RESULTS

Demographics and onset of luteal insufficiency

A total of 29 pregnancies were monitored in this cohort. Initial luteal function appeared clinically adequate in all subjects, as reflected by post-mating serum P4 concentrations measured 1–2 days after ovulation. Values ranged from 11.23 to 19.37 ng/mL.

The onset of hypoluteodism, defined as a decline in P4 requiring exogenous P4 supplementation, varied among individuals but followed a recognizable temporal pattern. Most cases demonstrated a critical decline in P4 concentrations between weeks 5 and 6 of gestation (Table 1).

Case IDᵃ | Breed | Baseline P4 (ng/mL) Ovulation | Baseline P4 (ng/mL) 1ˢᵗ mating | Diagnosis of hypoluteodism ᵇ Onset week | Diagnosis of hypoluteodism ᵇ P4 level (ng/mL) | Management MPA withdrawalᶜ | Outcome | Neonatal defects/ virilization
D1 | SS | N/A | 17.00 | 6 | 7.86 | −2 days | Live birth(3M, 3F) | None
D2 | SS | N/A | 19.37 | 5 | 7.34 | −1 day | Live birth(4M) | None
D3 | SS | 7.83 | N/A | 6 | 8.68 | −2 days | Live birth(3M, 6F) | None
D4 | SS | 7.66 | N/A | 6 | 4.43 | – | Abortion atWk 8 | N/A
D5-Aᵈ | SS | 8.31 | N/A | 5 | 8.70 | – | Abortion atWk 8 | N/A
D5-Bᵈ | SS | 6.32 | N/A | 5 | 7.80 | – | Abortion atWk 8 | N/A
D5-Cᵈ | SS | 8.95 | N/A | 4 | 7.43 | −1 day | Four mummified fetuses | None (mummified)
D6 | SS | 7.55 | N/A | 6 | 8.62 | −2 days | Live birth(2M, 3F) | None
D7 | SS | 5.18 | N/A | 7 | 5.40 | −2 days | Live birth(2M, 1F) | None
D8 | SS | N/A | 12.04 | 7 | 5.07 | −2 days | Live birth(3M, 1F) | None
D9 | SS | 7.51 | 12.08 | 5 | 7.06 | −1 day | Live birth(2M, 2F) | None
D10-Aᵈ | MAS | N/A | 12.54 | 5 | 8.80 | −1 day | Live birth(3M, 3F) | None
D10-Bᵈ | MAS | N/A | 12.20 | 7 | 5.65 | −2 days | Live birth(3M, 4F) | None
D10-Cᵈ | MAS | 10.50 | N/A | 5 | 6.53 | −2 days | Live birth(3M, 2F) | None
D11 | SS | 6.98 | N/A | 5 | 8.70 | −1 day | Live birth(2M, 1F) | None
D12 | SS | N/A | 13.32 | 5 | 7.70 | −1 day | Live birth(4F) | None
D13 | SS | 9.14 | N/A | 6 | 9.91 | −2 days | Live birth(4M) | None
D14 | SS | 6.75 | N/A | 5 | 8.12 | −1 day | Live birth(4M, 2F) | None
D15 | SS | N/A | 14.20 | 5 | 8.05 | −1 day | Live birth (2F) | None
D16 | SS | 8.76 | N/A | 5 | 7.05 | −2 days | Live birth(3M, 2F) | None
D17 | SS | 8.13 | N/A | 5 | 9.87 | −1 day | Live birth(2M, 1F) | None
D18 | Pom | 7.67 | N/A | 5 | 7.85 | −2 days | Live birth (1M) | None
D19 | SS | N/A | 11.79 | 7 | 5.35 | −2 days | Live birth(3M, 3F) | None
D20 | Pom | 5.09 | N/A | 6 | 8.51 | −2 days | Live birth(1M, 1F) | None
D21 | SS | 10.71 | N/A | 6 | 8.90 | −2 days | Live birth(4M, 1F) | None
D22 | SS | 7.53 | N/A | 4 | 7.85 | −2 days | Live birth (1M) | None
D23 | MAS | N/A | 12.55 | 5 | 9.70 | −1 day | Live birth(3M, 1F) | None
D24 | SS | 9.41 | N/A | 5 | 8.52 | −2 days | Live birth(3M, 2F) | None
D25 | SS | N/A | 11.23 | 5 | 7.57 | −1 day | Live birth(4M, 2F) | None

