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. Author manuscript; available in PMC: 2023 Apr 1.
Published in final edited form as: Theriogenology. 2022 Jan 31;182:53–62. doi: 10.1016/j.theriogenology.2022.01.033

Limitations in use of ovarian reserve biomarkers to predict the superovulation response in small ovarian reserve heifers

Kaitlin R Karl 1, Janet LH Ireland 1, Zaramasina L Clark 1, Robert J Tempelman 1, Keith E Latham 1, James J Ireland 1,2
PMCID: PMC8863639  NIHMSID: NIHMS1777168  PMID: 35123311

Abstract

High FSH doses during superovulation of heifers with a small ovarian reserve increase the number of dysfunctional ovulatory-size follicles that do not ovulate in response to human chorionic gonadotropin (hCG). Thus, anti-Müllerian hormone (AMH) and antral follicle count (AFC), two well-established biomarkers of responsiveness of individuals to superovulation, are hypothesized to be positively linked to number of dysfunctional ovulatory-size follicles developing in response to superovulation with high FSH doses. To test this hypothesis, heifers with a small ovarian reserve were stimulated beginning on Day 1 of the estrous cycle with twice daily treatments for 4 days with each of four Folltropin-V (FSH) doses (35 IU, 70 IU (industry standard), 140 IU, or 210 IU) followed by prostaglandin F2α to regress corpora lutea (CL) from the previous estrous cycle and hCG to induce ovulation. Ovulatory-size follicles were classified as functional or dysfunctional based on whether they ovulated and formed CL in response to hCG. FSH dose did not impact the relationship between AMH, AFC and the number of functional or dysfunctional ovulatory-size follicles developing in response to superovulation. Thus, data from the four superovulations were averaged for each heifer. AMH and AFC were positively associated with the subsequent number of functional and dysfunctional ovulatory-size follicles and the proportion of ovulatory-size follicles that are dysfunctional after superovulation. Because measurements of AMH concentration and AFC predict the number but not functionality of ovulatory-size follicles, which may also impact oocyte quality, these ovarian reserve biomarkers are concluded to be unlikely useful to improve IVF or embryo transfer outcomes in heifers with a small ovarian reserve.

Keywords: anti-Mϋllerian hormone, small ovarian reserve, superovulation, dysfunctional ovulatory-size follicles, bovine, biomarkers

1. Introduction

A major pitfall of FSH stimulation protocols during assisted reproductive technologies (ART) is the unpredictability [1–3] and inherent high variability in response of both women [4] and cattle [5, 6] to superovulation. This variability in the superovulation response results in a high rate of oocyte and embryo wastage resulting in costly unsuccessful ART outcomes [1–3]. Thus, discovery of biomarkers predictive of the donor’s response to superovulation should enable practitioners to better predict poor-, good-, and hyper-responders, and thereby improve current superovulation and embryo transfer protocols to optimize ART outcomes [7]. Such biomarkers could include circulating concentrations of anti-Müllerian hormone (AMH) or antral follicle count (AFC).

Anti-Müllerian hormone concentrations are widely used to predict responsiveness of donors to superovulation protocols. Anti-Müllerian hormone concentration is static during reproductive cycles in cattle [8–11] and women [12, 13] and positively correlated with size of the ovarian reserve (total number of morphologically healthy follicles in ovaries) in cattle [11, 14], women [15] and mice [16], and with number of antral follicles growing during ovarian follicular waves in cattle [8]. Cattle with greater circulating AMH concentrations have higher AFCs and correspondingly larger ovarian reserves, greater numbers of healthy growing large preantral and small antral follicles [17], and display better responses to superovulation compared with their age-matched counterparts with low circulating AMH concentrations [18]. These observations explain why numerous studies in women [19, 20] and cattle [14, 21–24] also report a positive correlation between circulating AMH concentrations, AFC, and subsequent individual response to superovulation. However, few if any studies have evaluated the association of AMH and AFC prior to superovulation with subsequent number and function (e.g., capacity to ovulate in response to LH or LH-like stimulus) of the ovulatory-size follicles developing in response to superovulation with FSH. Moreover, whether the positive association between the ovarian reserve biomarkers, AMH and AFC, with response to superovulation also exists among individuals with a relatively small ovarian reserve has not been examined. Consequently, the reliability of AMH and AFC measurements to predict number and function of ovulatory-size follicles developing in response to superovulation of individuals with a small ovarian reserve is unknown.

