Introduction
Anti-Müllerian hormone (AMH), secreted by the fetal testis to direct male reproductive differentiation, is also secreted by ovarian granulosa cells postnatally, though at much lower concentrations than those seen in males.1 Assessing AMH has become a valuable diagnostic tool in veterinary practice for determining gonadal status, specifically ovarian remnant syndrome and cryptorchidism in dogs2,3 and other species.4 In females, AMH is generally considered to be secreted at a fairly constant rate in a variety of species. Compared with other gonadal hormones, especially those secreted by granulosa cells such as inhibin-A and inhibin-B,5 AMH concentrations vary little by cycle stage on the basis of observations in mares,6,7 cows,8,9 and women.10,11 Anti-Müllerian hormone declines significantly only with age and reproductive senescence.12,13 The relative secretory consistency of AMH is likely because the primary source of the circulating hormone in these species is periantral (pre- and early postantral) ovarian follicles.1,14 Periantral follicle populations are recruited continuously and independent of gonadotropic influence, and their numbers vary relatively little with cycle stage.15 This contrasts with the larger, more gonadotropin-dependent follicles that grow and regress with cycle stage. These follicles secrete and largely determine systemic estrogen concentrations but are generally considered to contribute little to systemic AMH in livestock.1 However, reproductive cycle characteristics and physiology differ markedly among species.16 Notably, although the bitch ovulates spontaneously,17 as livestock and many rodents do,18 both canine proestrus and estrus are prolonged in comparison to many species, each lasting up to 9 to 10 days or more in the bitch.19 Estrus is sometimes almost as long in domestic queens, which are inducible ovulators.20,21 Given such differences in cyclicity, perhaps it should not be surprising that, in contrast to the fairly steady secretion seen in other species investigated to date, AMH concentrations can fluctuate substantially during estrous cycles of both bitches and queens.22–25 Inhibins are additional secretory products of maturing follicles that vary cyclically in some species,5 and vary closely with AMH in queens,22 but have never been investigated in cyclic bitches.
In clinical contexts, it is especially relevant to recognize that, in addition to apparent differences in secretory profiles of AMH among species, different immunoassay platforms exhibit varying degrees of specificity across species as well as in the sensitivity of AMH detection.2,26,27 Apparent concentrations (assay estimates) of AMH in the same samples from whatever species can also vary markedly, depending in part on the calibrators used in the assay standard curve.26 This can compromise comparisons of data from published studies (in any particular species) in which different assay platforms have been employed. To the best of our knowledge, only 1 commercially available AMH immunoassay utilizes homologous canine AMH calibrators at this point in time, an innovation that was incorporated into this assay platform early in 2020. This refinement rendered obsolete previously published population-based reference intervals (RIs) for intact bitches in breeding programs utilizing an earlier version of this commercial platform28 because they were established with nonhomologous, rat promature AMH calibrators.29
With these considerations in mind, the primary goals of the current studies were 3-fold: (1) to establish statistically validated, normal RIs30 for AMH in intact and neutered bitches by use of population-based sampling and in a small number of females before and after ovariohysterectomy (OVH); (2) to examine fluctuations in AMH concentrations in samples taken longitudinally during the clinical management of breeding cycles, together with inhibin-B and progesterone to better characterize the magnitude of possible change during cycles; and (3) to retrospectively analyze clinical data on the likely existence of ovarian tissue in samples from bitches that were submitted for assessment of their gonadal status. It was hypothesized that there would be a significant decline in AMH as bitches progressed from proestrus to estrus and, based on the results of studies of cats,22 AMH and inhibin-B would be positively correlated during canine breeding cycles. Archival clinical data were evaluated in the context of the newly established population-based normal RI in intact and spayed bitches to better interpret information for clinical case submissions in practice.
