Intra-amniotic Route Research Articles

Prenatal Zika virus exposure is associated with lateral geniculate nucleus abnormalities in juvenile rhesus macaques.

Zika virus' neural tropism causes significant neural pathology, particularly in developing fetuses. One of the consistent findings from humans and animal models is that prenatal exposure to Zika virus (ZIKV) causes pathology in the eyes and visual pathways of the brain, although the extent to which this pathology persists over development is not clear. In the present report, we build upon our previous work which demonstrated that full-term rhesus monkey ( Macaca mulatta ) fetuses who were exposed to ZIKV early in gestation had significant pathological abnormalities to the organization of the lateral geniculate nucleus (LGN), a major hub of the visual network. The objective of the present work was to replicate those LGN findings and determine whether such pathology persisted across childhood development. We carried out histological analyses of the LGNs of two juvenile rhesus monkeys who were prenatally exposed to ZIKV and two age-matched controls. Pregnant rhesus monkeys were infected with ZIKV via the intravenous and intra-amniotic routes and tracked across development. Following sacrifice and perfusion, brains were subjected to quantitative neuroanatomical analyses with a focus on the size and structure of the LGN and its composite layers. Early fetal ZIKV exposure resulted in developmental abnormalities within the brains' visual pathway: specifically disorganization, blending of layers, laminar discontinuities, and regions of low cell density within the LGN. These abnormalities were not observed in the control animals. Our findings demonstrate that the ZIKV's damage to the LGN that occurs during fetal development persists into childhood.

Nanoparticles for delivery of agents to fetal lungs

Fetal treatment of congenital lung disease, such as cystic fibrosis, surfactant protein syndromes, and congenital diaphragmatic hernia, has been made possible by improvements in prenatal diagnostic and interventional technology. Delivery of therapeutic agents to fetal lungs in nanoparticles improves cellular uptake. The efficacy and safety of nanoparticle-based fetal lung therapy depends on targeting of necessary cell populations. This study aimed to determine the relative distribution of nanoparticles of a variety of compositions and sizes in the lungs of fetal mice delivered through intravenous and intra-amniotic routes. Intravenous delivery of particles was more effective than intra-amniotic delivery for epithelial, endothelial and hematopoietic cells in the fetal lung. The most effective targeting of lung tissue was with 250nm Poly-Amine-co-Ester (PACE) particles accumulating in 50% and 44% of epithelial and endothelial cells. This study demonstrated that route of delivery and particle composition impacts relative cellular uptake in fetal lung, which will inform future studies in particle-based fetal therapy.

Methods for the Administration of EDAR Pathway Modulators in Mice.

Genetic deficiency of ectodysplasin A (EDA) causes X-linked hypohidrotic ectodermal dysplasia, a congenital condition characterized by the absence or abnormal formation of sweat glands, teeth, and several skin appendages. Stimulation of the EDA receptor (EDAR) with agonists in the form of recombinant EDA or anti-EDAR antibodies can compensate for the absence of Eda in a mouse model of Eda deficiency, provided that agonists are administered in a timely manner during fetal development. Here we provide detailed protocols for the administration of EDAR agonists or antagonists, or other proteins, by the intravenous, intraperitoneal, and intra-amniotic routes as well as protocols to collect blood, to visualize sweat gland function, and to prepare skulls in mice.

Intravenous and Intra-amniotic <em>In Utero</em> Transplantation in the Murine Model

In utero transplantation (IUT) is a unique and versatile mode of therapy that can be used to introduce stem cells, viral vectors, or any other substances early in the gestation. The rationale behind IUT for therapeutic purposes is based on the small size of the fetus, the fetal immunologic immaturity, the accessibility and proliferative nature of the fetal stem or progenitor cells, and the potential to treat a disease or the onset of symptoms prior to birth. Taking advantage of these normal developmental properties of the fetus, the delivery of hematopoietic stem cells (HSC) via an IUT has the potential to treat congenital hematologic disorders such as sickle cell disease, without the required myeloablative or immunosuppressive conditioning required for postnatal HSC transplants. Similarly, the accessibility of progenitor cells in multiple organs during development potentially allows for a more efficient targeting of stem/progenitor cells following an IUT of viral vectors for gene therapy or genome editing. Additionally, IUT can be used to study normal developmental processes including, but not limited to, the development of immunologic tolerance. The murine model provides a valuable and affordable means to understanding the potential and limitations of IUT prior to pre-clinical large animal studies and an eventual clinical application. Here, we describe a protocol for performing an IUT in the murine fetus through intravenous and intra-amniotic routes. This protocol has been used successfully to elucidate the necessary conditions and mechanisms behind in utero hematopoietic stem cell transplantation, tolerance induction, and in utero gene therapy.