Anti-Müllerian hormone is secreted primarily by the granulosa cells of morphologically healthy growing large preantral and small antral follicles [18, 23, 25], which undergo atresia during reproductive cycles unless they are stimulated with FSH to develop into ovulatory-size antral follicles during ART. Typically, a wide range of FSH doses are used by ART practitioners to enhance the superovulatory response in women with a small ovarian reserve [4]. However, FSH doses used during ART are inversely associated with oocyte recovery [26] and live birth rate independent of age, body mass index or health of donors [4]. These correlative findings in women are supported by dose response studies in cattle showing that high FSH doses decrease estradiol production and ovulation rate by ovulatory-size follicles developing response to superovulation [7], and reduce fertilization rate [27–29], embryo yield [29, 30] or quality [27, 29], and number of transferable embryos [28–31]. Taken together, these results support the overall working hypothesis for our studies that high FSH doses during superovulation are detrimental to ovulatory follicle function, oocyte quality and embryo survival in cattle.

To examine the impact of high FSH doses during superovulation on ovulatory follicle function, we used the small ovarian reserve heifer model. This model mimics key characteristics of women with small ovarian reserves including a low AFC [32], hypersecretion of FSH [32–34], low circulating AMH concentrations [8] during the reproductive cycle, and poor response to superovulation [32]. In our recent study [7], a Williams Latin Square Design was used to rigorously evaluate the effects of different FSH doses during superovulation of the same group of heifers with a small ovarian reserve on ovarian function. Results showed that the use of high FSH doses to improve the superovulatory response did not alter the circulating AMH concentrations or number of ovulatory-size follicles but did have a detrimental impact on the capacity of ovulatory-size follicles to produce estradiol and ovulate. Taken together, these observations support the hypothesis that AMH and AFC, which are well-established predictors of responsiveness of individuals to superovulation, are positively associated with number of dysfunctional ovulatory-size follicles developing in response to superovulation with high FSH doses.

To test this hypothesis, we examined the relationship between circulating AMH concentrations and AFC with number of dysfunctional and functional ovulatory-size follicles developing in response to superovulation of heifers with a small ovarian reserve with four different FSH doses. Ovulatory-size follicles were defined as dysfunctional or functional based on whether they ovulated and formed a CL in response to hCG. We find that the ovarian reserve biomarkers, circulating AMH concentrations and AFC, prior to superovulation are positively linked to number of both dysfunctional and functional ovulatory-size follicles developing in response to superovulation independent of the FSH dose. Because AMH and AFC measurements prior to superovulation predict total number but cannot distinguish between functional and dysfunctional ovulatory-size follicles with potentially poor-quality oocytes developing in response to superovulation, these ovarian reserve biomarkers may not be consistently useful to improve IVF or embryo transfer outcomes in cattle with a small ovarian reserve.

2. Materials and Methods

The experimental design for the present study, which was previously described in detail, will be briefly explained here. The Institutional Animal Care and Use Committee at Michigan State University sanctioned all procedures involving cattle.

2.1. Selection of heifers with a small ovarian reserve

We established that 11- to 12-month-old heifers with a low AFC (≤15 follicles of ≥3 mm in diameter) during ovarian follicular waves (15% to 20% of a herd) also have 80% smaller ovarian reserves and a reduced response to superovulation compared with age-matched counterparts with a high AFC (≥ 25 follicles) [7, 8, 32, 34–37]. In our previous study [7], serial ovarian ultrasonography was used to identify the nine, 11- to 12-month-old Holstein heifers of similar weights with a low AFC and small ovarian reserve used in the present study.

