Volume 51, Issue 6 - Highlight Issue: Stem Cells in Perinatal Medicine; Issue Editor: Wolfgang Holzgreve

Within the fast-growing field of regenerative medicine stem-cell therapy is well established in various hematologic and immunologic diseases and has received a recent substantial boost from the introduction of gene editing and gene transfer technologies. In neonates, for example, regenerative medicine may benefit those with congenital or acquired disease due to prematurity or perinatal hypoxia-ischemia. We compare and contrast the two main approaches – autologous vs. allogeneic – and summarize the recent advances and applications of interventional stem-cell research in perinatally acquired disorders such as intraventricular hemorrhage, hypoxia-ischemia and stroke. After discussing stem-cell sources and routes of administration, we conclude by highlighting the key opportunities and obstacles in this exciting field.

Recreating human cell and organ systems in vitro has tremendous potential for disease modeling, drug discovery and regenerative medicine. The aim of this short overview is to recapitulate the impressive progress that has been made in the fast-developing field of cell programming during the past years, to illuminate the advantages and limitations of the various cell programming technologies for addressing nervous system disorders and to gauge their impact for perinatal medicine.

Cerebral palsy, the most common disability in childhood, is a devastating non-progressive ailment of the infants’ brain with lifelong sequelae, e.g., spastic paresis, chronic pain, inability to walk, intellectual disability, behavioral disorders, for which there is no cure at present. CP may develop after pediatric brain damage caused, e.g., by hypoxic-ischemia, periventricular leukomalacia, intracranial hemorrhage, hypoxic-ischemic encephalopathy, trauma, stroke, and infection. About 17 million people worldwide live with cerebral palsy as a result of pediatric brain damage. This reflects both the magnitude of the personal, medical, and socioeconomic global burden of this brain disorder and the overt unmet therapeutic needs of the pediatric population. This review will focus on recent preclinical, clinical, and regulatory developments in cell therapy for infantile cerebral palsy by transplantation of cord blood derived mononuclear cells from bench to bedside. The body of evidence suggests that cord blood cell therapy of cerebral palsy in the autologous setting is feasible, effective, and safe, however, adequately powered phase 3 trials are overdue.

Perinatal brain damage is still one of the leading contributors to perinatal death and postnatal disability worldwide. However, the term perinatal brain damage encompasses very different aetiological entities that result in an insult to the developing brain and does not differentiate between the onset, cause and severity of this insult. Hypoxic-ischemic encephalopathy (HIE), intraventricular haemorrhage, periventricular leukomalacia and perinatal stroke are often listed as the major aetiologies of perinatal brain damage. They differ by type and timing of injury, neuropathological and imaging findings and their clinical picture. Along the timeline of neurodevelopment in utero , there appears to be a specific “window of vulnerability” for each type of injury, but clinical overlap does exist. In the past, peripartum acute hypoxia was believed to be the major, if not the only, cause of perinatal brain damage, but intrauterine inflammation, prematurity, chronic hypoxia/growth retardation and genetic abnormalities appear to be at least equally important contributors.

To date, four cases illustrate that stem cell transplantation (SCT) remains an option for cure of HIV in only few cases. Long-term follow-up data on viral shedding of reservoirs are still needed. So far, interruption of ART is the only way to validate long-term HIV remission.

The mammalian retina lacks regenerative potency to replace damaged or degenerated cells. Therefore, traumatic or genetic insults that lead to the degeneration of retinal neurons or retinal pigment epithelium (RPE) cells alter visual perception and ultimately can lead to blindness. The advent of human stem cells and their exploitation for vision restoration approaches has boosted the field. Traditionally, animal models – mostly rodents – have been generated and used to mimic certain monogenetic hereditary diseases. Of note, some models were extremely useful to develop specific gene therapies, for example for Retinitis Pigmentosa, Leber congenital amaurosis and achromatopsia. However, complex multifactorial diseases are not well recapitulated in rodent models such as age-related macular degeneration (AMD) as rodents lack a macula. Here, human stem cells are extremely valuable to advance the development of therapies. Particularly, cell replacement therapy is of enormous importance to treat retinal degenerative diseases. Moreover, different retinal degenerative disorders require the transplantation of unique cell types. The most advanced one is to substitute the RPE cells, which stabilize the light-sensitive photoreceptors. Some diseases require also the transplantation of photoreceptors. Depending on the disease pattern, both approaches can also be combined. Within this article, I briefly feature the underlying principle of cell replacement therapies, demonstrate some successes and discuss certain shortcomings of these approaches for clinical application.

