Society for Maternal-Fetal Medicine Special Statement: Beyond the scalpel: in utero fetal gene therapy and curative medicine.

With the recent advances in gene editing with systems such as CRISPR-Cas9, precise genome editing in utero is on the horizon. Sickle cell disease is an excellent candidate for in utero fetal gene therapy, because the disease is monogenic, causes irreversible harm, and has life-limiting morbidity. Gene therapy has recently been proven to be effective in an adolescent patient. Several hurdles still impede the progress for fetal gene therapy in humans, including an incomplete understanding of the fetal immune system, unclear maternal immune responses to in utero gene therapy, risks of off-target effects from gene editing, gestational age constraints, and ethical questions surrounding fetal genetic intervention. However, none of these barriers appears insurmountable, and the journey to in utero gene therapy for sickle cell disease and other conditions should be well underway.

Cumulative outcome of pre-implantation genetic diagnosis for sickle cell disease: a 5-year review.

To review the cumulative outcome of pre-implantation genetic diagnosis (PGD) cycles performed for prevention of sickle cell disease (SCD). Couples referred for PGD for SCD between April 2012 and October 2017 were included. Ovarian stimulation was performed using a short gonadotrophin-releasing hormone (GnRH) antagonist protocol and follicle-stimulating hormone injections. The GnRH agonist was used to trigger oocyte maturation. Oocytes were fertilised using intracytoplasmic sperm injection. Trophectoderm biopsy was performed on day 5 or 6 followed by vitrification. Genetic testing was done using pre-implantation genetic haplotyping. A total of 60 couples started 70 fresh PGD cycles (mean 1·2 cycles/couple) and underwent a total of 74 frozen-embryo-transfer (FET) cycles (mean 1·3 FET/couple). The mean (SD) female age was 33 (4·4) years and the mean (SD) anti-müllerian hormone level was 22·9 (2·8) pmol/l. The cumulative live-birth rate was 54%/PGD cycle started and 63%/couple embarking on PGD. The rate of multiple births was 8%. The cumulative outcome of PGD treatment for prevention of SCD transmission is high and PGD treatment should be offered to all at-risk couples.

Novel and known microsatellite markers within the ß-globin cluster to support robust preimplantation genetic diagnosis of ß-thalassemia and sickle cell syndromes.

Preimplantation genetic diagnosis (PGD) for ß hemoglobinopathies has become the most common application among monogenic disorders. We present the identification of microsatellite markers [short tandem repeats (STRs)] closely linked to the ß-globin gene for incorporation within PGD protocols, with the aim of increasing the number of transferable embryos. Nine candidate STRs were identified in-silico, of which three were selected based on rate-of-heterozygosity, polymerase chain reaction (PCR) efficiency and size. The multiplex reaction (ß-globin gene and selected STRs, all within <0.4 Mb from the ß gene) was optimized in single lymphocytes, and subsequently applied in 38 PGD cycles in couples at-risk for transmitting ß hemoglobinopathies. In conclusion, incorporation of closely linked polymorphic microsatellite markers <0.4 Mb from the ß-globin gene, facilitates robust assignment of ß hemoglobinopathy genotypes, increasing the number of transferrable embryos otherwise rejected due to allele-drop-out (ADO), at the mutation-specific locus, compared to results based on disease-mutation genotyping alone (p < 0.001).

Prenatal diagnosis of sickle syndromes in India: dilemmas in counselling.

OBJECTIVES: The sickle gene is prevalent in the scheduled caste and tribal populations in India. The clinical presentation of sickle cell disease is extremely variable, and there are no neonatal screening programmes. This is the first report on prenatal diagnosis of sickle syndromes in 85 couples at risk (sickle cell anemia-69; sickle thalassemia-16) from different regions in India. Most of the couples were from a low socioeconomic group and their decisions were entirely dependent on the local counselling given. We have evaluated the acceptability of prenatal diagnosis and the dilemmas faced in counselling these families. METHODS: Chorion villus sampling was done in the first trimester and DNA analysis using reverse dot blot hybridization or restriction enzyme digestion with Dde1 in 65 cases. Cordocentesis was done in the second trimester and fetal blood analyses by automated HPLC in 20 cases who came late. RESULTS: 32.9% of couples came prospectively for diagnosis. 23.5% of fetuses were affected (sickle cell anemia-18, sickle thalassemia-2). The beta-thalassemia mutation in both cases was IVS 1-5(G->C). All the couples with an unfavourable diagnosis opted for termination of pregnancy. CONCLUSION: Sickle cell anemia has a relatively benign clinical course in some tribal groups in India. This raises a dilemma whether we are justified in advising prenatal diagnosis in all such cases.

Early prenatal diagnosis by celocentesis.

BACKGROUND: Celocentesis is the ultrasound-guided aspiration of fluid from the extra-amniotic cavity at 7-8 weeks of gestation. This paper reports on the clinical application of celocentesis for early prenatal diagnosis. METHODS: Celocentesis was successfully performed in nine pregnancies and 1-2 mL of fluid were obtained after one needle insertion. The indications were prenatal diagnosis of beta-thalassemia or sickle cell disease (n = 6), Marfan syndrome (n = 1) and paternity testing (n = 2). Molecular biological techniques were used to analyze the celomic fluid and this was successfully carried out in all cases. RESULTS: In two cases pregnancy termination was performed at the request of the mother because in one case the fetus was found to have sickle cell anemia and in the second case paternity testing demonstrated that the father was not the woman's husband. In both cases the results were confirmed using the placental samples collected after pregnancy termination. In six of the seven pregnancies with desirable results, amniocentesis was performed at 16 weeks and the results were concordant with those obtained from celocentesis. All pregnancies were uneventful and resulted in the delivery of healthy and appropriately grown babies. CONCLUSION: Celocentesis may be a viable alternative to the currently used tests of chorionic villus sampling and amniocentesis.