2.2. Superovulation protocol, blood sampling and ovarian ultrasonography

To synchronize estrous cycles for superovulation prior to FSH administration, these heifers were treated twice with prostaglandin-F2α (PG, 12.5mg PG/mL im, Lutalyse HighCon, Zoetis) 10 d apart followed by a 3rd PG 12 h after the 2nd PG. Each heifer was subjected to daily ovarian ultrasonography to detect ovulation and emergence of the first follicular wave. The 1st treatment of Folltropin-V (porcine pituitary extract containing primarily FSH with <1% LH contamination, Vetoquinol USA Inc) began 36 h after the last PG, which was ±1 d from ovulation and initiation of the first follicular wave in all heifers. Folltropin-V is hereafter referred to as FSH. The superovulation regimen in our previous study consisted of a total of eight im treatments of FSH at 12-h intervals. To regress the CL of the previous estrous cycle during superovulation, three additional PG treatments were given 12 h apart starting at the time of the 7th FSH treatment (about Day 4 to 5 of estrous cycle). To induce ovulation of the ovulatory-size follicles developing in response to superovulation with FSH, heifers were treated with a 2,500 IU of human chorionic gonadotropin (hCG, Chorulon HCG 10,000 IU im, Merck Animal Health, USA), which is sufficient to ovulate up to 40 to 60 follicles in heifers [38], coincident with the 3rd PG (which is 12 h after the last (or 8th) FSH treatment). As explained in our previous study [7], serial ovarian ultrasonography was used daily to monitor alterations in AFC (follicles ≥3 mm in diameter), number of ovulatory-size follicles (follicles ≥10 mm in diameter) and CL beginning at the time of the 1st FSH treatment and ending nine days after the hCG for each superovulation regimen. In addition, blood samples (10 ml) were taken from the tail vein of each heifer once or twice daily beginning and ending concurrently with ultrasonography. A previously validated [8] commercially available AMH ELISA kit for cattle (MOFA Global, Verona, WI), which does not cross-react with other members of the transforming growth factor beta (TGFβ) superfamily including TGFβ, bone morphogenic factor-4 (BMP4), inhibin or activin [16], was used to measure circulating AMH concentrations in duplicate 20 μl serum samples per kit instructions [7]. The inter- and intra-assay coefficients of variation for the AMH assay (n = 6 plates) were 5.4% and 5.6%, respectively [7]. To measure circulating estradiol concentrations, serum estradiol-17β concentrations were determined by RIA as previously published [39, 40]. Inter- and intra-assay coefficients of variation for estradiol-17β assays (n = 15 plates) were 7.6% and 3.7%, respectively [7].

2.3. Experimental design

The Williams Latin Square Design [41, 42] was used in our previous study [7] to minimize animal numbers, potential carry-over effects, and confounding variables such as environment and aging that could contribute to variability in responsiveness to superovulation [41, 42]. Each of the nine heifers used in the present study were superovulated as explained above a total of four times with an interval of approximately 4 weeks between each superovulation regimen [7]. Each superovulation regimen used a total of eight equal FSH dose treatments of each different FSH dose: 35 IU (20mg NIH-FSH-P1/ml), 70 IU (40mg/2ml), 140 IU (80mg/4ml), or 210 IU (120mg/6ml). These FSH doses ranged from 60% lower and 240% higher than the Vetoquinol recommended dose per treatment of 87.5 IU. Hereafter, dose of FSH is referred to as IU per treatment.

The data used in the present study to evaluate the association of AMH and AFC with number of dysfunctional and functional ovulatory-size follicles developing in response to superovulation with the different FSH doses was generated in our previous study [7] as follows.

Anti-Müllerian hormone concentrations were determined from blood samples taken immediately prior to the 1st FSH treatment for all nine heifers subjected to each of the four FSH doses used during the superovulation regimens [7]. The AFCs for the heifers were determined by use of ovarian ultrasonography to count follicles ≥3 mm in diameter 1 to 2 h before the 1st FSH treatment for each of the four different FSH doses used during the superovulation regimens [7].

To determine number of ovulatory-size follicles that developed in response to superovulation with the different FSH doses and the corresponding circulating estradiol concentrations, the total number of follicles ≥10 mm in diameter for each heifer were determined at the time of the hCG treatment for each different superovulation regimen [7]. Time of hCG was chosen to determine number of ovulatory-size follicles because it coincides with the maximum number of ovulatory-size follicles that developed during each superovulation regimen in our previous study [7].

Capacity to ovulate in response to a preovulatory LH surge is a well-established characteristic of normal functioning ovulatory follicles in cattle [43–47]. Thus, the total number of ovulatory-size follicles determined at time of the hCG treatment was separated into two different categories (functional, dysfunctional) based on whether the ovulatory-size follicles formed CL in response to the hCG treatment after each superovulation regimen. Number of functional ovulatory-size follicles was therefore defined as the total number of CL counted on Day 7 post-hCG [7] whereas the number of dysfunctional ovulatory-size follicles not ovulating in response to the hCG treatment was determined by subtracting the number of CL (or number of functional ovulatory-size follicles) from the total number of ovulatory-size follicles determined at time of the hCG treatment.

2.4. Statistical analysis

Box and whisker plots were used to show the distribution of all variables (AMH concentrations, AFC, total number of ovulatory-size follicles, number of dysfunctional and functional ovulatory-size follicles, proportion of total number of ovulatory-size follicles that were dysfunctional) before these data were transformed for statistical analyses. A Shapiro-Wilk test for normality of data distribution indicated all data for each FSH dose were normally distributed (P>0.05) after transformations. Anti-Müllerian hormone measurements were subjected to Log10 transformation whereas all other variables were subjected to square root transformation before correlation and linear regression analysis.