Prevalence of Maternal Genital Tract Colonization by Group B Streptococcus From Western Province, Taif, Saudi Arabia

Background: Group B streptococcus (GBS) or Streptococcus agalactiae are members of the normal flora of the female genital tract. Maternal colonization has been found to be a major risk factor for invasive GBS disease within 6 days of birth. GBS has become the major cause of bacterial infections in the perinatal period, including bacteremia, amnionitis, endometritis, and urinary tract infection in pregnant women as well as sepsis and meningitis in neonates and young infants. Infection of the new born may be acquired by the intra-amniotic route or directly during passage through the birth canal. This study was undertaken to determine the prevalence of GBS colonization in pregnant women attending the antenatal clinic of Al-Hada Armed Forces Hospital in Western province, Taif, Saudi Arabia. This paper was the first data on incidence of GBS in pregnant women at Western province, Taif, Saudi Arabia. Methods: A total of 2,632 pregnant women were screened for GBS colonization between January and December 2014. Standard microbiological methods were used to isolate and identify GBS from vaginal and anorectal swabs obtained from study subjects. An antimicrobial susceptibility test was performed for all GBS isolates according to the criteria of the Clinical and Laboratory Standards Institute (CLSI) by disk diffusion method. Results: A total of 632 out of 2,632 (24%) pregnant women were colonized by GBS. Statistically significant association was observed for GBS colonization with any of socio-demographic characteristics of the study subjects including age, occupation, number of antenatal clinic visits, and type of gravida. All GBS strains were susceptible to penicillin, ampicillin, vancomycin and gentamicin, erythromycin, tetracycline, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, and norfloxacin. Resistance was observed in some strains against clindamycin (0.18%). The data from the present study showed that incidence of GBS in Saudi pregnant women increased rapidly. These results are the first record of the database in Saudi Arabia at western province with high prevalence of GBS in pregnant women. Conclusion: This study showed that prevalence of GBS colonization was 24% among the study subjects. The finding of this study was comparable with findings reported from developed and developing countries. However, further epidemiological investigations should be done in different parts of the country (all provinces) in order to know the actual GBS colonization rate in pregnant women and to consider the use of intrapartum antibiotics prophylaxis for prevention of early onset GBS-neonatal diseases, considering the accelerated demand for reducing neonatal morbidity and mortality due to GBS. J Clin Gynecol Obstet. 2015;4(3):258-264 doi: http://dx.doi.org/10.14740/jcgo341w

Ultra-high dose of intra-amniotic or direct fetal intramuscular betamethasone for lung maturation in the preterm goat model

To evaluate the effects of intra-amniotic (IA) and fetal injections of a single ultra-high dose of betamethasone (BM) 48 h before preterm delivery on neonatal pulmonary function, using an experimental goat model. Eighteen date-mated singleton pregnant Hair goats were randomized into four groups. At gestational day 118 (alveolar phase, term 150-155 days) after obtaining a sample of amniotic fluid, fetuses in group 1 (n=5) received 8 mg/kg IA BM, and in group 2 (n=5) 4 mg/kg fetal IM BM. In group 3 (n=4) (0.3mg/kg/day) maternal BM was administered at day 118 and 119 with a 24h interval; control fetuses (n=4) received 1 mL/kg of IA saline at day 118. At gestational day 120, after obtaining second sample of amniotic fluids 18 kids were delivered by preterm cesarean section, entubated, weighed, and mechanically ventilated for 15 min. Arterial blood gas samples and deflation/inflation lung pressure-volume measurements were obtained. After sacrifice, lungs were removed, weighed, gross examined and processed for further histological and immunohistochemical (IHC) evaluations. On hematoxylin and eosin (HE) stained slides, presence and severity of lung emphysema was evaluated; slides stained for surfactant proteins, and caspases were used for semi-quantitative evaluation of lung maturation. Kruskal-Wallis, Mann-Whitney, Wilcoxon signed rank, and chi-square tests were used for comparisons. IA BM was associated with increased number of stillbirths (60% vs. 0% in control) (p=0.06) and emphysematous changes. Bodyweight-adjusted pressure-volume measurements were improved after maternal, but not IA or fetal, BM (p=0.06). Following mechanical ventilation, arterial blood gas parameters did not significantly alter across maternal and fetal administrations. However, pH was significantly lower (p<0.05) and carbon dioxide partial pressure was higher (p<0.05) in the control group, indicating hypercapnic acidemia in non-treated pregnancies. None of the treatments induced measurable alterations in amniotic fluid lecithin/sphingomyelin (L/S) values. IA and fetal routes were associated with decreased surfactant protein expressions and increased apoptotic activity in alveolar and bronchio-alveolar epithelial cells. Ultra-high dose IA and fetal IM BM is not superior to the standard dose and maternal way of administration in our experimental design.