Apparently, there is a broad scope of legal issues affecting the work with embryonic stem cells. I cannot address them all. I would like to focus on two fields of law and discuss, rather in the overview, some salient issues of German constitutional and administrative law framing the scientific research process. Compared to the sophisticated scientific work we discuss here, legal arguments and operations remain blunt and awkward, addressing rather the identity of a society than the fluid frontiers of science and technology.

Objectives Early diagnosis and treatment of intra-amniotic infection is crucial. Rapid pathogen identification allows for a definite diagnosis and enables proper management. We determined whether the 16S amplicon sequencing performed by a nanopore sequencing technique make possible rapid bacterial identification at the species level in intra-amniotic infection. Methods Five cases of confirmed intra-amniotic infection, determined by either cultivation or 16S rDNA polymerase chain reaction (PCR) Sanger sequencing, and 10 cases of women who underwent mid-trimester genetic amniocentesis were included. DNA was extracted from amniotic fluid and PCR was performed on the full-length 16S rDNA. Nanopore sequencing was performed. The results derived from nanopore sequencing were compared with those derived from cultivation and Sanger sequencing methods. Results Bacteria were successfully detected from amniotic fluid using nanopore sequencing in all cases of intra-amniotic infection. Nanopore sequencing identified additional bacterial species and polymicrobial infections. All patients who underwent a mid-trimester amniocentesis had negative cultures, negative 16S PCR Sanger sequencing and nanopore sequencing. Identification of the microorganisms using nanopore sequencing technique at the bacterial species level was achieved within 5–9 h from DNA extraction. Conclusions This is the first study demonstrating that the nanopore sequencing technique is capable of rapid diagnosis of intra-amniotic infection using fresh amniotic fluid samples.

Objectives To investigate the adherence of German perinatal specialist units and those of basic obstetric care to the national guideline we compared data from a nation-wide survey on the practice of maintenance tocolysis, tocolysis in preterm premature rupture of membranes and in the perioperative setting of cervical cerclage, and bedrest during and after tocolysis with recommendations from the current German Guideline 015/025 “Prevention and Treatment of Preterm Birth”. Methods A total of 632 obstetric clinics in Germany were approached and received a link to an online questionnaire. Data were descriptively analyzed by performing measures of frequency. To compare two or more groups Fisherʼs exact test was used. Results The response rate was 19%; 23 (19.2%) of respondents did not perform maintenance tocolysis, while 97 (80.8%) conducted maintenance tocolysis; 30 (25.0%) of obstetric units performed cervical cerclage without tocolysis and 90 (75.0%) combined cervical cerclage with tocolysis; 11 (9.2%) of respondents did not use tocolytics in patients with preterm premature rupture of membranes, while 109 (90.8%) conducted tocolysis in these patients; 69 (57.5%) of obstetric units did not recommend bed rest during tocolysis, whereas 51 (42.5%) favored bedrest. Perinatal care centers of basic obstetric care recommend bed arrest during tocolysis statistically significant more often to their patients than those of higher perinatal care levels (53.6 vs. 32.8%, p=0.0269). Conclusions The results of our survey are in accordance to others from different countries and reveal considerable discrepancies between evidence-based guideline recommendations and daily clinical practice.