To further assess variability in the biomarkers used in this study, repeatability of measurements of AMH and AFC within individuals was determined for the nine heifers used in the present study. AMH and AFC measurements were made in the individual heifers four different times 1.5 to 2 months apart, which is the interval between each superovulation regimen with the four different FSH doses. Repeatability of each AMH or AFC measurement within individuals was determined using transformed data as previously reported [34]. Repeatability (range 0–1, where 1 = perfect) is defined as the proportion of the total variance attributable to animal variance, which is [48] calculated as σ2 animal/(σ2 animal + σ2 error) [49]. Variance components and repeatability estimates were determined using the PROC MIXED model approach of SAS [50], where heifers were accounted for as a source of variation and a common residual variance was used for all response variables across FSH doses.

Linear regression analysis was performed using Statistical Analysis System (SAS 9.4 Institute, Cary, NC) with PROC GLIMMIX [50] to determine if a statistically significant (P≤0.05) linear relationship existed between AMH and AFC measurements before each superovulation with corresponding alterations in total and number of dysfunctional and functional ovulatory-size follicles in response to the different FSH doses during each superovulation regimen. PROC GLIMMIX [50] was then used to determine if the means and slopes generated during linear regression analysis differed (P≤0.05) among the four FSH doses used to superovulate heifers.

Statistical Analysis System (SAS 9.4 Institute, Cary, NC) was also used to conduct a Pearson Correlation with PROC CORR [50] to determine if a statistically significant (P≤0.05) correlation existed between AMH concentrations and AFC measurements before each superovulation with corresponding total number of ovulatory-size follicles, number of functional and dysfunctional ovulatory-size follicles, and proportion of total number of ovulatory-size follicles that were dysfunctional.

3. Results

3.1. Individual variability in circulating AMH concentration and AFC prior to superovulation with FSH and the corresponding variability in the endpoints used to measure response of each heifer to the different superovulation regimens.

The nine heifers in this study were of the same age, breed, weight, and all had a low AFC (≤15 follicles ≥3 mm in diameter). These selection criteria could have resulted in a relatively narrow degree of variability in the data collected from the nine heifers, thereby hindering interpretation of the linear regression and correlation results in our present study [51]. To examine this possibility, box and whisker plots [52] are provided to depict the distribution of the values examined in the present study, which included AMH (Fig. 1A), AFC (Fig. 1B), total number of ovulatory-size follicles (Fig. 3A), number of functional ovulatory-size follicles (Fig. 3B), number of dysfunctional ovulatory-size follicles (Fig. 5A), and proportion of total number of ovulatory-size follicles that were dysfunctional (Fig. 5B) for the nine heifers treated with each of the four FSH doses (35 IU, 70 IU, 140 IU, 210 IU) used during each superovulation regimen.

Figure 1:

Figure 1:

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Box and whisker plots showing the distribution of AMH concentrations and AFC prior to superovulation of nine heifers with small ovarian reserves with different doses of Folltropin-V (FSH) A.

A The same group of Holstein heifers (11- to 12-months old) were superovulated four times with four different FSH doses (n = 9 heifers per dose) followed by a single treatment of hCG 12 h after the last FSH treatment for each superovulation regimen as explained in Methods. AMH concentration and AFC prior to each superovulation among the nine heifers were determined for each different FSH dose.

Figure 3:

Figure 3:

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Box and whisker plots showing the distribution of the number of ovulatory-size follicles and the number of functional ovulatory-size follicles observed following superovulation of nine heifers with small ovarian reserves with different doses of Folltropin-V (FSH) A.

A The same group of Holstein heifers (11- to 12-months old) were superovulated four times with four different FSH doses (n = 9 heifers per dose) followed by a single treatment of hCG 12 h after the last FSH treatment for each superovulation regimen as explained in Methods. The number of ovulatory-size follicles was determined at time of hCG. The number of functional ovulatory-size follicles was determined by counting the number of ovulatory-size follicles that had formed CL 7 d after the hCG.

Figure 5:

Figure 5:

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Box and whisker plots showing the distribution of the number of dysfunctional ovulatory-size follicles and proportion of total number of ovulatory-size follicles that were dysfunctional following superovulation of nine heifers with small ovarian reserves with different doses of Folltropin-V (FSH) A.

