later implanting embryo (long O–I interval) was smaller than expected, by almost exactly the number of days of variance about the median O–I interval, independent of first‐trimester CRL growth rate. Moreover, first‐trimester fetal growth was not influenced by O–I interval. This is the first study to prospectively investigate the impact of biological variation in ovulation and implantation timing on GA assessment in natural conception. The Robinson and Fleming CRL charts were derived from CRL measurements plotted against GA deduced from LMP in women with regular menstrual cycles, and remain to this day the standard method of assigning GA in the first trimester6. However, these charts do not account for a woman's ovulation day. As this is reported to occur later than day 14 of the menstrual cycle in most women9, 10, such charts are likely to lead to a systematic overestimation of postimplantation GA. This has been hypothesized though not shown by others4; that ovulation occurs at a median of 16 days after LMP is the most plausible explanation for the observed 1.3 days overestimation from GACRL compared with GAOV. We found an 11‐day range of implantation timing in viable pregnancies. Interestingly, a late‐implanting embryo was smaller in size at 10–14 weeks. It has previously been shown that first‐trimester fetal size predicts subsequent birth weight. This is based on studies in which a single first‐trimester CRL measurement was deemed to be small in relation to the expected size based on known conception timing in a cohort of assisted‐conception pregnancies14 or known ovulation timing in pregnancies conceived naturally15, 16. Although the possibility of GA misclassification based on implantation timing was raised in these studies16, they were unable to take into account implantation timing. Further, first‐trimester growth was extrapolated from a single CRL measurement, though in fact it was size that was being measured14-16. Our findings could underlie the observed variation in first‐trimester fetal size, and also explain why embryos with larger than expected CRL are more likely to be large at term15. We did not find a significant relationship between first‐trimester growth rate and CRL Z‐score based on LMP at 10–14 weeks. The CRL growth rate is, however, related to the difference between observed and expected CRL adjusted for ovulation and implantation timing. Put simply, first‐trimester CRL is a composite of two variables: (1) when the embryo implants and (2) its subsequent growth rate. Thus, the contribution of implantation timing may explain the discrepancy in fetal size at 10–14 weeks seen in previous studies14, 15, rather than this being principally due to first‐trimester intrauterine growth restriction. This alternative explanation is supported by studies showing no relationship between first‐trimester growth or first‐trimester CRL and birth weight24, 25. The 95% LoA of differences between GACRL and GALMP reduced from 21.9 days to 10.2 days by adjusting for ovulation and to 8.9 days by adjusting for implantation day. This implies that the precision in dating a pregnancy by CRL improves greatly when taking into account ovulation, and improves even further when taking into account implantation date. Thus, CRL charts derived from a knowledge of implantation day are likely to be considerably more accurate than those derived from gestation assumed from LMP. A potential criticism may be made of the assumption that implantation day is equivalent to the day of first detectable hCG using urinary testing kits and that of missing implantation earlier than day 8 using our urinary testing protocol. A large study of O–I interval in human pregnancy reported the day of first detection of hCG using sensitive urine assays as the ‘implantation day’13. Although earliest implantation was observed on day 6 from the time of ovulation in that study, this was not observed in our study, as no participant who performed urinary testing as per the protocol had a positive hCG at the time of the first test on day 8. We observed that delayed implantation is associated with pregnancy loss before 6 weeks, as previously reported13. We show that the hitherto unexplored contribution of ovulation and implantation timing is important in determining fetal size, and hence in influencing estimation of GA. Reliable information on implantation timing is more important than is certain LMP or known ovulation date in natural conception and in IVF pregnancies. In IVF pregnancies GA is often estimated from the egg collection date, adjusting for day 14 ovulation. This is based on the assumption that median ovulation timing is day 14, and that implantation timing is either constant or has no effect on embryonic size. Variation in implantation timing may however explain the variability of embryonic or fetal ultrasound appearances compared with those expected from known LMP or IVF dates. Furthermore, it might explain the recent observation that isolated measurements of mean sac diameter or embryo size are unreliable when establishing a diagnosis of miscarriage early in pregnancy2. Where there is late implantation, even though the LMP and probable conception date may be known, a perfectly healthy early fetus might appear to be significantly smaller than expected, or embryonic structures may not be visible on ultrasound scan, giving a false impression of a pregnancy of unknown viability. Moreover, we challenge the assumption that small first‐trimester CRL equates to fetal growth restriction. We do not discount a contribution of fetal growth rate in explaining observed fetal size differences from expected CRL. However, these measurements are predominantly explained by variability in implantation timing, the influence of which on embryonic and fetal size has not previously been considered.

Acknowledgments The ovulation and pregnancy test kits were provided free of cost by Swiss Precision Diagnostics, GmbH (SPD) Bedford, UK. We wish to thank all the sonographers of the Early Pregnancy Unit in the Rosie Hospital for helping with early pregnancy scans and all the women who participated in the study. A.A.M. is supported by Cambridge Fetal Care and Flexibility and Sustainability Funding (FSF) from the National Institute of Health Research (NIHR), UK. T.R.E. is funded by Evelyn Trust. I.B.W. and C.M.M. are both British Heart Foundation Fellows and are supported by the Cambridge Biomedical Research Centre (NIHR) and the Comprehensive Local Research Network (CLRN), UK. T.B. is supported by Imperial Healthcare NHS Trust NIHR Biomedical Research Centre. Cambridge University Hospital NHS Trust acted as a sponsor for this study.