Among the monitored pregnancies, luteal insufficiency was detected in 6.9% (2/29) of cases during week 4. The highest incidence occurred during week 5, affecting 55.2% (16/29) of pregnancies, followed by week 6, in which 24.1% (7/29) of cases required treatment. Late-onset luteal insufficiency during week 7 was observed in 13.8% (4/29) of pregnancies. Overall, most cases (79.3%) exhibited a critical decline in P4 concentrations between weeks 5 and 6 of gestation, demonstrating a highly consistent temporal pattern of luteal failure.

Treatment efficacy and pregnancy outcomes

The MPA protocol maintained pregnancy to term in 86.2% (25/29) of cases. Longitudinal P4 profiles showed a mean decline from 15.3 ± 2.1 ng/mL at week 4 to 7.8 ± 1.2 ng/mL at the time of diagnosis during weeks 5–6. All successful pregnancies resulted in healthy puppies delivered by elective cesarean section according to standardized obstetrical protocols.

Litter characteristics showed considerable variability, with litter sizes ranging from 1 to 9 puppies and an average of approximately 4.4 puppies. The sex distribution was male-biased, comprising 63 M and 47 F.

Notably, the planned withdrawal of MPA 1–2 days before elective cesarean section in all 25 successful pregnancies resulted in the absence of premature labor or fetal compromise. These findings provide systematic evidence supporting the safety and effectiveness of this timed-withdrawal approach in a relatively large cohort.

Reproductive failures

Reproductive failure was documented in 13.8% (4/29) of the monitored pregnancies despite routine P4 surveillance and timely clinical intervention.

Abortion: Three pregnancies (subjects D4, D5-A, and D5-B) resulted in abortion. In these cases, serum P4 concentrations declined to 4.43, 8.70, and 7.80 ng/mL during weeks 6, 5, and 5 of gestation, respectively, coinciding with pregnancy loss.

Fetal mummification: Subject D5-C experienced an additional reproductive failure characterized by four mummified fetuses. This outcome occurred despite the early initiation of MPA treatment during week 4, when the P4 concentration had decreased to 7.43 ng/mL.

Neonatal outcomes and maternal safety: Among the 25 successful pregnancies, a total of 110 puppies (63 M and 47 F) were born with birth weights consistent with breed standards, and 94.5% (104/110) survived until weaning. No maternal complications, including clinical diabetes mellitus, mammary hyperplasia, or lactation failure (agalactia), were documented, indicating overall reproductive success.

However, one subject (D5-C) experienced fetal mummification during week 4. Although this event could not be definitively attributed to MPA administration, it may have reflected an unfavorable uterine environment.

In addition to survival outcomes, all neonates were assessed using a modified Apgar score at 0 and 5 min after delivery. Overall, 92% of puppies achieved scores >7, indicating satisfactory neonatal health. Mean birth weights were consistent with breed standards, with SS puppies weighing 185 ± 25 g on average.

Detailed perineal examinations of the 47 female puppies revealed no clitoral hypertrophy, reduced anogenital distance, or other signs of virilization. Follow-up evaluations until weaning at 6 weeks of age showed no evidence of MPA-associated congenital abnormalities, including facial clefts, as reported in previous high-dose studies.

Maternal follow-up revealed that all successful dams resumed normal estrous cycles within 5–7 months after whelping. Furthermore, no cases of secondary insulin resistance or mammary tumors were observed during the 6-month postpartum period.