A The same group of Holstein heifers (11- to 12-months old) were superovulated four times with four different FSH doses (n = 9 heifers per dose) followed by a single treatment of hCG 12 h after the last FSH treatment for each superovulation regimen as explained in Methods. The number of dysfunctional ovulatory-size follicles that did not ovulate and form CL in response to hCG was determined by subtracting the number of CL formed 7 d after the hCG (number of functional ovulatory-size follicles) from the total number of ovulatory-size follicles observed at time of hCG. The proportion of the total number of ovulatory-size follicles that were dysfunctional was determined by dividing the number of dysfunctional ovulatory-size follicles by the total number of ovulatory-size follicles at time of hCG.

Extensive variability existed in circulating AMH concentrations and AFC prior to superovulation and in all variables measured as endpoints of the response to the different superovulation regimens among the nine heifers. However, some variables at the different FSH doses (e.g., AFC at the 30 IU and 70 IU doses in Fig. 1B) were skewed (horizontal line within box representing the median value does not divide box into two equal halves) as depicted by the data points (solid dark circles) within boxes and by the whiskers (capped line running through the center of each box showing lowest and highest data point values) for each box in Figs. 1, 3 and 5. Thus, all data shown in Fig. 1, 3 and 5 were transformed (e.g., as depicted in Figs 2, 4, 6) to ensure normality before statistical analyses were conducted. As shown in the Box and Whisker Plots (Figs 1, 3, 5), the variability in both AMH concentrations and AFC observed among the nine heifers with a small ovarian reserve was sufficiently widespread to reliably evaluate not only if AMH concentration and AFC were repeatable within individual heifers, but also to determine if size of the ovarian reserve as measured by circulating AMH concentrations and AFC prior to superovulation with FSH was linked to the subsequent number and functionality (capacity to ovulate in response to hCG) of the ovulatory-size follicles developing in response to superovulation with different FSH doses.

Figure 2:

Figure 2:

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Regression analysis and correlation coefficients for the relationship between concentrations of anti-Mϋllerian hormone (AMH) and antral follicle count (AFC) before superovulation of heifers with a small ovarian reserve.

A The same group of Holstein heifers (11- to 12-months old) were superovulated four times with four different FSH doses (n = 9 heifers per dose) followed by a single treatment of hCG 12 h after the last FSH treatment for each superovulation regimen as explained in Methods. Because there was no FSH dose effect, data were pooled. Thus, each symbol represents the mean ±SEM for the four superovulations for each heifer. Concentrations of AMH and AFC (≥3 mm in diameter) were determined 1 to 2 h before the first treatment of FSH. Asterisks indicate level of statistical significance (** = P<0.01).

Figure 4:

Figure 4:

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Regression analysis and correlation coefficients for the relationship between concentrations of anti-Mϋllerian hormone (AMH) and antral follicle count (AFC) before superovulation with total number of ovulatory-size follicles and with number of functional ovulatory-size follicles following superovulation of heifers with a small ovarian reserveA.

A The same group of Holstein heifers (11- to 12-months old) were superovulated four times with four different FSH doses (n = 9 heifers per dose) followed by a single treatment of hCG 12 h after the last FSH treatment for each superovulation regimen as explained in Methods. Because there was no FSH dose effect, data were pooled. Thus, each symbol represents the mean ±SEM for the four superovulations for each heifer. Concentrations of AMH and AFC (≥3 mm in diameter) were determined 1 to 2 h before the first treatment of FSH. The total number of ovulatory-size follicles (≥10 mm) and number of functional ovulatory-size follicles was determined as explained in the legend for Fig. 3. Plus signs or asterisks indicate level of statistical significance (++ = P≤0.07, +++= P≤0.06, * = P<0.05, ** = P<0.01, *** = P<0.001).

Figure 6:

Figure 6:

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Regression analysis and correlation coefficients for the relationship between concentrations of anti-Mϋllerian hormone (AMH) and antral follicle count (AFC) before superovulation with number and proportion of ovulatory-size follicles that were dysfunctional following superovulation of heifers with a small ovarian reserveA.

A The same group of Holstein heifers (11- to 12-months old) were superovulated four times with four different FSH doses (n = 9 heifers per dose) followed by a single treatment of hCG 12 h after the last FSH treatment for each superovulation regimen as explained in Methods. Because there was no FSH dose effect, data were pooled. Thus, each symbol represents the mean ±SEM for the four superovulations for each heifer. Concentrations of AMH and AFC (≥3 mm in diameter) were determined 1 to 2 h before the first treatment of FSH. The number and proportion of dysfunctional ovulatory-size follicles were determined as explained in the legend for Fig. 5. Asterisks indicate level of statistical significance (* = P<0.05, ** = P<0.01, *** = P<0.001).