Methods
Animals
Residual serum was recovered from samples submitted for routine, presurgical blood chemistry analysis taken from 102 intact bitches prior to undergoing OVH at the University of California-Davis School of Veterinary Medicine between December 2021 and November 2022. All were determined to be healthy at the time of sampling. Additional residual samples were obtained from 78 reportedly spayed bitches (for analyses unrelated to reproduction) that were submitted to the central clinical pathology laboratory at the Colorado State University College of Veterinary Medicine. Information on the mix of breeds, age, and body weight was often unavailable and therefore incomplete. Samples were received between December 2022 and October 2023, stored at 4 °C for 72 hours, then frozen and stored at –20 °C or below until assayed. A third set of residual sera included longitudinally collected samples from 24 bitches, 4.5 (1.0) samples per bitch, in proestrus and estrus, undergoing routine assessment of progesterone concentration for breeding management during normal estrous cycles from March to October, 2023. Finally, paired serum samples were collected before and (a variable number of days) after surgery from 8 bitches undergoing OVH from May 2019 to January 2020 at the Society for the Prevention of Cruelty to Animals of Tompkins County on behalf of the Cornell University College of Veterinary Medicine. The samples collected at each site, with the exception of those noted earlier, were stored at 4 °C until aliquots were removed, frozen, and stored at –20 °C until they were analyzed. Save for the samples collected at Cornell, only residual serum samples were utilized, and therefore they were exempt from IACUC approval. The Cornell samples were collected under IACUC protocol No. 2007-0146.
Assays
Serum was analyzed for AMH with the use of a canine-specific ELISA, revised in 2020 to include homologous canine AMH calibrators, following the manufacturer’s recommended protocol (AMH [canine/feline] ELISA; Ansh Labs). The platform uses a double monoclonal sandwich with a reported sensitivity of 0.015 ng/mL (mean + 2 SDs of 16 replicates of the negative control). The intra- and interassay coefficients of variation (CVs) were 6.2% and 4.1% (n = 18 and 26 replicates, respectively) for a kit control sample included in all assays of 4.6 ng/mL (0.3 ng/mL). Samples from a canine serum pool measuring 0.25 ng/mL (0.07 ng/mL) that were also included in all assays yielded an intra-assay CV of 7.1% and interassay CV of 3.9% (n = 19 and 223 replicates, respectively). Inhibin-B was assessed by ELISA (inhibin-B [equine/canine/mouse] ELISA; Ansh Labs), an assay previously validated and used in studies of horses5 and cats22 with a sensitivity of 2.3 pg/mL and intra- and interassay CVs of 1.8% and 12.8% (n = 18 and 26, respectively). All AMH and inhibin-B assays were conducted in the University of California-Davis Clinical Endocrinology Laboratory. If duplicates differed by > 10%, the samples were assayed again. Progesterone concentrations were determined in the clinic by one of the authors (BWC) using an automated immunoassay analyzer (AIA-360; Tosoh Bioscience Inc) according to the manufacturer in samples from bitches presented for breeding management.
Statistical analysis
The 95% RIs for AMH were determined with the Reference Value Advisor plug-in for Excel (Microsoft Corp)31 as recommended30 after Box-Cox transformation of the data with the nonparametric method, first on those collected from intact bitches and subsequently on samples from the reportedly spayed group. Outliers were identified with Dixon-Reed and Tukey tests.31 These were retained in the final analysis of the intact group, absent of any rationale to exclude them. Outliers identified in the spayed group were reanalyzed to confirm the results. Concentrations of AMH in pre- and post-OVH sample pairs were analyzed with a paired t test. Results from 3,193 clinical submissions, received and assayed between September 2021 and July 2024, by use of the same assay platform with canine calibrators, were also reassessed retrospectively with RIs determined from the intact and reportedly OVH sample groups. Samples submitted for any investigation of suspected reproductive conditions were excluded. Each sample represented a single bitch and was not known to be represented more than once in the data subjected to analysis.
The longitudinal sample sets from bitches presented in proestrus for breeding management through estrus were aligned to the day that progesterone concentrations exceeded 3 ng/mL,32 arbitrarily designated day 0, and then analyzed for AMH and inhibin-B concentrations as a function of time (mixed model with bitch as a random factor; lmer function; The R Project for Statistical Computing).33 Hormone concentrations were log (AMH and progesterone) or arcsin (inhibin-B) transformed to improve the homogeneity of variance and normalize the data before further analysis as appropriate, and means were represented graphically without transformation. Correlations between AMH and inhibin-B were calculated with untransformed data. Sample days and frequency varied, but each bitch contributed at least 3 consecutive samples until they were inseminated and ovulation was confirmed by further elevated progesterone concentrations. All were confirmed to be pregnant subsequent to that breeding.
