MATERIALS AND METHODS

Ethical approval

This retrospective study was reviewed and approved by the Animal Research Ethics Committee of Mahanakorn University of Technology, Bangkok, Thailand (Approval No. ACUC-MUT-2025/012). Because this study was entirely based on the review of pre-existing clinical records and routine reproductive data, all procedures were conducted in strict accordance with institutional guidelines for animal welfare and ethical standards for veterinary clinical research. Written informed consent had been previously obtained from all owners through signed participation agreements permitting the use of their animals’ medical and reproductive data for research purposes.

Study period and location

The study was conducted from January 2024 to December 2025 at the Clinic of Obstetrics, Gynecology and Animal Reproduction, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok, Thailand. Clinical records from pregnancies managed at the referral clinic during the study period were retrospectively reviewed. Cases originated from privately owned breeding dogs that underwent routine reproductive management and pregnancy monitoring.

Study design

This study consisted of a retrospective review of clinical records from 25 bitches comprising 29 pregnancies diagnosed with hypoluteodism. The study population included three breeds: Shetland Sheepdog (SS; n = 21; 24 pregnancies), Miniature American Shepherd (MAS; n = 2; 3 pregnancies), and Pomeranian (Pom; n = 2; 2 pregnancies). The population was heavily skewed toward SS, suggesting a potential breed predisposition.

The mean age of the cohort was 4.2 ± 1.8 years (range: 2–8 years), with a mean parity of 1.5 ± 0.8 previous litters. Notably, 80% (20/25) of bitches had a documented history of at least one prior unexplained pregnancy loss. Body weights ranged from 2.4 to 18.2 kg (mean 9.8 ± 2.4 kg), allowing precise calculation of the MPA dosage for each individual. Breeding management was standardized, and all bitches underwent fresh-semen artificial insemination (n = 29) following ovulation confirmation.

Monitoring and diagnosis criteria

Ovulation timing was determined using serial P4 measurements, with ovulation defined as the point at which concentrations reached 4–10 ng/mL. After mating was confirmed, pregnancy status and fetal development were monitored using transabdominal ultrasonography [15–17].

Surveillance for hypoluteodism was conducted through weekly or biweekly serum P4 assessments beginning during the fourth week of gestation. Hypoluteodism was diagnosed based on two criteria: (1) a decline in serum P4 to <10 ng/mL or (2) a rapid decrease of >5 ng/mL within a 48–72-h interval, even when the absolute value remained slightly above the traditional threshold of 5 ng/mL.

All P4 measurements were performed using a Vcheck V200 analyzer (Bionote Co., Ltd., Hwaseong, South Korea), a validated point-of-care fluorescent immunoassay showing a high correlation with reference laboratory chemiluminescence methods [15]. Subsequent references to the analyzer are designated as Bionote.

Therapeutic and safety protocol

When luteal insufficiency was diagnosed, pregnancy maintenance therapy was initiated using MPA (Provera®, Pfizer Inc., New York, NY, USA). Treatment commenced immediately after the detection of subthreshold P4 concentrations, as indicated by the asterisks in Table 1. A base dosage of 0.1 mg/kg once daily was administered orally, with adjustments made according to the clinical response when necessary.

During treatment, dams were monitored every 2 weeks using transabdominal ultrasonography to assess fetal heart rates (target >200 beats/min) and placental integrity. Maternal safety was evaluated through physical palpation of the mammary chains to detect mammary hyperplasia or fibroadenomas.

A key refinement of this study was the implementation of a standardized withdrawal protocol. To support appropriate fetal maturation and avoid excessively prolonged gestation, a recognized limitation of long-acting progestins, MPA treatment was discontinued 1–2 days before the planned cesarean section. The procedure was scheduled at 63 ± 1 days after ovulation, corresponding to the expected gestational length in dogs. This timing was intended to align with the pharmacokinetic profile of oral MPA in canines, thereby allowing sufficient clearance of exogenous progestins before surgical intervention.

Monitoring of maternal and fetal safety

Following the initiation of MPA treatment, dams were monitored weekly for evidence of insulin resistance using urine glucose dipsticks and for mammary gland abnormalities. Neonatal safety was assessed immediately after cesarean section.

A systematic physical examination was performed to identify congenital abnormalities, including facial clefts, and signs of virilization in female puppies, such as clitoral hypertrophy or reduced anogenital distance. Neonatal viability was monitored until weaning at 6 weeks of age.

Statistical analysis

Data collected included ovulation-phase P4 concentrations, timing of hypoluteodism onset, P4 concentrations at diagnosis, pregnancy outcomes (abortion versus live birth), litter size, and sex distribution. Descriptive statistical analyses were applied to characterize temporal patterns of luteal decline and evaluate the clinical effectiveness of the treatment protocol.