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. 2021 Jul;246(14):1668-1679.
doi: 10.1177/15353702211003508. Epub 2021 Apr 1.

Strategies for intra-amniotic administration of fetal therapy in a rabbit model of intrauterine growth restriction

Mari Kinoshita  1   2 , Fàtima Crispi  1   2   3 , Carla Loreiro  1   2 , Eduard Gratacós  1   2   3 , Míriam Illa  1 , Mònica Zamora  1   2
Affiliations

Strategies for intra-amniotic administration of fetal therapy in a rabbit model of intrauterine growth restriction

Mari Kinoshita et al. Exp Biol Med (Maywood). 2021 Jul.

Abstract

Intrauterine growth restriction affects up to 10% of all pregnancies, leading to fetal programming with detrimental consequences for lifelong health. However, no therapeutic strategies have so far been effective to ameliorate these consequences. Our previous study has demonstrated that a single dose of nutrients administered into the amniotic cavity, bypassing the often dysfunctional placenta via intra-amniotic administration, improved survival at birth but not birthweight in an intrauterine growth restriction rabbit model. The aim of this study was to further develop an effective strategy for intra-amniotic fetal therapy in an animal model. Intrauterine growth restriction was induced by selective ligation of uteroplacental vessels on one uterine horn of pregnant rabbits at gestational day 25, and fetuses were delivered by cesarean section on GD30. During the five days of intrauterine growth restriction development, three different methods of intra-amniotic administration were used: continuous intra-amniotic infusion by osmotic pump, multiple intra-amniotic injections, and single fetal intraperitoneal injection. Technical feasibility, capability to systematically reach the fetus, and survival and birthweight of the derived offspring were evaluated for each technique. Continuous intra-amniotic infusion by osmotic pump was not feasible owing to the high occurrence of catheter displacement and amnion rupture, while methods using two intra-amniotic injections and one fetal intraperitoneal injection were technically feasible but compromised fetal survival. Taking into account all the numerous factors affecting intra-amniotic fetal therapy in the intrauterine growth restriction rabbit model, we conclude that an optimal therapeutic strategy with low technical failure and positive fetal impact on both survival and birthweight still needs to be found.

Keywords: FGR; Fetal growth restriction; nutritional therapy; prenatal intervention; surgical model; transamniotic.

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Conflict of interest statement

DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and publication of this article.

Figures

Figure 1.
Figure 1.
Experimental design: Exploring three different strategies for fetal therapy. The diagram presents the general study design including IUGR induction in the experimental animals, administration of nutritional solution, and sample size (number of pregnant rabbits) for each of the three methods. Different stages for the development of fetal therapy were designed: Phase I evaluated the feasibility of the technical procedures and Phase II evaluated the therapeutic effect of the treatment using the established techniques. Phase I: Strategies using osmotic pump or two IA injections were first evaluated for technical feasibility in control fetuses without nutritional solution. The dotted bar marked with † represents our previous experience with IA injections, which is why we shortened Phase I in this study. Only the treatment with two IA injections passed on to the next stage. Phase II: A strategy using two IA injections was then evaluated for therapeutic effect in control and IUGR fetuses with nutritional solution. Also, as an extension of the two-injection technique, one IP injection was evaluated for therapeutic effect in Phase II. As in the treatment with two IA injections, we abbreviated Phase I of the treatment with one IP injection owing to our previous experience with ultrasound-guided fetal injections in the experimental setting. IA: intra-amniotic; GD: gestational day; IP: intraperitoneal; IUGR: intrauterine growth restriction.
Figure 2.
Figure 2.
Illustrative images of Strategy 1: Continuous IA infusion by osmotic pump. The diagram presents the procedural flow. (a–e) During surgery on GD25: catheter is inserted into the amniotic cavity using a 14-gauge needle with external guiding sheath (a, b), the guiding sheath is removed (c), and the insertion point is closed with a suture (d). (e) After completing the procedures for IA administration and before returning the uterus into the maternal abdominal cavity with catheters and pumps. (f–j) During cesarean section on GD30: technical failures included the catheter being completely pulled out (f, white arrow), the amnion completely missing around the fetus (g, white arrow), and when the catheter could be fixed in place (h, white arrow) but the tip was outside the amniotic cavity (i, white arrow). The technique was successful when the catheter tip was positioned under the amnion and confirmed to be inside the amniotic cavity (j, white arrow). GD: gestational day. (A color version of this figure is available in the online journal.)
Figure 3.
Figure 3.
Continuous IA infusion by an osmotic pump reaches the fetus and its internal organs. (a) The diagram presents the procedural flow. BrdU solution was continuously infused from the osmotic pump into the amniotic cavity until the fetus was delivered. (b) Cryosections of jejunum and heart immunostained against BrdU were captured at ×20 magnification. Black arrows point to one of the many BrdU-positive nuclei stained in brown. The fetus from which the tissues for the pictures were obtained did not complete the therapy as planned: the catheter connected to the osmotic pump was completely pulled out on GD30, so it was unclear when the last BrdU entered the amniotic cavity. Nonetheless, BrdU had entered the amniotic cavity while the catheter was in place, and was then swallowed and absorbed by the fetus and passed into the fetal circulation. BrdU: bromodeoxyuridine; PBS: phosphate buffered saline; IA: intra-amniotic; GD: gestational day. (A color version of this figure is available in the online journal.)
Figure 4.
Figure 4.
Illustrative images of Strategy 2: Two IA injections and tracing of administered solution. (a) The diagram presents the procedural flow: in Phase I, PBS was administered as the first injection into the amniotic cavity of control fetuses on GD25, and PBS with EB dye was administered as the second injection on GD27; in Phase II, sham or nutritional solution was administered into the amniotic cavity of both control and IUGR fetuses on GD25 and 27. (b) Phase I fetuses immediately after birth on GD30, showing apparent difference in skin color from blue to pale blue to pink. The variability between fetuses is apparent: all fetuses in the picture had received the two-injection therapy in the same way. (c) Macroscopic staining of internal organs (H: heart; L: liver; Je: jejunum): for the three fetuses in (b) that were not completely blue, only the gastrointestinal tract was stained blue (right picture) in contrast to the fetuses that were completely blue inside and outside (left picture). (d) During Phase I, jejunum, liver, and heart from a fetus that had not received any injections (upper row) are compared with those of a completely blue-skinned fetus (lower row). The scale bar on the lower right of each image represents 2 mm. IA: intra-amniotic; EB: Evans blue; GD: gestational day; IUGR: intrauterine growth restriction. (A color version of this figure is available in the online journal.)
Figure 5.
Figure 5.
Illustrative images of Strategy 3: One fetal IP injection. (a) The diagram presents the procedural flow. (b) The fetus within the uterus was gently handled to set its position under the ultrasound probe. Abundant warm gel was applied to the uterine wall while determining the target point of injection under ultrasound view of the fetal IP cavity, and the syringe holder was set in place. (c) The syringe holder was manipulated to move the syringe at a precise angle toward the target point, and IP injection was administered while the fetus was held in place. (d) Representative echography image during fetal IP injection: the white arrow points to where the needle punctures the gestational sac to pass through the amniotic cavity and into the fetal IP space. IP: intraperitoneal; GD: gestational day; IUGR: intrauterine growth restriction. (A color version of this figure is available in the online journal.)
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