Results
The population-based RIs of AMH and inhibin-B in intact and owner-reported OVH bitches (n = 102 and 78 samples, respectively) are shown in Table 1. The mean (SD) of AMH concentration in intact females was 1.32 ng/mL (1.55 ng/mL). There were 2 outliers identified statistically in the final dataset (4.39 and 14.0 ng/mL). The lower limit of the intact RI was calculated to be 0.11 ng/mL. The vast majority of samples in the presumptive OVH group had undetectable concentrations of AMH, nominally assigned the concentration of the negative control, 0.02 ng/mL. The analysis of OVH samples also identified 7 statistical outliers with high AMH concentrations of 0.30 ng/mL (0.20 ng/mL) that were above the lower limit of the RI for intact bitches. Reference intervals determined by use of data from the OVH group that included the outliers overlapped considerably with the intact group (0.11 to 0.38 ng/mL). Excluding the outliers from the OVH data established RI that did not overlap. Inhibin-B was detectable in only 10% of samples in both the intact and OVH groups of samples so that population-based RIs could not be determined for either group.
Canine anti-Müllerian hormone (AMH; ng/mL) 95% reference intervals (RIs) were determined with the use of serum samples from intact bitches between December 2021 and November 2022 and reportedly spayed bitches between December 2022 and October 2023.
| Including outliers in spayed group | Excluding outliers in spayed group | |||
|---|---|---|---|---|
| Gonadal status | AMH (ng/mL) | Sample No. | AMH (ng/mL) | Sample No. |
| Ovarian tissue | 0.11–3.91 | 102 | 0.11–3.91 | 102 |
| Inconclusive | 0.11–0.38 | 0.05–0.10 | ||
| Spayed | 0.02–0.38 | 78 | 0.02–0.04 | 71 |
Nonparametric intervals were calculated from raw concentration data with Box-Cox transformation in Reference Value Advisor in Excel (Microsoft Corp). Statistical analysis identified 7 outliers with apparently elevated AMH concentrations in the spayed group. Reference intervals were established with outliers included in the calculation (second and third columns), in which case the concentration overlap between the groups was designated as inconclusive. Estimated intervals, after being excluded from the calculation, did not overlap (fourth and last columns); in this case, concentrations exceeding the spayed range but that were less than the lower limit of the intact range were designated as inconclusive.
The concentrations of AMH in bitches before and after OVH were also assessed (Supplementary Table S1). The AMH concentrations in pre- and postsurgical samples (n = 8 sample pairs) were 1.75 (0.97) ng/mL and 0.18 (0.19) ng/mL (P < .001), declining nearly 10-fold after undergoing OVH. The concentrations of AMH decreased in all cases after OVH regardless of the time interval between surgery and the postsurgical sampling date. Pre- and postsurgery AMH concentrations were significantly correlated (P = .014; r = +0.81). For example, the highest presurgical AMH concentration was also the highest after surgery (3.6 and 0.67 ng/mL, respectively). However, there was no significant correlation between postsurgery AMH concentrations and the number of days elapsed before the postsurgical sample was taken.
Records were analyzed retrospectively on 3,193 submissions from bitches for which AMH testing was requested to aid in determining gonadal status with the intervals established for intact and OVH groups and expressed as a percentage of the total. Applying the RI determined on all samples in the intact and OVH groups, including the OVH outliers, resulted in classifying 1,888 of 3,193 (59% [37% + 7% + 15%]) of submitted samples as spayed and 1,305 of 3,193 (41% [32% + 9%]) as having evidence of ovarian tissue (Table 2). Defining intervals after excluding the outliers resulted in classifying 1,180 of 3,193 (37%) samples as spayed, 1,787 of 3,193 (56%) as having ovarian tissue, and 226 of 3,193 (7%) as inconclusive.