Outcomes of universal antenatal screening for haemoglobinopathies.

OBJECTIVE: To evaluate universal antenatal screening for haemoglobinopathies. SETTING: District general hospital serving a London borough with 45% ethnic minorities. METHODS: Retrospective cohort study of 1444 women referred in 1688 pregnancies and 95 tertiary referrals during 101 pregnancies. RESULTS: Unselected women at risk for sickle cell disease booked 2.7 weeks (95% confidence interval (CI) 0.14 to 5.1) later in gestation than those at risk for beta thalassaemia were less likely to attend counselling (83% v 93%, relative risk (RR) 0.89; 95% CI 0.85 to 0.94), their partners were less likely to be tested (77% v 95%, RR 0.81; 0.77 to 0.83), and they were less likely to accept prenatal diagnosis (22% v 90%, RR 0.37; 0.24 to 0.57). Over 99% of tertiary referrals attended counselling and had their partners tested. There were no significant differences in acceptance of prenatal diagnosis between those at risk of sickle cell disease and beta thalassaemia (55% v 67%). Unselected women at risk of sickle cell disease were significantly less likely to have their partner tested or to accept prenatal diagnosis than tertiary referrals, but not those at risk of beta thalassaemia. 80% of beta thalassaemia and 16% of SS births were prevented. CONCLUSIONS: Uptake of prenatal diagnosis among unselected women at risk of beta thalassaemia is similar to that reported by tertiary centres. It is considerably lower for sickle cell disease but could increase considerably if screening occurred earlier in gestation. Acceptance of counselling is universally high, suggesting that informed choices are made, and indicating a need to measure these outcomes for cost effectiveness studies.

In utero transplantation for thalassemia.

In utero hematopoietic stem cell transplantation is a promising approach for the treatment of a variety of congenital hematologic diseases. Although the approach has been successful for immunodeficiency syndromes, attempts thus far to treat the hemoglobinopathies have failed. In most of these cases the late gestational age at transplantation, source of donor cells, or procedure-related complications, provide an explanation for failure. Nevertheless the biology of thalassemia, in the context of prenatal transplantation, requires examination. In contrast to postnatal bone marrow transplant regimens, engraftment after in utero transplantation requires donor cells to effectively complete for developing receptive sites in the recipient hematopoietic microenvironment. Effective prenatal treatment of thalassemia will depend on the ability of normal cells to engraft and complete in the thalassemic microenvironment. Clinical observations after bone marrow transplantation of amelioration of anemia in beta-thalassemia by relatively low degrees of mixed chimerism, and the apparent selective advantage observed for donor erythropoiesis, suggest prenatal transplantation could succeed. Prenatal strategies involving multiple transplants, donor-specific tolerance induction, and postnatal same-donor transplants should be considered.

Experience with 500 prenatal diagnoses of sickle cell diseases: the effect of gestational age on affected pregnancy outcome.

Prenatal diagnosis of sickle cell diseases is obtained rapidly and precisely by polymerase chain reaction (PCR) with Ddel restriction analysis and dot-blotting with allele-specific oligonucleotides (ASO). Prenatal diagnosis of HgbSS and HgbSC was performed in 500 pregnancies, 196 by Southern blot and 304 by PCR. PCR drastically shortened the interval from sampling to reporting, allowing acceptance even of samples with unknown paternal phenotype, and resulted in an overall four-fold increase in diagnoses. In 108 pregnancies, the diagnosis was an affected fetus; 25 were HgbSC: 3 (12 per cent) were terminated; 83 were HgbSS: four ended in miscarriage; 40/79 (51 per cent) were terminated. The gestational age at the time of report to the mother appeared to be a major outcome determinant when the fetal diagnosis was HgbSS. The change-point in the maternal decision was found at 20 weeks of gestation. Before the 20th week, most mothers (64 per cent) chose termination; thereafter, the majority (72 per cent) chose continuation. The odds ratio of termination in earlier relative to later reporting was 4.7. In order to offer a choice to the mothers at risk of delivering a fetus affected by sickle cell disease, the diagnosis should be reported before the 20th week of gestation.

Sonographic anatomy of the fetal cerebral ventricles, with reference to the early diagnosis of hydrocephaly.

Real time ultrasound was used to evaluate the anatomy of the fetal brain at different gestational ages. Anatomical correlation with the gross brain was utilized for more accurate identification of the neuroanatomical structures. The normal growth of the ventricular system was studied. Transaxial measurements of the anterior horn (AH) and maximum ventricular length (MVL) and width (MVW) were made, and enlarged as pregnancy progressed. The ratios of MVW/MVL, MVL/BPD, and MVW/BPD provide guides to the early diagnosis of hydrocephalus and intracranial abnormalities. Specific measurements of the cerebral ventricles at various gestational ages may be made accurately by utilizing the anatomical landmarks. The anterior horn and midbrain measurements are of little value in the early diagnosis of hydrocephaly. Maximum ventricular length and width are the most useful determinants of hydrocephaly, even as early as 20 weeks. A set of discordant twins in which twin B was found to be hydrocephalic by these studies in the twentieth week is presented with serial measurements for both twins. Multiple measurements of the cerebral ventricular system in utero permit early and precise diagnosis of fetal hydrocephaly.