Targeted Gene Transfer to Fetal Rat Lung Interstitium by Ultrasound-guided Intrapulmonary Injection

In utero gene transfer to the developing lung may have clinical or research applications. In this study, we developed a new method for specifically targeting the fetal rat lung with adeno and lentiviral vectors encoding the enhanced green fluorescence protein (EGFP) marker gene at E15.5 using ultrasound biomicroscopy (UBM). Survival rate, morphometric parameters, viral biodistribution, and lung transduction efficiency were analyzed and compared to the intra-amniotic route of administration. Expression of EGFP started as early as 24 and 72 h after the injection of adenoviral and lentiviral vectors, respectively. Both vectors transduced lung parenchyma with gene expression limited to interstitial cells of the injected region, in contrast to intra-amniotic injection, which targeted the pulmonary epithelium. Expression of EGFP was most intense at E18.5 and E21.5 for adenoviral and lentiviral vectors, respectively. In contrast to lentivirus, adenoviral expression significantly declined until final analysis at 1 week of age. This study demonstrates the feasibility of targeting the fetal rat lung interstitium with viral vectors under UBM guidance during the pseudoglandular stage. This model system may facilitate in vivo studies of dynamic lung morphogenesis and could provide insight into the efficacy of prenatal gene transfer strategies for treatment of specific lung disorders.

Versatile roles of docosahexaenoic acid in the prenatal brain: From pro- and anti-oxidant features to regulation of gene expression

Docosahexaenoic acid (DHA) is the most ubiquitous polyunsaturated fatty acid (FA) in brain tissue. It is selectively esterified to amino phospholipids (PL) and therefore it is highly prevalent at the cytofacial site of the plasma membrane where it may specifically participate in intracellular events. A highly selective DHA accumulation prior to birth is the result of maternal supply via the placenta through a bio-magnification process. Supplements of DHA via the intra-amniotic route to the fetal rat increase brain DHA levels and also confer neuroprotection to fetuses subjected to global ischemic stress. The protective effect has been attributed to an enhanced free radical scavenging capacity of DHA. Dietary deprivation of linolenic acid (LNA) during the perinatal life on the other hand, resulted in losses of DHA from cerebral PLs [M. Schiefermeier, E. Yavin, n-3 deficient and DHA-enriched diets during critical periods of the developing prenatal rat brain, J. Lipid Res. 43 (2002) 124–131]. LNA deprivation also caused changes in a number of gene markers the identification of which was attained by a labor-intensive suppression subtractive hybridization protocol using mRNA from 2-week-old postnatal brains [E. Yakubov, P. Dinerman, F. Kuperstein, S. Saban, E. Yavin, Improved representation of gene markers on microarray by PCR-select subtracted cDNA targets, Mol. Brain Res. 137 (2005) 110–118]. Most notable was a remarkable elevation of dopamine (DA) receptor (D1 and D2) genes as evaluated by quantitative RT-PCR, SDS-PAGE gel electrophoresis and immunochemical staining [F. Kuperstein, E. Yakubov, P. Dinerman, S. Gil, R. Eylam, N. Salem Jr., E. Yavin, Overexpression of dopamine receptor genes and their products in the postnatal rat brain following maternal n-3 FA dietary deficiency, J. Neurochem. 95 (2005) 1550–1562]. Over-expression of DA receptors has been attributed to a compensatory mechanism resulting from impairment in DA neurotransmitter production, storage and processing. In conclusion, DHA is a versatile molecule with a wide range of actions spanning from participation in cellular oxidative processes and intracellular signaling to modulatory roles in gene expression and growth regulation.