Objectives Cervical insufficiency (CI) is a condition consistent with painless cervical dilatation that can lead to preterm delivery. Cervical cerclage is a procedure in which cervical suture is performed for preventing preterm labor in several indications. Late emergency cerclage is technically more challenging compared to elective cerclage, performed earlier during pregnancy, prior to cervical changes. Pregnancy outcomes with emergency cerclage were found to be improved in previous reports, but there is still inconclusive data. To assess the effectiveness and safety of emergency cerclage vs. conservative management with progesterone and/or bed resting, in preventing preterm birth and improving neonatal outcomes in women with clinically evident cervical insufficiency. Methods This is a retrospective cohort study conducted on all women diagnosed with cervical insufficiency between the 16th and 24th gestational week who met the inclusion criteria, from January 2012 to December 2018. Obstetric and neonatal outcomes: time from diagnosis to delivery, duration of pregnancy, birth weight and Apgar score, were compared between women who underwent cerclage and those who treated conservatively. Results Twenty eight women underwent emergency cerclage (cerclage group) and 194 managed with a conservative therapy, progesterone and/or bed rest (control group). Time from diagnosis to delivery 13 weeks vs. 8 weeks and birth weight 2,418 g vs. 1914 g were significantly higher in the first cohort. Average pregnancy duration was three weeks longer in the cerclage group, but that was not significant. No complications occurred in the cerclage group and no difference in mode of delivery were found. Conclusions Emergency cerclage is an effective and safe procedure in preventing preterm birth and prolongation of pregnancy, in women with cervical insufficiency in the late second trimester.

Objectives To determine the effect of gestational age at delivery on maternal and neonatal outcomes in preterm prelabor rupture of membranes (PPROM) and assess various predictors of neonatal and infant mortality in these pregnancies. Methods United States birth data from CDC-National Center for Health Statistics natality database for years 2004–2008 was used to identify singleton pregnancies with PPROM and delivery from 32 0/7 to 36 6/7 weeks. Controls were singletons at 37–40 weeks, without PPROM. Maternal and neonatal complications reported by all states were analyzed along with neonatal outcomes such as chorioamnionitis and hyaline membrane disease, reported by a subgroup of states. OR (95% CI) were calculated after adjusting for preeclampsia, diabetes, chronic hypertension, maternal race, and infant sex. Results There were 134,502 PPROM cases and similar number of controls. There was a significant decrease in need for prolonged ventilation, hyaline membrane disease, 5 min Apgar score <7, and NICU admission with advancing gestational age. Placental abruption decreased and chorioamnionitis and cord prolapse were not different between 34 and 37 weeks. We found reductions in early death, neonatal death, and infant mortality with advancing gestational age (p<0.001 for each). Gestational age at delivery was the strongest predictor for early death, neonatal death, and infant mortality in PPROM. These differences persisted after adjusting for antenatal steroid use. Conclusions We provide population-based evidence showing a decrease in neonatal complications and death with advancing gestational age in PPROM. Gestational age at delivery in pregnancies with PPROM is the strongest predictor of mortality risk.

Objectives Open spina bifida (OSB) is the most common neural tube defect. Prenatal repair reduces the need for ventriculoperitoneal shunting (VPS) due to hydrocephalus from 80–90% to 40–50%. We aimed to determine which variables work as risk factors for VPS at 12 months of age in our population. Methods Thirty-nine patients underwent prenatal repair of OSB by mini-hysterotomy. The main outcome was occurrence of VPS in the first 12 months of life. Logistic regression was used to estimate the odds ratios (OR) between prenatal variables and the need for shunting. Results VPS at 12 months occurred in 34.2% of the children. Larger ventricle size before surgery (62.5% ≥15 mm; 46.2% between 12 and 15 mm; 11.8% <12 mm; p=0.008), higher lesion level (80% >L2, vs. 17.9% ≤L3; p=0.002; OR, 18.4 [2.96–114.30]), and later gestational age at surgery (25.25 ± 1.18 vs. 24.37 ± 1.06 weeks; p=0.036; OR, 2.23 [1.05–4.74]) were related to increased need for shunting. In the multivariate analysis, larger ventricle size before surgery (≥15 mm vs. <12 mm; p=0.046; OR, 1.35 [1.01–1.82]) and higher lesion level (>L2 vs. ≤L3; p=0.004; OR, 39.52 [3.25–480.69]) were risk factors for shunting. Conclusions Larger ventricle size before surgery (≥15 mm) and higher lesion level (>L2) are independent risk factors for VPS at 12 months of age in fetuses undergoing prenatal repair of OSB by mini-hysterotomy in the studied population.