3.2. Repeatability of circulating AMH concentration and AFC within individual heifers prior to superovulation with the different FSH doses.

Circulating AMH concentrations and AFC were highly variable among the individual heifers (Fig. 1). In addition, a total of 8 months was required to complete the four superovulation regimens with the four different FSH doses (9 heifers per dose [7]). Thus, the high variability in AMH and AFC prior to superovulation with FSH among heifers, coupled with potential decline in the ovarian reserve during aging in cattle [53], could contribute to the high variability of responsiveness of heifers to superovulation depicted in Fig. 1. However, we have shown that circulating AMH concentrations and AFC are reliable biomarkers for size of the ovarian reserve in heifers [8]. Thus, to examine the potential age-related changes in size of the ovarian reserve, the four different measurements of AMH concentrations and AFC at approximately 2-month intervals prior to each superovulation regimen for each heifer were used to determine repeatability of circulating AMH concentrations and AFC within individuals.

Although circulating AMH concentrations varied 41-fold between individuals ranging from 0.42 to 701 pg/ml (Fig. 1A), AMH concentrations were highly repeatable within individuals (0.94, 1 = perfect). In addition, AFC varied 4.7-fold between individuals ranging from 3 to 14 (Fig. 1B) and repeatability of AFC within individuals was moderate (AFC = 0.39).

3.3. The linear relationship and correlation of circulating AMH concentrations with AFC prior to superovulation of heifers with the different FSH doses.

The linear regression analysis indicated that the overall means and linear relationships (slopes) between AMH concentrations with AFC prior to superovulation with the four different FSH doses were similar (P>0.10) among heifers (data not shown). Thus, the average for circulating AMH concentrations (n = 4 measurements per heifer) or AFC (n = 4 measurements per heifer) for the nine heifers was determined. These data were then re-analyzed by linear regression and correlation analyses to further evaluate the association of AMH concentrations with AFC. A positive linear relationship and positive correlation existed between circulating AMH concentrations with AFC prior to superovulation of heifers with FSH (Fig. 2).

3.4. The linear relationship and correlation between AMH concentration or AFC prior to superovulation with FSH with the endpoints used to measure the response of heifers to the different superovulation regimen.

The linear regression analysis indicated that the overall means and linear relationships (slopes) between AMH or AFC prior to superovulation with FSH with the endpoints used to measure the response of heifers to the four different superovulation regimens were similar (P>0.10, data not shown). Because there was no evidence that FSH dose during superovulation impacted the relationship between AMH and AFC with the response to superovulation, data collected prior to and during the four superovulation regimens were pooled and averages for each variable (AMH, AFC, total and number of functional and dysfunctional ovulatory-size follicles, proportion of total number of ovulatory-size follicles that were dysfunctional) were determined for each heifer. These data for the nine heifers were then re-analyzed by linear regression and correlation analyses to further evaluate the association between the ovarian reserve biomarkers, AMH and AFC, prior to superovulation with FSH, with the subsequent number and functionality of the ovulatory-size follicles developing in response to superovulation. A positive linear relationship and positive correlation existed in heifers between circulating AMH concentrations and AFC with the subsequent total number of ovulatory-size follicles (Fig. 4) and with number of functional (Fig. 4) and dysfunctional (Fig. 6) ovulatory-size follicles and proportion of total number of ovulatory-size follicles that were dysfunctional (Fig. 6) developing in response to superovulation with FSH during the superovulation regimen.

4. Discussion

The goal of the present study was to determine if circulating AMH concentrations and AFC, which are well-established ovarian reserve biomarkers predictive of the responsiveness of individuals to superovulation, were positively associated with number of dysfunctional ovulatory-size follicles developing in response to superovulation with high FSH doses. The most significant finding of this study is that measurements of circulating AMH concentrations and AFC prior to superovulation with FSH were highly predictive of total number of ovulatory-size follicles developing in response to superovulation. However, the differences in AMH concentrations and AFC prior to superovulation with FSH were positively linked to an increased number of both dysfunctional and functional ovulatory-size follicles as well as an increased proportion of ovulatory-size follicles that were dysfunctional independent of the FSH dose used during superovulation. Because measurements of AMH concentration and AFC predict total number but not functionality of the ovulatory-size follicles developing in response to superovulation, which may also impact oocyte quality, these biomarkers are unlikely to be consistently useful to improve IVF or embryo transfer outcomes in heifers with a small ovarian reserve.