Retrospective analysis of results of AMH concentrations (ng/mL) determined in 3,193 samples from individual bitches using RIs on the basis of analysis of verified intact and presumptively ovariohysterectomized (OVH) bitches.
| AMH concentration intervals (ng/mL) | No. of submissions | Percentage of total (n = 3,193) |
|---|---|---|
| 0.02–0.04 | 1,180 | 37.0 |
| 0.05–0.10 | 226 | 7.1 |
| 0.11–0.38 | 482 | 15.1 |
| 0.39–3.9 | 1,014 | 31.8 |
| ≥ 4.0 | 291 | 9.1 |
| Total | 3,193 | 100 |
The results utilized in the retrospective analysis were from sera submitted to the Clinical Endocrinology Laboratory between September 2021 and July 2024. Anti-Müllerian hormone RIs were established from 102 intact (0.11 to 3.91 ng/mL) and 78 reportedly OVH (0.02 to 0.38 ng/mL) bitches that overlapped (0.11 to 0.38 ng/mL) because of high concentrations detected in 7 statistical outliers. Excluding those 7 outliers in the OVH group (with AMH concentrations in the intact interval) narrowed the OVH interval for AMH (0.02 to 0.04 ng/mL) that did not overlap with the intact interval. This established a nonoverlapping interval of inconclusive results (0.05 to 0.10 ng/mL). Several submissions had AMH concentrations that exceeded the upper limit of the intact interval (≥ 4.0 ng/mL). The number of submissions within the intervals so defined are presented as a percentage of the total number analyzed.
Analysis of AMH in bitches during breeding cycles demonstrated that mean concentrations varied significantly over days in each individual bitch (P < .001; Figure 1). Despite being undetectable in the vast majority of the samples from intact and neutered females that were used in RI determinations, inhibin-B was readily detectable in the proestrous and estrous samples from breeding bitches. Consequently, data on both AMH and inhibin-B were aligned to the day progesterone concentrations reached 3 ng/mL (denoted day 0). After alignment, AMH concentrations decreased from a peak of 1.2 ng/mL on day –8 to a nadir of just under 0.4 ng/mL on day +2, rising steadily thereafter to over 0.7 ng/mL on day +6. Similarly, and in concert with AMH, inhibin-B concentrations decreased from around 27 pg/mL on day –7 to just over 10 pg/mL on day +1. Thereafter, inhibin-B increased again transiently, then declined to about 10 pg/mL on day +6. Concentrations of AMH and inhibin-B were positively correlated over this period (r = +0.58; P < .001).
Mean anti-Müllerian hormone (ng/mL) and inhibin-B (pg/mL) concentrations measured in serum from 24 bitches sampled longitudinally over days before and after insemination during breeding cycles managed between March and October, 2023. The graphed concentrations represent means normalized to the initial rise in mean progesterone (> 3 ng/mL) designated at day 0. All breedings resulted in established pregnancies.
Citation: Journal of the American Veterinary Medical Association 263, 2; 10.2460/javma.24.06.0405
Discussion
The utility of AMH as a diagnostic aid in determining gonadal status in female dogs and cats is hampered in the absence of reliable, truly population-based RIs. Valid RIs for reproductive hormones in females are particularly difficult to establish because of the major physiological shifts associated with puberty, cyclicity, and pregnancy,17 apart from day-to-day pulsatile and even diurnal secretory rhythms.34 Moreover, clinical submissions often lack accurate information on the age and breed of animals, as well as their likely reproductive stage or state. This is especially problematic in small animal practice because, unlike other studied species,6–11 AMH concentrations vary with cycle stage23,24 (and breed28) in the bitch and in queens.22 The current studies were conducted to reestablish population-based RIs for intact and, in addition, OVH bitches with a commercial canine AMH platform that recently incorporated homologous canine AMH calibrators. Concentrations of AMH and inhibin-B were also determined from a large number of bitches sampled longitudinally during managed breeding cycles normalized to progesterone. This enabled a more definitive estimate than has been reported previously23,24 of the degree to which concentrations of AMH can vary within cycles or with cycle stage. Redefining RIs and better defining cyclic variation reaffirms the optimum time for sampling and facilitates the interpretation of AMH concentrations in determining the gonadal status of bitches in which cycle stage remains unknown.