Species-specific but not genotype-specific primary and secondary isotype-specific NSP4 antibody responses in gnotobiotic calves and piglets infected with homologous host bovine (NSP4[A]) or porcine (NSP4[B]) rotavirus

Using recombinant baculoviruses expressing rotavirus NSP4 [A], [B], [C], and [D] genotypes of bovine, porcine, human, simian, or murine origin, we analyzed serum antibody responses to NSP4s in gnotobiotic calves and piglets infected by the oral/alimentary or intraamniotic route with bovine (NSP4[A]) (Wyatt, R.G., Mebus, C.A., Yolken, R.H., Kalica, A.R., James, H.D., Jr., Kapikian, A.Z., Chanock, R.M., 1979. Rotaviral immunity in gnotobiotic calves: heterologous resistance to human virus induced by bovine virus. Science 203(4380), 548–550) or porcine (NSP4[B]) (Hoshino, Y., Saif, L.J., Sereno, M.M., Chanock, R.M., Kapikian, A.Z., 1988. Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen. J. Virol. 62(3), 744–748) rotaviruses. Following primary infection and challenge with virulent rotaviruses, the animals developed higher or significantly higher antibody titers to homologous host homotypic NSP4s than to heterologous host homotypic or heterologous host heterotypic NSP4s, indicating that antibody responses were species specific rather than genotype specific. Antibody responses to NSP4s corresponded closely with the phylogenetic relationships of NSP4s within a species-specific region of amino acids (aa) 131–141. In contrast, NSP4 genotypes determined by amino acid full-length sequence identity predicted poorly their “serotypes”. In piglets, antibodies to NSP4 induced by previous oral infection failed to confer protection against challenge from a porcine rotavirus bearing serotypically different VP4 and VP7 but essentially identical NSP4 to the porcine rotavirus in primary infection. Thus, in an approach to immunization with a live oral rotavirus vaccine, the NSP4 protein does not appear to play an important role in protection against rotavirus disease and infection.

Successful treatment of fetal anemia via the intraamniotic route at 110 and 125 days gestation in the ovine fetus

Successful treatment of fetal anemia via the intraamniotic route at 110 and 125 days gestation in the ovine fetus

The Feasibility of Early Second Trimester Pregnancy (12 to 14 weeks) Termination with Single Intra‐Amniotic Injection of 15‐Methyl PGF2α

Prostaglandin analogues have proven to be safe and effective for second trimester pregnancy terminations when given by various routes. Of these, the intra-amniotic (IA) route has proven to be safer, especially in women with medical disorders, as it causes the least occurrence of systemic side-effects. Previously, blind transabdominal IA instillation of the analogue 15-methyl PGF2 alpha was routinely possible only after 16 weeks' gestation. With the advent of ultrasound-guided techniques, it has now become possible to use the IA route for very early second trimester pregnancies. This study examines the use of single 1.5 mg 15-methyl PGF2 alpha IA injection for termination of 12-14 weeks pregnancies. Single injection resulted in successful abortion in 76.5% of women and, in the unsuccessful cases, additional prostaglandin injection resulted in successful abortion in all women within 48 hours without problems. It is therefore feasible to use ultrasound-guided IA prostaglandin injection for successful termination of early second trimester pregnancies.

Experience with prostaglandins for therapeutic abortion in Norway. Their need and their benefits.

Since 1977 the number of therapeutic abortions has been decreasing in Norway, a trend which is probably due to improved contraceptive practice. The results with PGs used for cervical dilatation in late first trimester and induction of abortion in the second trimester are reported. Pretreatment overnight or for 3-4 hours simplified the surgical procedure and Sulprostone (Schering AG) caused fewer side effects than 15(S) 15 Me F2 alpha. In the second trimester 15(S) 15 Me F2 alpha was administered by different routes and in different doses. 16,16 diMe trans delta 2E2 was given only as vaginal suppositories. Side effects were fewer by the intraamniotic route than by the other routes. The need for and benefits of PGs for therapeutic abortions are discussed.