Another key finding of the present study showed that, despite use of 11- to 12-month-old heifers with a low AFC and small ovarian reserve, circulating AMH concentrations and AFC and correspondingly size of the ovarian reserve, varied extensively among animals but were highly or moderately repeatable, respectively, within individuals and positively associated with each other during the 8 months required to complete the different superovulation regimens. While use of the ovarian reserve biomarkers, AMH and AFC, may be useful to select a more uniform group of heifers (with similar AMH concentrations, AFC, and ovarian reserves) that also respond similarly to superovulation, small-ovarian-reserve-specific superovulation protocols designed to optimize development of functional ovulatory-size follicles will also likely be necessary to enhance ART outcomes.

The highest FSH doses used to superovulate heifers in the present study did not impact the relationship between AMH concentrations and AFC with number and functionality of the ovulatory-size follicles developing in response to superovulation. This finding was surprising because the highest FSH doses during superovulation of the same heifers in the present study negatively impacted estradiol production and ovulation rate in our previous study[7]. However, we recognize that only nine animals were used per FSH dose in the present study, thus larger animal numbers may be necessary to further assess effects of high FSH doses on the biomarker-superovulation response relationship. Nevertheless, differences in size of the ovarian reserve, as measured by AMH concentrations and AFC in the heifers in the present study, were positively linked, independent of the FSH dose, with the total number of ovulatory-size follicles and with number of functional (or number of CL post hCG) ovulatory-size follicles developing in response to superovulation. These positive relationships have also been reported by others using cattle [21, 54–56] and women [57, 58] with mixed ages and unknown ovarian reserves. Taken together, these observations support the value of both biomarkers, AMH and AFC, to not only predict the number of ovulatory-size follicles developing in response to superovulation but also the number of functional ovulatory-size follicles capable of ovulation in response to hCG (number of CL). Using reliable biomarkers such as AMH or AFC to predict responsiveness of individuals to superovulation enables ART practitioners to improve superovulation protocols for example to avoid excessive use of FSH, estimate number of oocytes potentially available for IVF or number of transferable embryos, and better inform patients/clients of expected ART outcomes. Nevertheless, the ovarian reserve biomarkers, AMH and AFC, were also positively associated with number of dysfunctional ovulatory-size follicles that did not ovulate in response to an hCG treatment and the proportion of total number of ovulatory-size follicles that were dysfunctional. The capacity to ovulate in response to a preovulatory LH surge is well-recognized by reproductive biologists as a physiologically indispensable criteria for a normal functioning ovulatory follicle with high quality oocytes during reproductive cycles of most mammals, including cattle. Thus, the strong positive correlation between both ovarian reserve biomarkers, AMH and AFC, with number of dysfunctional ovulatory-size follicles, as observed in the present study, implies that size of the ovarian reserve is positively linked not only with number of functional (capable of ovulation and forming CL or number of CL) but also with number of dysfunctional (incapable of ovulation) ovulatory-size follicles independent of the FSH doses used to superovulate heifers with a small ovarian reserve.

The reason circulating AMH concentrations and AFC are positively linked with number of dysfunctional ovulatory-size follicles is unknown. Nevertheless, previous studies show that superovulation diminishes capacity of bovine oocytes to develop into blastocysts [59, 60] and an inverse relationship exists between responsiveness to superovulation and proportion of ovulatory-size follicles with high quality oocytes in cattle [27, 29, 30] and women [4, 26, 61]. For example, superovulation of heifers with a high compared with a low AFC result in a greater number of CL, embryos/oocytes recovered and transferable embryos per animal but a lower proportion of transferable embryos and higher proportion of fertilized non-transferable embryos [8, 32]. In addition, studies in cattle with unknown AFC report that the proportion of high-quality embryos transferred is inversely linked to the responsiveness of superovulation [32, 62, 63], and that the number of transferable embryos declines as the number of oocytes recovered increases in women [61]. Taken together, these results indicate that responsiveness of individuals to superovulation is positively linked to increased number of both functional and dysfunctional ovulatory-size follicles with corresponding differences in oocyte quality. Whether oocyte quality is also impaired in the putative dysfunctional ovulatory-size follicles was beyond the scope of the present study. These questions, however, are important to answer to establish new methods to minimize formation of dysfunctional ovulatory-size follicles, reduce oocyte wastage, and improve efficiency of superovulation protocols to improve ART outcomes.

