RELAXIN AS A MARKER OF PREGNANCY IN CANINES

Pashu Sandesh, 02 July 2018

Dr. Mohit Gautam, Dr.Irfan Ahmad Bhat, Dr. Sriti Pandey, Dr Abhishek Rajput

Relaxin is a heterodimeric polypeptide, a member of the insulin-like superfamily and a hormone of pregnancy in numerous species shown to affect the reproductive tissues of mammals, most commonly“cervicalextensibilityanduterinecontractibility”.In the dog, relaxin is undetectable in the serum of cycling, pseudopregnant or hysterectomized bitches and male dogs. Thesourceofrelaxin synthesis varies among species, but the predominant sites are the corpus luteum, placenta, and uterus. Depending on the species, detection of relaxin in peripheral blood is not always restricted to pregnant females; however in the domestic dog it has been established as a pregnancy-specific hormone. The site of synthesis in the bitch has been elucidated and primarily ascribed to the placenta, although the hormone can also be traced in the ovary and uterus. These latter tissues may be areas influenced by the paracrine deposition of relaxin.  

Many mammalian species are facing extinction due to problems created by human encroachment, agriculture, pollution, and willful slaughter. Conservation groups try to save species in the wild by preserving habitat and limiting animal-human conflicts, often with limited success. Another alternative is to preserve the extant gene pool through captive breeding as a hedge against extinction. Measurement of circulating reproductive hormones is impractical for most wildlife species; determination of urinary or fecal hormone metabolites provides a more viable approach. The serum relaxin reliably distinguishes between pregnancy and pseudopregnancy in bitches, relaxin measurement might also provide a method for detecting a successful pregnancy in endangered species. Appropriate immunoassay reagents have enabled the estimation of relaxin concentrations in the serum of elephants and rhinos and the determination of pregnancy establishment and the outcome. Relaxin was also detected in panda serum and urine. However, the extreme variability of the time between observed mating and parturition and the confounding factors of delayed implantation, pseudopregnancy, and frequent fetal resorptions made it impossible to use the panda relaxin data as a specific marker of pregnancy.

Current methods of pregnancy diagnosis

In the bitch, Physical examination, vaginal exfoliative cytology  and ultrasonography remain the most common and effective method of pregnancy diagnosis at about 3-4 weeks of gestation.Abortion is often difficult to diagnose as the bitch rarely show well defined clinical signs (often no signs at all or just a short lasting malaise or anorexia). Further, the clinical signs of pseudo pregnancy can confuse even an experienced breeders/clinician about the pregnancy. 

Relaxin as pregnancy specific protein

• During the luteal phase, or “diestrus,” where the hormone progesterone is produced at elevated levels, whether or not the animal is pregnant. Hence, progesterone may not be a useful hormone for pregnancy diagnosis in bitches.
• Prolactin and LH due to diurnal and stress related variations are unreliable for pregnancy diagnosis.
• There are reports of elevated levels of acute phase proteins in pregnancy but elevated acute phase protein levels are also seen in most instances of inflammatory diseases and tumor cases.
• No pregnancy specific proteins are reported in bitches except relaxin. There are several reports of peptide hormone called relaxin to be exclusively produced in pregnant bitches and not by non-pregnant and pseudo pregnant bitches.

During the approximately 60 days of gestation, immunoreactiverelaxin is first detected in the serum by day 18 of gestation and exhibits the highest serum relaxin concentrations of any species reported to date, reaching peak levels at 6–8 wk of gestation.Relaxin is regarded as a specific marker of pregnancy in dogs. Upon parturition or shortly before spontaneous abortion, relaxin serum concentrations decrease to undetectable levels, implicating relaxin as a potentially useful indicator for monitoring the onset of parturition in this species.Also, the amino acid sequence of the A and B domains of canine relaxin have been determined. Employing solid-phase peptide synthesis and sequential site-directed disulfide bond formation, we have produced synthetic bioactive dog relaxin, but the procedure proved to be difficult and inefficient, yielding only low amounts of relaxin.

Structure of relaxin

Relaxin is a 6 kDa polypeptide hormone with structural homology to insulin and insulin like growth factors. The amino acid sequence of relaxin is highly variable with homology between species is ranging from 38% to 98%. The structural variation is that much it results in minimal or no cross reactivity with antisera rised in other species. It has two chains A & B with 24 & 35 residues, respectively and connected by interchain (two) and intrachain (one) disulfide bonds. The amino acid motif Arg-X-X-X-Arg-X-X-Ile present in B chain necessary for bioactivity of relaxin.

Source of relaxin

The major sources of circulating relaxin is different among species.

• Corpus luteum in case of mouse, rat, human, pig, and cow.
• Placenta in hamster, rabbit, cat and mare.
• Endometrial glands of uterus in guineapig.
• Little amount is produced from testis and prosrtate in males.
• Non reproductive organs also produce relaxin- kidney and heart.
• In the dog, placenta and ovary are reported to secrete relaxin  with the placenta being the major source.

Function of relaxin

• Relaxin causes extensibility of connective tissue of cervix and pelvic ligament
• Remodeling of uterine stroma and vasculature & production of factors that promote trophoblast adherence to & invasion of endometrium.
• Inhibition of myometrial contractility.
• Stimulatory effect on the follicular growth and CL function.
• In the rat, RLX promotes uterine growth and softens the uterine cervix before labor.

Relaxin in pregnancy

A RIA for relaxin that uses an antiserum against synthetic canine relaxin was validated for felid urine and shown to detect relaxin immunoreactivity in pregnant cat urine subjected to acid-acetone extraction. In the cat, urinary relaxin was first detected between days 21 and 28 of gestation; levels peaked at 42–49 days, and the concentrations then declined over 2 wk prior to parturition. The urinary relaxin profiles of the cat mirrored those in serum. In the leopard, urinary relaxin was first detected at day 25–28 of gestation; levels peaked at day 60–64 and declined in the last 3–4 wk of pregnancy. These results indicate that measurement of urinary relaxin in the cat and leopard provides a reliable method for pregnancy determination from as early as 3–4 week of gestation. This method of pregnancy diagnosis and monitoring may prove useful in the breeding management of domestic cats and other felid and canid species, and provides a foundation for future studies on pregnancy in captive exotic carnivores.

The diagnosis of pregnancy in the domestic dog (Canisfamiliaris) often employs specialized equipment, experienced staff, and the cooperation of the bitch. These procedures can be challenging when the subject is a wild canid, particularly in a field setting. In addition, reproductive hormone assays are unreliable as a diagnostic tool because the estrous profiles of pregnant and pseudopregnant canines are similar. However, research has demonstrated that the hormone relaxin can be detected in maternal blood after embryonic implantation, but remains negligible in non-pregnant females.

Relaxin kit available in market

Canine pregnancy test kit (symbiotics)

• Early detection of planned or unplanned pregnancy
• Early detection of false pregnancy
• No refrigeration required
• Detect relaxin hormone in biological samples as early as 22 – 27 days after implantation of a fertilized egg for detection of pregnancy.
• A small blood sample will provide almost instant results

FASTestRelaxin-

• Rapid immune-chromatographic assay for the detection of relaxin in the plasma or serum of dog and cat.
• Offers a simple, reliable, inexpensive means of early pregnancy detection
• An indicator for a pregnancy and for monitoring pregnancy status, if there is concern about spontaneous abortion or to exclude pseudopregnancy.