To date, the report by Hollinshead et al28 is the only one to have established a valid RI for intact bitches from a large population. The concentration intervals reestablished in the current study with homologous canine AMH calibrators were markedly lower, by 10-fold, than those reported previously. This serves to emphasize once again how alternative assay platforms that rely on different calibrators will generate different apparent hormone concentrations.26,27 Whether results from different platforms are proportional across the entire working range of concentrations depends on the linearity of the calibration curves of the assays in question. Results cannot be compared directly between studies in which different assay platforms have been employed. The earlier study by Hollinshead et al28 had the benefit of extensive information on breed, age, and even parity, enabling detection of an age-related decline in AMH as well as a significantly lower average AMH concentration in giant versus other canine breeds. No similarly complete information was available for analysis of the samples comprising the current study. However, a large sampling of reportedly OVH bitches enabled the determination of RIs for both intact and OVH populations and thereby inconclusive intervals, also for the first time. On the basis of these intervals, a retrospective analysis of clinical samples indicated that more than half of those submitted to determine gonadal status have evidence of the presence of gonadal tissue.
Anti-Müllerian hormone has several unusual characteristics as a reproductive biomarker. It is a large homodimeric glycoprotein hormone but, unlike luteinizing hormone and follicle-stimulating hormone, is not secreted in a pulsatile fashion and is not a component of the hypothalamic-pituitary feedback axis.4 This explains in part the relative stability of AMH concentrations with cyclicity or pregnancy, at least in large animals.6,8,9 Stable secretion and lack of feedback are also why AMH has been used as a proxy of ovarian follicular reserve35 and why AMH concentrations almost exactly halve after hemiovariectomy in young and old mares.13 This suggests that AMH concentrations likely reflect not only follicle populations but also perhaps the amount of actual ovarian tissue remaining if ovariectomy is not complete. That implies the smaller an ovarian remnant remaining in situ, the greater the likelihood of a false negative result with AMH testing. In addition, AMH has a longer circulating half-life in women (> 1 day36) than luteinizing hormone and follicle-stimulating hormone with half-lives of 2 to 6 hours on average.37 In other species studied to date, AMH half-lives have been estimated to be 1.5 to 2.5 days in horses,6,38,39 over 2 days in cattle,40 and 2.5 days in dogs.41 Not surprisingly, there was a significant decrease in AMH concentration after OVH, but a slow rate of systemic clearance likely explained lingering concentrations of AMH for many days after surgery in the bitches studied here. The analysis of AMH in postsurgical serum can be used to confirm whether remnant tissue has been completely removed, but waiting at least 3 weeks after surgery may be necessary to allow hormone concentrations to clear completely and reestablish a definitive baseline.
Secretory variability is an important consideration for interpreting hormone concentrations in a clinical context for any species, and this is as true for AMH as for any other reproductive hormone. For instance, higher AMH concentrations have been observed in females over 6 months of age than those younger in age, suggesting perhaps that AMH concentrations increase as the reproductive axis matures.42 Lower AMH concentrations in prepubertal than in proestrous or estrous females have also been reported.25 Additional observations in 5 postpubertal, cyclic bitches sampled longitudinally from late anestrus through estrus indicated that AMH concentrations increase in proestrus but then decrease as estrus progresses toward ovulation in individual bitches.23 Bitches in late anestrus and estrus have lower AMH concentrations than in proestrus in studies utilizing a cross-sectional sampling regimen.24,25 Finally, suppression of gonadotropin secretion in bitches, by use of the gonadotropin-releasing hormone receptor antagonist acyline, decreased AMH.43 As in the bitch, downregulation of the gonadotropic axis by use of gonadotropin-releasing hormone implants in female cats44 and cheetahs45 was also associated with lower AMH concentrations. Therefore, systemic AMH concentrations in bitches (and queens22) appear to be determined primarily by secretion from gonadotropin-dependent follicles (in proestrus and estrus) rather than preantral follicles that are likely to be less dependent on gonadotropic support. These physiological fluctuations in AMH in female dogs23–25 (and cats22) can potentially complicate the interpretation of AMH concentration as a biomarker of their gonadal status.46 The present data collected during breeding cycles confirm that AMH concentrations decline by two-thirds from a peak at proestrus to a nadir just as progesterone rises around the time of ovulation. This confirms that false negative results are more likely during estrus and less likely during proestrus. If spotting occurs in a supposedly spayed bitch, or if there is a history of spotting, samples for AMH are best taken as early as possible once discharge is noticed or left until after estrus ends when AMH concentrations begin to rise again (Figure 1). Preliminary data also indicate that AMH concentrations increase considerably in bitches around the time of expected puberty (Wilborn and Conley, personal communication). This would also be expected if AMH concentrations were determined by cohorts of actively growing follicles; therefore, false negative results may be more likely in prepubertal bitches, as has been previously suggested.25,42
