Fetal fluid centesis in the bitch

Introduction

Breeders invest much time and effort in designing the best breeding protocols to ensure healthy litters, but there is currently very little information relating to the immediate environment of the canine fetal “unit”. The fetal membranes contain the developing puppy throughout pregnancy and, along with the placenta, play an integral role in fetal growth and progression 1, as they are essential for the metabolic, gaseous, and hormonal exchanges between dam and fetus 2. The fetal membranes of the canine species are the allantois, amnion, chorion and yolk sac 3, but the ones pertinent to fetal fluid sampling in this species are the amnion and the allantois (Figure 1).

What are fetal fluids?

The amniotic cavity provides physical protection for the developing fetus within a liquid medium 4, the amniotic fluid. This is a complex biofluid which allows both mechanical and antimicrobial protection for the fetus as well as lubrication, nutrition, and growth factors, all of which are important for normal development 5. Amniotic fluid is formed with contributions from oral, nasopharyngeal, tracheal and pulmonary secretions, and urine excretion from the conceptus 6.

The allantoic fluid in carnivores accumulates within the allantoic membrane surrounding the amnion 7. The allantoic sac grows to line the chorionic sac almost entirely and persists until parturition. It communicates with the urinary apparatus of the fetus and acts as a waste reservoir for fetal metabolic products 4. The main mechanisms for the initial accumulation of allantoic fluid are probably the transmembrane transport and the secretory activity of the extra-embryonic membranes, and – later in pregnancy – from the mesonephros, metanephros and kidney secretions 8.

As a result, fetal fluid can be collected from both amniotic and allantoic cavities in the dog (Figure 2). Any change in the volume and composition of either fluid may indicate the metabolic, pathologic or developmental status of the fetus at a particular gestational stage 8. Fetal fluid collection in the bitch is feasible from the second half of pregnancy onwards, as there is sufficient fluid and the amniotic and allantoic membranes are discernible on ultrasound scan 9. Centesis is performed using a needle via a transabdominal approach and under continuous ultrasound guidance 10.

Why perform fetal fluid centesis?

The main reason for collecting fluid is to allow ante-partum assessment of fetal viability and detection of disease 11,12,13. Analysis of amniotic biofluid is used extensively in human clinical medicine for diagnosis of chromosomal abnormalities or single gene disorders, as well as for the evaluation of fetal lung maturity, microbial invasion of the amniotic cavity, intra-amniotic inflammation, fetal infections, and gender determination 14,15,16. In veterinary medicine, early detection of abnormalities is important for the welfare of the animals, and is also advantageous for the breeder, both in terms of producing healthy puppies (and keeping the dam healthy), and from a financial perspective. So far, only a few studies have analyzed fetal fluids from at-term bitches 17,18; one case study has shown the feasibility of performing gender analysis from amniotic cells of a singleton canine pregnancy 19. In addition, puncture of the amniotic or allantoic cavities have been used in the canine species for research purposes 20,21, gene therapy and induction of abortion 22, and there is interest in how the technique may contribute towards “One Health” – given that dogs and their owners share the same habitat and are exposed to the same environmental conditions, dogs may make a good model for studying certain environmental effects, such as toxins (Figure 3).

Going forward, fetal fluid collection is essential in order to establish a database to characterize the amniotic and allantoic fluids in healthy pregnant bitches; this will enable the determination of biomarkers that can be used as predictors of perinatal outcome. 

When should centesis be done?

An efficient and viable technique for fetal fluid centesis in pregnant bitches during the second half of pregnancy has been established by the author. The procedure is conducted in a similar fashion to the one used in humans, using guided ultrasound 9. Prior to day 35 post-conception, the volume of fetal fluids in the bitch is low, and it can be assumed that the potential risk for injuring the conceptus is higher. Fetal fluid collection via needle puncture from after day 35 to the end of the pregnancy is expected to be a safe procedure 17,19.

Methodology

A bitch scheduled for fetal fluid sampling should undergo a full general and gynecological examination, including at least urinalysis, blood tests (complete blood count and biochemistry panel) and abdominal ultrasound, in order to ascertain a baseline for her health and the pregnancy status.

Prior to sampling, the abdomen should be clipped and disinfected using povidone iodine surgical scrub solution and 70% ethanol solution. 30-60 minutes prior to the procedure, topical local anesthetic cream (lidocaine/prilocaine) is applied to the skin at a maximum 0.4 mL/kg over the area into which the needles are to be inserted. The treated skin is then covered by an adhesive bandage until collection commences 23.

There are two options for sampling, both of which employ a disposable spinal needle (18-21G x 3½ inch/9 cm).

Freehand technique

This method is performed without a needle guide. The uterine and gestational cavity, as well as the fetus, are first examined via ultrasonography, and a site that appears to be optimal for fluid collection without injuring the fetus or the fetal membranes is selected. At the same time the location of the fetus (in the left or right uterine horn) and the collection site (allantois or amnion) are estimated. The spinal needle is then inserted at an angle of almost 90° through the abdominal wall and advanced firstly into the uterine cavity and then into the gestational sac (amnion or allantois). Both amniotic and allantoic membranes are often visible on the scan, but it is not always possible to identify the exact position of the needle tip (Figure 4). Once the needle is in position, fluid is aspirated using a 3 or 5 mL syringe and immediately transferred to a sterile tube and frozen at -20^oc or -80^oc for future analysis.

Needle-guided technique 

This method involves a needle-guiding device which can be attached to the curved array ultrasound transducer. Prior to collection, the guide must be sterilized in an autoclave, and (when undertaking multiple samplings from one bitch) should be disinfected with 70% ethanol between sampling each gestational sac. The guide is attached to the probe according to the manufacturer’s instructions, and the spinal needle inserted through a small hole in a preset position on the guide (Figure 5). Penetration of the fetal membranes, fluid collection and processing then follow the same procedure as described for the “free hand” technique. 

Using a guide may be a safer and easier option for less experienced operators, although the main drawback is the need to sterilize the apparatus between multiple fetal fluid samplings from different conceptuses in one bitch during the procedure. With experience, the “free hand” technique is preferable for experienced operators.

The amount of fluid retrieved from each conceptus varies between 0.5-5.0 mL, depending on the size of the bitch, the day of pregnancy, and the position of the fetus. Following sampling, the volume of fluid removed should be replaced by an equal volume of sterile 0.9% sodium chloride solution prior to removal of the spinal needle. After collection, the bitch should be kept in a quiet environment for at least 24 hours in order to prevent any possible short-term complications.

What are the risks?

Potential complications of amniocentesis in human medicine include infection of the amniotic sac, induction of preterm labor, respiratory distress in the mother, fetal deformities, alloimmunization, and failure of the puncture wound to heal properly 24. Although very uncommon in human medicine, fetal injury can occur secondary to trauma from the needle. To date, the author has not detected any macroscopic fetal injury after performing centesis in bitches, although it is possible that microscopic injuries may occur – it is not impossible that some microscopic puncture sites in the uterus, fetal membranes or in the fetus itself have gone undetected, although it can be assumed that if they had occurred they were not of clinical relevance 9.

Conclusion

Although still in its infancy, fetal fluid collection in the bitch is a technique that can offer various benefits to small animal veterinarians, breeders and owners alike in the years ahead. A skilled clinician should be able to collect either allantoic or amniotic fluid without too much difficulty and with minimal risk to the conceptus. Analysis of the fluid should assist in early detection of abnormalities and gene disorders, placental defects, microbial infection of the dam or fetus, and gender determination.