Use of intra- and extra-amniotic prostaglandins for the termination of pregnancies — Report of multicentric trial in India

The Indian Council of Medical Research initiated a, multicentric trial with prostaglandins in 1976 to assess the safety and efficacy of their use in midtrimester abortions, PGF 2αand 15-Me-PGF 2α were compared using the intra-amniotic (I.A.) route, 15-Me-PGF 2α was also evaluated by extra-amniotic (E.A.) route. With intra-amniotic instillation, success rate was 88.1 per cent with PGF 2α and 93.0 per cent with 15-Me-PGF 2α within 48 hours and by the extra-amniotic route it was 78.1 per cent within 36 hours. The mean induction-abortion interval was 19 hours with I.A. and 14.8 hours with E.A. Abortions were complete in 48.8 per cent of the women following I.A. PGF 2α, 56.0 per cent following I.A. 15-Me-PGF 2αand only 23.0 per cent following E.A. administration. Vomiting and diarrhoea were the most commonly reported side effects. Cervical injuries were 4.7 per cent with I.A. PGF 2α, 1.4 per cent with I.A. 15-Me-PGF 2α and only 0.6 per cent with E.A. route.

Hormone changes in relation to the time of fetal death after prostaglandin-induced abortion

The changes in unconjugated estradiol-17β and estriol, progesterone and chorionic somatomammotropin (HCS) in peripheral plasma have been studied in 18 women at 30-minute intervals following intra-uterine prostaglandin E 2 administration for therapeutic termination of second trimester pregnancy. The hormonal changes were related to the time of fetal death detected by the disappearance of fetal heart pulsations. Prostaglandin E 2 was given by the intra-amniotic route with urea (5 patients) or with intravenous oxytocin (5 patients), or by the extra-amniotic route with intravenous oxytocin (8 patients). Fetal death occurred rapidly with intra-amniotic PGE 2, but usually at a late stage with extra-amniotic PGE 2. Three fetuses in the extra-amniotic group died at or just before abortion. A variety of fetal heart changes were noted and the time of fetal death did not appear to influence the time of abortion within each treatment subgroup. Estradiol and estriol showed a slight but persistent fall over 24 hours prior to induction of abortion. A more rapid fall usually occurred after induction, with a consistent fall around the time of fetal death. Progesterone and HCS usually fell much less before and immediately after fetal death. A marked rise in estradiol sometimes occurred before fetal death, particularly in the intra-amniotic PGE 2 and urea subgroup. Estriol levels declined more rapidly before than after fetal death, whereas fetal death had less consistent effects on the other hormones. All hormones had usually fallen considerably at the time of abortion, and in some individuals marked fluctuations in hormone levels were seen.

Intraamniotic and Intramuscular Administration of 15‐Methyl Prostaglandin F2α for Midtrimester Abortion

Two series of midtrimester abortion inductions are compared. In one series of 68 cases of midtrimester pregnancies (12-24 weeks), legal abortion was induced by one intraamniotic injection of 2.5 mg 15-methyl prostaglandin F 2alpha. Fetus was expelled in 67 cases (98.5%) after a mean time of 18.4 hours. One case with duplex failed to abort (1.5%). Abortion was complete in 54% of the aborted cases. In the second series of 93 cases abortion was induced by intramuscular injection of 300 microgram 15-methyl PGF 2alpha every third hour (the first dose was 200 microgram) during 30 hours. Fetus was expelled in 79 cases (85%) after a mean time of 16.7 hours. Failure occurred in 14 cases (15%). Abortion was complete in 57% of the aborted cases. Side effects (vomiting and diarrhea) were more frequent in the intramuscular series and very inconvenient to many of the patients. Excessive bleeding occurred more often in the intraamniotic series. A small rupture of the cervix was noted once (primigravida) in the intramuscular group. It is concluded that the intramuscular way of administration is a simple method of second trimester pregnancy termination with small bleedings but that otherwise it is inferior to the intraamniotic route.