To the best of our knowledge, this study was the first to report concentrations of inhibin-B in dogs. Inhibin-B concentrations were positively correlated with AMH concentrations in longitudinal sampling of bitches during breeding cycles (as in cyclic queens22), suggesting that systemic levels of both hormones are highest during follicle maturation in proestrus. These data suggest that inhibin-B concentrations may be a useful biomarker of follicular growth in the bitch if sampled frequently enough during estrous cycles. The pattern of secretion observed in the bitch was consistent with previously reported observations of inhibin concentrations during ovulatory cycles in blue fox vixens.47 However, inhibin-B was also detected in multiple samples from the OVH group, all of which had undetectable AMH consistent with a lack of ovarian tissue. A similar proportion of samples from the intact reference group had detectable inhibin-B concentrations, but the vast majority did not, and inhibin-B was not correlated with AMH concentration in the intact reference group. Therefore, inhibin-B is not an alternative marker for gonadal status, and detection of inhibin-B in spayed bitches suggests that, unlike AMH, ovarian follicles are not the only possible source of the hormone in dogs. Elevated concentrations of inhibins in canine Cushing disease48 is consistent with the ability of the adrenal cortex to secrete some form of inhibin, at least in spayed or neutered dogs.
There were notable limitations to this study. First, the samples in the OVH group were only reportedly so, but this could not be confirmed. The 7 outliers identified in this group with AMH concentrations in the intact range point to an obvious lack of certainty. The RI for this group was calculated with and without these outliers to enable individual assessments to be made as to which is more reliable. Second, the health status of the animals in this group was unknown. To the best of our knowledge, the only condition that has been associated with altered AMH in the bitch would be a granulosa cell tumor.49 However, an elevation would be interpreted as indicative of the presence of ovarian tissue in such a case and most likely recommend surgery, which would be warranted. Third, the samples used in this study came from several different sources and were therefore handled differently according to variable protocols at these sites. Storage at 4 °C was the usual clinical practice at all sites and only extended beyond a few minutes or hours for the OVH-group samples, which were stored at 4 °C for 4 days. This likely had minimal effects on AMH concentrations on the basis of prior studies of human serum held at the same temperature for that many days.50 All of the above represent potential sources of error of unknown magnitude. Nonetheless, sampling under such conditions is more representative of many clinical samples routinely submitted for AMH determinations without histories or clinical presentation and handled more variably than otherwise might be expected of a controlled study. Therefore, they arguably have more relevance in a clinical context.
In conclusion, these data provide a firm foundation for the interpretation of AMH concentrations, as an aid in determining gonadal status in bitches with unknown or uncertain histories upon clinical presentation, by use of a commercially available ELISA with homologous canine calibrators.
Supplementary Materials
Supplementary materials are posted online at the journal website: avmajournals.avma.org.
Acknowledgments
The authors are profoundly grateful to Dr. Lane Johnson (and the Gourley Clinical Teaching Center staff) for the help in collecting residual serum from discarded blood samples drawn from bitches for routine presurgical biochemistry analysis prior to ovariohysterectomies. The authors are equally grateful for the help from colleagues who organized residual samples submitted to the Colorado State University College of Veterinary Medicine from ovariohysterectomized bitches, colleagues who collected and prepared samples at the Society for the Prevention of Cruelty to Animals of Tompkins County and Cornell University Animal Health Diagnostic Center, and Anna Wilson for organization of samples from bitches during breeding cycles.
Disclosures
The Clinical Endocrinology Laboratory in the School of Veterinary Medicine, University of California-Davis, is a self-funded, not-for-profit laboratory. The authors have no financial interests to disclose.
No AI-assisted technologies were used in the generation of this manuscript.
Funding
Funded in part by the Clinical Endocrinology Laboratory, School of Veterinary Medicine, University of California-Davis.
ORCID
A. J. Conley https://orcid.org/0000-0003-3798-7390
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