Objectives To describe prevalence, outcome, color Doppler, and HDlive Flow (Silhouette) with spatiotemporal image correlation (STIC) features of isolated tortuous ductus arteriosus in Japanese fetuses at 28–31 + 6 weeks of gestation. Methods During a 37-month period, 3,150 fetal screenings were performed at 28–31 + 6 weeks of gestation. Isolated tortuous ductus arteriosus was evaluated using color Doppler and HDlive Flow (Silhouette) with STIC. Prevalence and outcomes were also assessed. Results There were 22/3,150 fetuses (0.7%) had isolated tortuous ductus arteriosus at 28–31 + 6 weeks of gestation. According to color Doppler and HDlive Flow findings, two types of isolated tortuous ductus arteriosus (Right-angled and Loop shapes) were classified. There were 20 Right-angled and 2 Loop shapes. One fetus with a Right-angled shape was associated with aortic tortuosity. HDlive Flow with STIC revealed spatial relationships of tortuous ductus arteriosus, aortic arch, and descending aorta. All fetuses with isolated tortuous ductus arteriosus had good neonatal outcomes. Conclusions The incidence of isolated tortuous ductus arteriosus in Japanese fetuses was low compared with those in previous reports. HDlive Flow with STIC provides precise information for spatial recognition and differentiation of isolated tortuous ductus arteriosus, resulting in two type classifications. Isolated tortuous ductus arteriosus in a fetus may be a transient, benign finding in utero .

Objectives To determine reference curves for fetal aortic and pulmonary valve annulus area by three-dimensional ultrasonography using the spatio-temporal image correlation (STIC) in the rendering mode, and to ascertain its applicability in congenital heart disease (CHD). Methods We performed a retrospective cross-sectional study of 328 normal fetuses and 42 fetuses with CHD between 20 and 33 weeks 6 days of gestation. The outflow plane view of the great vessels was used to measure the areas of the valvar annuli, and the measurements were performed in systole. A linear regression model adjusted according to the determination coefficient (R 2 ) was utilized to construct the reference intervals. The concordance correlation coefficient (CCC) was used to calculate the reproducibility of the mitral and tricuspid valve areas. Results The mean ± standard deviation (SD) of the aortic and pulmonary valve annulus areas ranged from 6.6 ± 1.2 to 32.9 ± 1.1 mm 2 and 10.7 ± 1.3 to 40.3 ± 1.2 mm 2 , respectively. We observed a linear relationship and strong positive correlation between the area of the aortic and pulmonary valve annuli with r=0.97 and 0.96, respectively. Good intra (CCC=0.99) and interobserver agreement (CCC=0.98) was observed for the measurement of the aortic valve annulus area. A good intra (CCC=0.99) and interobserver (CCC=0.97) agreement was also observed for the measurement of the pulmonary valve annulus area. The mean ± SD of the difference of the areas of the aortic and pulmonary valve annuli between the normal fetuses and those with CHD were −1.801 ± 1.429 mm 2 (p=0.208) and −1.033 ± 1.467 mm 2 (p<0.0001), respectively. Conclusions The reference curves for the areas of the aortic and pulmonary valve annuli of fetal hearts were determined, and showed good inter and intraobserver reproducibility. The constructed reference curves showed applicability in different types of CHD.

Objectives To assess the feasibility of identifying fetal brain structures and anatomic landmarks included in the anterior complex (AC) and posterior complex (PC), as well as the proximal hemisphere (PH). Methods This was a prospective observational multicenter study of healthy pregnant women evaluated by ultrasound screening at 24 to 36 + 6 weeks’ gestation. Six physicians performed transabdominal ultrasound, to obtain the planes required to visualize the AC, PC, and PH. Blind analysis by an expert and non-expert operator in fetal neurosonography was used to assess the structures included in each plane view. Results In the population studied (n=366), structure detection rates for AC were over 95 %, with an agreement of 96 % when comparing expert and non-expert examiners. Visualization of the corpus callosum crossing the midline was detected in over 97 and 96 % of cases for the AC and PC, respectively, with an agreement of over 96 %. The PH plane, particularly through the posterior access via the mastoid fontanelle, enabled visualization of the proximal anatomical structures in almost 95 % of cases. Detection of the corpus callosum through the AC and PC, both proximal/distal germinal matrix (AC) and proximal Sylvian fissure through the anterior access (PH) in the 24–25 + 6, 26–31 + 6 and 32–36 + 6 weeks’ gestation groups were successful in over 96 % of cases with high level of agreement. Conclusions Inclusion of AC, PC, and PH later in pregnancy proves feasible with a high level of agreement between both expert and non-expert operators.