Circulating AMH concentrations and AFC in the heifers with small ovarian reserves were positively correlated with each other prior to superovulation with FSH thus confirming results of our previous study that used the mixed group of heifers with low, intermediate or a high AFC [11, 36] as well as results of others using cattle [14, 22, 23, 64, 65] and women [66–68] with a mixed AFC and different ages. In addition, during the 1.5- to 2-month intervals between superovulation regimens in the present study, AMH concentration at emergence of the first follicular wave for each different estrous cycle was highly repeatable (0.91) within individuals. This observation indirectly implies that, despite having a low AFC and small ovarian reserve, growth of the large preantral and small antral follicles potentially responsive to the FSH treatment during each superovulation regimen was also relatively constant at the beginning of each follicular wave within individuals in the present study. Thus, the high variation in response to superovulation observed here is more likely attributable to differences in size of the ovarian reserve among individuals rather than erratic growth of follicles during follicular waves within individuals.

Unlike circulating AMH concentrations, AFC reflects the total number of antral follicles ≥3 mm in diameter during a follicular wave and thus is a direct measurement of follicles potentially responsive to superovulation. However, in contrast to AMH concentrations, repeatability of AFC during the first follicular waves within the same individual heifers in the present study was moderate (0.39) and lower than repeatability (0.85 to 0.95) of peak AFC during follicular waves within individuals determined in our previous studies which used heifers with a low, intermediate, or high peak AFC [34]. The precise reason for the disparity in repeatability of AFC within individuals between our studies is unknown. However, FSH secretion is inversely associated with AFC [25, 34] and factors such as heat stress [69] and photoperiod [70] alter FSH secretion and can negatively impact repeatability of AFC in cattle. Whether FSH secretion and, in turn, growth of the larger antral follicles (≥3 mm in diameter) during follicular waves is less repeatable for individual heifers with a small ovarian reserve, as used in our present study, compared with age-matched individuals with a higher AFC is unknown. Alternatively, it is also possible that determination of peak AFC as in our previous study [34] is more repeatable within individuals than determination of AFC at an unknown stage of a follicular wave as in the present study. Nevertheless, grouping individual heifers based on both circulating AMH concentrations and AFC, although time consuming, may be necessary to produce more uniform responses of individuals to superovulation.

5. Conclusion

In summary, availability of multiple, developmentally normal ovulatory-size follicles following superovulation is the pivotal physiological event to ensure enough high-quality oocytes are available for successful in vivo or in vitro fertilization and embryo transfer in cattle. Results of the present study, however, showed that the ovarian reserve biomarkers, AMH concentration and AFC, were positively associated with total number of ovulatory-size follicles, number of functional and dysfunctional ovulatory-size follicles, and proportion of total number of ovulatory-size follicles that were dysfunctional developing during superovulation with FSH. Because AMH concentration and AFC predict subsequent number but not functionality of ovulatory-size follicles developing in response to superovulation, which may also impact oocyte quality, measurement of these ovarian reserve biomarkers prior to superovulation with FSH is concluded to unlikely be consistently useful to improve IVF or embryo transfer outcomes in heifers with a small ovarian reserve. However, ovarian-reserve-specific superovulation protocols that optimize number of functional ovulatory follicles capable of ovulation in response to an LH or LH-like stimulus may be necessary to improve efficiency of superovulation procedures and reliability of circulating AMH and AFC to predict ART outcomes in individual heifers with a small ovarian reserve.

Future studies will be necessary to determine if oocyte quality is also compromised in the dysfunctional ovulatory-size follicles that do not ovulate in response to hCG and whether the link between the ovarian reserve with increased number of dysfunctional ovulatory-size follicles is unique to cattle with a low AFC and small ovarian reserve.

Highlights:

  • Unpredictable response to superovulation – biomarkers needed for consistency

  • Superovulation dose has no impact on link amid AMH, AFC & ovulatory-size follicles

  • AMH & AFC linked to ratio of dysfunctional ovulatory-size follicles post-treatment

  • AMH & AFC as biomarkers – probably not useful for improving reproductive outcomes

Acknowledgment

We thank Green Meadow Farms Inc for allowing us to identify and purchase the small ovarian reserve heifers for our study and the Beef Cattle Research and Teaching Center at Michigan State University for use of animal handling equipment, maintenance, and care of heifers.

Funding:

This study was supported by the Agriculture and Food Research Initiative Competitive USDA-NIH Dual Purpose Program Grant no. 2017–67015-26084 from the USDA National Institute of Food and Agriculture, the Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health under Award Number T32HD087166, MSU AgBio Research, and Michigan State University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes

Declaration of Interest: None

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