A New Method For Evaluate The Fetal Conus Medullaris Position By Ultrasound And Explore Copy Number Variants In Tethered Cord Fetuses

Objective:Conus medullaris?CM?was the end of the spinal cord,abnormal position of the conus medullaris are closely related of the tethered spinal cord syndrome?TCS?.The general congenital diseases that lead to TCS include spina bifida,myelomeningocele,intraspinal tumors?cyst,teratoma,lipoma?,scoliosis,etc.TCS was subtle lesion,there were no obvious symptoms in the early,and often referred to motion and sensory obstruction or dysfunction of lower limb and bladder function.As infants express ability is limited,which often led to severe nerve injury and missed the best time for treatment.Serious cases lead to paralysis or incontinence,and surgery is ineffective,multiple sequelae,and unsatisfactory rehabilitation,which seriously affected the quality of life of children and increased the economic burden of the family.Therefore,early detection and early treatment were of great significance.In the past,abnormal conus medullaris position was very difficult to be found in the fetal period,as the improvement of resolution of ultrasonic equipment and the rapid development of prenatal ultrasound diagnosis subject,the attention to the CM have greatly increased,and the CM position have became the research hotspot in recent years,and the detection of tethered cord in fetal began to increase.Detection of a low-lying CM is essential in diagnosing tethered conus syndrome.If the presence of low-lying CM could be detected,this might raise suspicion for these associated abnormalities.But the CM position was not fixed during fetal period.Therefore,identification of the normal CM position and recognition of abnormal position on the prenatal ultrasound are necessary.In previous studies,there were several methods had been reported,and these methods have some limitation,because the number of sacral vertebrae and ascension of the CM show individual variability during the fetal period.The most accurate method has not been identified.In the first part of this study,we used the physiological curvature of the lumbosacral spinal to identify the first sacral vertebra?S1?by two-dimensional?2D?ultrasound,and then used S1 as a fixed reference point to count the vertebral body or disc space level corresponding to the CM.And compared with the three-dimensional?3D?ultrasound imagine.To explore the feasibility of this method in location of the fetal conus medullaris,and to summarize the rising regular of the CM during fetal period.In the second part of this study,we used a measurement between the CM and the central points of S1 in normal fetuses,to quantitatively evaluate the CM position,and the correlation between CM location and gestational age,and established the normal reference range,and compared with 17 fetuses with tethered cord to explore this parameter might be helpful in the detection of tethered cord prenatally.Because TCS belongs to the neural tube defects?NTD?,especially the spinal bifida occult?SBO?.The exact pathological mechanism of NTD has not clear,and it is clear that the deficiency of folic acid is closely related to the NTD,some studies have shown that NTD is associated with gene mutation,chromosomal abnormalities,environmental factors,genetic factors and so on.With the rapid development of molecular genetics subject,genome-wide sequencing technology has been applied to the detection of congenital developmental disorder diseases,it is helpful to find the cause of disease.With the accumulation of experience in detection of hereditary diseases in children,genome-wide sequencing technology is gradually applied to prenatal diagnosis,providing a new way for prenatal diagnosis.The next generation sequencing?NGS?have advantages such as high throughout,high resolution,high accuracy and low cost,it can detect the copy number variations?CNV?including micro deletion and micro duplication,can analysis abnormal fragment origin,length,and pathogenic.In the third part of this study,a new high throughout sequencing technology was used to detect chromosome CNV in fetuses with tethered cord,so as to explore whether there is an obvious CNV can be detected than normal human,try to analyze whether some CNV fragment are pathogenic for tethered cord.To provide theoretical basis on chromosome level for further study about TCS.Methods:1.A total of 581 normal fetuses of 20-38 weeks of gestational age were included.Two-line intersection method of lumbosacral junction was used to identify S1and then count upward to locate the fetal conus medullaris relative to vertebral body.The distribution of conus medullaris position in each gestational age was observed.101 fetuses in different gestational age were randomly selected to compared with localization results within two operators and with three-dimensional imaging.And 30 fetuses were randomly selected to compare the time used for the two location methods.2.There were 581 structurally normal singletons between 20 and 38 weeks of gestation included.Sonographic evaluation of the CM position was performed using a quantitative measurement of the distance from the CM to the top of the first sacral vertebra?S1?in routine 2D scans.The correlation between the CM-S1 distance and GA was evaluated by linear regression analysis and calculated the normal reference ranges and the 5^th percentile values for 20-38 weeks'gestation respectively,Normal results were compared with 18 suspected tethered spinal cord cases.3.There were 16 cases of tethered cord fetuses,the test sample for NGS testing was amniotic fluid and umbilical cord blood or aborted fetal tissue.After extracted DNA,using NGS high-throughput sequencing technology to detect CNV,and to compare with the gene database,remove polymorphism,and analyze whether some CNV fragment are pathogenic for tethered cord.Results:1.530 fetuses can clearly identify S1 by two-line intersection method of lumbosacral junction and locate the conus medullaris easily.The conus medullaris gradually raised with the develop of gestational age,at 23 weeks all located above L3,more than 32 weeks 98.2%above L2-3,and more than 37 weeks about 95%above L2.The agreement of location by two-line intersection of lumbosacral junction within operators and with three-dimensional imaging were well?Kappa value was 0.94 and 0.92respectively?.The time used for location CM by 2D ultrasound was significantly shorter than the 3D ultrasound method.?P<0.01?2.In 89.5%of the 581 cases,we accurately identified the CM and S1.The CM-S1 increased with pregnancy progression.Linear regression analysis showed a significant association between the CM-S1 distance and GA?R²=0.895?.And obtained the normal reference ranges and calculated the 5^th percentile values for 20-38 weeks'gestation,respectively.The CM-S1 distance was well below the 5th percentile in the 18 abnormal cases?P<0.05?.3.A total of 16 cases were successful detected using NGS,there were 12?75%?cases were detected CNVs,and a total of 19 CNVs fragments were detected,9 deletion CNVs and 10 duplication CNVs were included,including 14 CNVs fragments with unknown clinical significance,12 in 14 fragments with polymorphism variation,and 2 in 14fragments contain pathogenic genes.3 CNVs were suspected benign fragment,and 2?12.5%?CNVs fragments with clear pathogenic.No CNVs were detected in the other 4?25%?cases.Conclusions:1.Two-line intersection method of lumbosacral junction can be used to identify S1 and then locate the fetal conus medullaris accurately,it can be used to determine position of the conus medullaris quickly and conveniently.2.The time used for location CM by 2D ultrasound was significantly shorter than the 3D ultrasound method.3.The conus medullaris gradually raised with the develop of gestational age,in the third pregnancy period,if CM position not lower than L3 level was generally considered normal.4.CM-S1 distance was significant correlated with GA.The CM-S1 increased with pregnancy progression and appeared good linear relationship.CM-S1 distance in tethered cords fetuses was significantly lower than that of normal fetus,and less than the 5^th percentile.5.A normal reference range of CM-S1 distance was established for normal fetuses at20-38 weeks,it is useful in the assessment of the CM position and in detecting tethered cords prenatally.6.NGS can detect CNV accurately,and some tethered cord related to CNV,some CNV fragments with clear pathogenic,the detection rate of CNV and pathogenic CNV was significantly higher than the normal population.