Results:
From January 2014 to June 2019, 1243 pregnant women between 24+0 and 39+4 gestational weeks underwent late amniocentesis in our hospital. They carried 1272 fetuses including 1154 singleton pregnancies, 5 monochorionic pregnancies with single puncture (monochorionic monoamniotic, MCMA), 44 monochorionic pregnancies with double puncture (monochorionic diamniotic, MCDA), 36 dichorionic twin pregnancies with double puncture (dichorionic diamniotic, DCDA) and 2 triplet pregnancies with triple punctures (trichorionic triamniotic, TCTA). Among them, 7 MCDA and 5 DCDA chose to conduct amniocentesis on the fetuses with abnormal ultrasound finding; 24 MCDA, 11 DCDA and 2 TCTA chose to conduct amniocentesis on the healthy fetuses because the abnormal ones had been terminated already.IndicationsTable 1 showed the late amniocentesis indications among women studied. It was observed that the most common cause was late detected aberrant ultrasound findings (or a combination of ultrasound findings with other earlier indications) (1123/1272 fetuses, 88.3%). These abnormal ultrasound findings included Central nervous system anomalies (183/1272, 14.4%), cardiovascular defects (161/1272, 12.7%) and urinogenital defects (157/1272, 12.3%). Another common cause of late amniocentesis was suspected prenatal diagnosis results (113/1272 fetuses, 8.9%). Other causes were advanced maternal age, abnormal childbearing history and monogenic disease in the family.ComplicationsCommon complications of amniocentesis include chorioamnionitis, preterm birth (PTB), intra-uterine demise (IUD) and trauma 12. In our study, only one chorioamnionitis was identified on the third day following the amniocentesis. A total of 38 PTBs (38/1243, 3.1%) and 21 IUDs (21/1272, 1.7%) were identified in our study. Among these PTB and IUD cases, 6 PTBs (15.8%) and 4 IUDs (19%) developed within the first week after amniocentesis. 8 PTBs (21.1%) and 5 IUDs (23.8%) developed within one month but before 37 gestational weeks after amniocentesis. The remaining PTBs (63.2%) and IUDs (57.1%) cases took place after one month of the amniocentesis. 89.7% (104/116) complication is associated with fetal abnormalities, and 7.8% (9/116) complication took place in fetuses with suspected prenatal screening tests. (Table S1)
PTBs occurred in 0.3% (4/1155) and 0.5% (6/1155) in singleton pregnancies, while that occurred in 2.4% (2/84) and 2.4% (2/84) in twin pregnancies (one week and one-month post-procedure, respectively). No PTB was observed in triplet pregnancies. The earliest PTB occurred in a singleton pregnancy at the third day after the operation. 89.5% PTBs (34/38) took place in fetuses with ultrasound anomalies. Near one-third (12/38,31.6%) of PTBs among our cases happened to twin pregnancies, as twin pregnancies are faced with a higher risk (56.6%) for PTB than singleton pregnancies (9.7 %) even without late amniocentesis13. Of note, all 12 twin pregnancies suffering PTBs have double punctures, including 8 MCDA and 4 DCDA.
For IUD, it took place in 1.1% (13/1154) singleton pregnancies and 5.9% (5/85) twin pregnancies. No IUD was observed in triplet pregnancies. The earliest IUD occurred in a singleton pregnancy with CNS anomalies on the next day after the operation. Only one of the 21 IUDs cases reported no fetal malformations and all 5 twin pregnancies suffering IUD have double punctures. It should be noted that among 5 IUD cases in twin pregnancies, 2 cases show 50% mortality rate, with one case having oligohydramnios and the other with hydramnios.Pathogenic findingsCMAIn our cohort, chromosomal disorders were identified in 133 (133/1272, 10.5%) fetuses. Sixty-six of them were aneuploidies (66/1272, 5.2%), including 35 trisomy 21, 9 trisomy 18 and 6 trisomy 13. Other aneuploidies included sex chromosomal abnormality (like XXX, XXY), trisomy 8 and trisomy 9. (Table S2) Pathogenic copy number abnormality were identified in 67 (67/1272, 5.3%) fetuses by CMA, while only two could be detected by karyotyping (a deletion of on chr18q22.3q2314 and a duplication of on 5q21.1q22.2). The other 24 CNVs (24/26, 92.3%), two trisomy 21 and one trisomy 18 from CMA could not be identified via karyotyping.ES (MES and WES)MES and WES were carried out in five and seven fetuses (parents and fetus) respectively.
Chromosomal disorders were identified in one fetus (1/12, 8.3%) whose CMA result was negative. Two copy number changes were considered likely pathogenic (2/12, 16.7%), while another two cases were likely benign (2/12, 16.7%).
However, 6 cases tested by exome sequencing showed different genetic results compared with tests by CMA. 4 cases tested by exome sequencing (1 case with pathogenic result, 2 cases with likely pathogenic result and 1 with likely benign result) were reported positive by CMA. 2 cases tested by exome sequencing (1 case positive and 1 likely benign) were reported aneuploidies by CMA. (Table 2)
Two ES reports were supported by the ultrasound findings. In the first case, the pregnant woman with pathogenic WES findings underwent amniocentesis due to FGR identified by ultrasound. Trio-exome sequencing showed a mutation c.625+1G>A in the SLC7A7 gene compatible with fetal Lysinuric protein intolerance (LPI) 15. The couple chose to continue the pregnancy, but IUD took place 2 months after the amniocentesis. In the second case, the couple was referred for genetic counseling during their fourth pregnancy due to fetal cenencephalocele, hydrocephalus and cerebellum dysplasia. They decided to terminate the pregnancy, although CMA showed normal result. The muscle tissue of the aborted fetus was then isolated for WES. ISPD gene mutations, c.674delC(p.A225Dfs*21) and c.1106T>G(p.V369G), associated with Walker-Warburg syndrome were identified16 which was consistent with the ultrasound findings
Pregnancy outcomeIn our cohort, 725 (57.0%) pregnancies resulted in live births, 451 (35.5%) pregnancies were terminated, 21 (1.7%) fetuses died in utero, and 75 (5.9%) fetuses were lost to follow up. (Table 3) Among the 451 terminated pregnancies, pathogenic gene was identified in 114 cases by one of CMA and ES (114/132, 86.4%). 310 couples terminated the pregnancy despite normal results of CMA and ES (310/1272, 24.4%) owing to the fetal abnormalities detected by ultrasound, especially urinogenital and cardiovascular malformations. 27 of 65 (41.5%) fetuses that have been diagnosed with a VOUS, likely pathogenic or likely benign in the report, decided to terminate the pregnancy owing to the abnormal finding on ultrasound examination. Others were live birth, except one was lost to follow up and the other two were IUD. In 66 fetuses with aneuploidy identified by CMA, 31/35 pregnancies with trisomy 21 (include the two pregnancies whose ES results showed normal and likely benign), 9/9 pregnancies with trisomy 18, 6/6 pregnancies with trisomy 13 and 13/16 other aneuploidy were terminated after receiving the genetic reports. Two fetuses with trisomy 21 had IUD and one had PTB took place before receiving the genetic result. The other 4 among the 66 women finally decided to give birth to the babies. 60 out of 67 pregnancies with pathogenic CNV identified by CMA were finally terminated. A woman with a CNV was IUD and another three was PTB before receiving the genetic result. The other 3 women chose to continue the pregnancy and gave birth to the fetuses finally.