Comparative effects of hyperosmolar urea administered by intra-amniotic, intravenous, and intraperitoneal routes in rhesus monkeys

Hypertonic urea solutions (58 per cent weight/volume) were injected rapidly into ten anesthetized rhesus monkeys at a dose of approximately 2 Gm. per kilogram by the intravenous, intraperitoneal, or intra-amniotic routes. An additional monkey received an intra-amniotic dose of 12.5 Gm. per kilogram. A tracer dose of 14C-urea was included for measuring absorption and elimination. The following parameters were monitored before and for four hours after urea injection: arterial and cerebrospinal fluid pressures, heart and respiration rates, urine flow, and urea clearance. Serum electolytes, hematocrit and white count, and cumulative urea excretion were measured for one week. Monkeys were observed for three to six months after injection. At doses up to approximately twice the human dose (on a body weight basis) there were no urea-related deaths and no serious side effects noted in any of the experiments. These results support the suggestion that urea is a relatively safe hyperosmolar agent for inducing midtrimester abortions, especially with regard to inadvertent systemic injection.

Substrate-induced acceleration of lactase synthesis in fetal rat intestine.

Twenty milligrams of lactose were injected into the amniotic sacs of fetuses in one uterine horn and 20 mg glucose were injected into the amniotic sacs of fetuses in the opposite uterine horn of six rats on days 17-19 of pregnancy. Two or 3 days later the pregnant animals were killed and segments of jejunum obtained from their fetuses were homogenized in water. Assays for lactase and protein determinations were performed on these homogenates. Lactase values were significantly higher in the lactose-injected group than in fetuses receiving glucose (Table 2), P less than 0.0005. Thus, fetal intestinal lactase activity can be increased by exposure to the substrate lactose during late fetal life.

The current status of prostaglandins as abortifacients

The present use and potential uses of prostaglandins as abortifacients are summarized. Pertinent history, chemistry, prostaglandins' possible role in physiologic and pathologic processes and pharmacologic actions are discussed. The results of natural prostaglandins and their analogues by systemic and intrauterine administration for the purposes of postcoital contraception, menstrual regulation, first- and second-trimester abortion, preoperative dilatation of the cervix, and delivery of patients with death in utero are presented. The only approved method of induction of abortion with prostaglandins, prostaglandin F2α by the intra-amniotic route for the induction of midtrimester abortion, is evaluated and compared to other methods of midtrimester abortion. It was concluded that: (1) the present use of prostaglandins is an important addition to the obstetrician's armamentarium, (2) more effective and/or convenient methods that are useful in patients over a wider gestational age appear to have been defined, and (3) the routine use of prostaglandins for postcoital contraception, menstrual regulation, and first-trimester abortion will require the development of analogues that are more specific as to their abortifacient actions than the natural prostaglandins and/or the development of improved delivery systems.

Early mid trimester abortion — By intramuscular 15 methyl prostaglandin E 2

Intramuscular administration of 15(s) 15 Methyl Prostaglandin E 2 was successful in terminating twelve of thirteen second trimester pregnancies. Two patients had an incomplete abortion and one case was classified a failure. Side effects were limited to nausea and vomiting in seven cases, diarrhea in eight cases, chills in twelve cases and and elevated temperatures in eight cases. The intramuscular route does offer an advantage in early second trimester abortions and in patients where the intra-amniotic route is not available.

Reassessment of systemic administration of prostaglandins for induction of midtrimester abortion

The present investigation was undertaken to evaluate the abortifacient efficacy of vaginal and intramuscular administration of different dose schedules of the 15-methyl analogues of prostaglandin F 2α. 1. Vaginal administration . Both 15-methyl-PGF 2α and 15-methyl-PGF 2α methyl ester can be absorbed from the vagina in sufficient amounts to induce abortion. The potency of the methyl ester was approximately twice that of the free acid. The most successful treatment schedule consisted of an initial dose of 0.5 mg of the methyl ester followed by 1.0 or 2.0 mg every third hour. On this treatment all patients aborted within 24 hours. 2. Intramuscular administration . Initially 200 μg of 15-methyl-PGF 2α was given. The dose was increased to 400 μg or occasionally to 500 μg depending on the effect and tolerance of the patient and repeated every third hour. The treatment schedule resulted in a 100 per cent abortion rate and the mean induction-abortion interval was 16.1 hours. Both routes were associated with a higher frequency of side effects than that reported for intra-amniotic administration of 15-methyl-PGF 2α. It seems justified to conclude that the intra-amniotic route is preferable after the 14th week when the uterine cavity is easy to puncture but that vaginal or intramuscular injections of the compounds could be an alternative in late first trimester and early second trimester cases.