We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared spectroscopy (FTIR) metabolomics to identify spectral models predictive of pregnancy outcome. Embryos collected on Day 6 from superovulated cows in 2 countries were individually cultured in synthetic oviduct fluid medium with BSA for 24 h before embryo transfer. Spent CM, blank controls, and plasma samples (Day 0 and Day 7) were evaluated using FTIR. The spectra obtained were analyzed. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the ROC curve (AUC). Endpoints considered were Day 60 pregnancy and birth. High AUC was obtained for Day 60 pregnancy in CM within individual laboratories (France
The current increase in the use of embryos selected for their genetic merit and the need for high fertility recipients [
Assessment of embryonic quality requests noninvasive or minimally invasive techniques that do not interfere with embryonic development to term. These conditions restrict the number of procedures with practical application for ET programs. In cattle, embryonic cell biopsy is barely used to evaluate incidence of chromosomal abnormality, while it is used to detect and quantify expression of some genes associated with developmental competence [
Together with the particular limitations exposed, the above methods are technically difficult to perform, time-consuming, and expensive or require qualified operators [
Classically less attention has been paid to recipient selection in the ET field. In practice, selection of recipients is based on assessment of corpus luteum (CL) function, by rectal palpation or ultrasonography, and progesterone (P4) measurement. Such selection procedures help to increase pregnancy rates but also often exclude too many intrinsically fertile animals [
A variety of spectral and analytical approaches may allow determination of the metabolites associated with embryo viability and pregnancy outcome [
By using another spectroscopy technique, Fourier transform infrared spectroscopy (FTIR), we recently developed a noninvasive system that successfully predicted embryonic sex [
In the present study we analyzed plasma by FTIR metabolomics to predict pregnancy viability in recipients of superovulated embryos. In vivo embryo transfer accounts for approximately 65% of the total embryos transferred worldwide (source: IETS Newsletter, Dec. 2013). Therefore improving pregnancy rates by a better selection of embryos and recipients may contribute to increasing significantly the efficiency of selection schemes. The “gold standard” in vivo embryos used were singly cultured in vitro for 24 h and their CM also analyzed, in order to compare the predictive value of recipients and embryos (i.e., plasma versus CM, resp.). On the basis of previous studies with human embryos replicated in different laboratories and culture conditions [
All experimental procedures were carried out in accordance with the European Community Directive 86/609/EC and were sanctioned by the Animal Research Ethics Committee of SERIDA (licensed 30/01/09).
Animal experiments with in vivo embryo production and ET were performed in independent laboratories from France (UNCEIA, Station Experimentale de Chateauvillain) and Spain (UTE-Bos).
All reagents were purchased from SIGMA (Madrid, Spain) unless otherwise stated.
In vivo embryo production methods differed between Spanish and French laboratories involved.
In UTE-Bos (Spain), cyclic, Holstein donor cows were synchronized in estrus. Briefly, a progestagen device (PRID; Ceva, France) was inserted in the vagina. On Day (−5), 8 FSH (Folltropin, Bioniche, Canada) decreasing doses were given every 12 h apart. On Days (−3) pm and (−2) am, cows received prostaglandin F2
In UNCEIA (France) cyclic, lactating Holstein donor cows housed in station were synchronized in estrus by using progestagen implants and prostaglandin F2
In both the UTE-Bos and UNCEIA laboratories embryo recovery was performed by flushing the uterine horns on Day 6 in the cycle. Recovered embryos were in vitro cultured in synthetic oviduct fluid containing amino-acids, citrate, and myo-inositol supplemented with 6 g/L BSA (SOFaaci) in single 12
In UTE-Bos, heifers were synchronized in estrus with intravaginal progestagen device (PRID ALPHA, Ceva, Barcelona, Spain) for 8 days and a PG analogue (Dalmazin) injected 24 h before progestagen removal. A GnRH analogue (Dalmarelin) was injected at the time of progestagen insertion and on Day 0.
In UNCEIA, heifers were synchronized in estrus with progestagen implant (Crestar, Intervet, France) for 10 days combined with a PG analogue (Estrumate, Intervet, France) injected 24 h before progestagen removal.
Spent culture media (10
Blood plasma samples from recipients were taken in EDTA-vacuum tubes from coccygeal vein puncture. Samples were taken at the time of standing estrus (Day 0) and on Day 7 (2–4 h before the ET time). Blood tubes were immediately refrigerated at +4°C, and centrifuged at 2,000 ×g, not later than 30 min after recovery. Supernatant plasma was aliquoted and stored at −80°C up to FTIR analysis.
In both places and all recipients, pregnancy was diagnosed by transrectal ultrasound scanning on Days 60 ± 2, and birth date registered.
Spent CM and blank samples were analyzed using a Golden-Gate ATR device (diamond crystal) mounted on a Varian 620-IR FTIR spectrophotometer running Varian Resolutions Pro software version 5.0.0.700. 5
The FTIR spectra obtained from CM and blood plasma were uploaded to Matlab programming environment (R2011b; The MathWorks, Natick, MA) for data analysis and predictive model development. Two separate datasets were generated considering Day 60 pregnancy and birth endpoints. In each dataset, samples were labeled as 1 and −1 associated with positive and negative outcomes, respectively. Binary (two-class) classification experiments were performed for model development.
The overall study population included spectra obtained from CM of embryos transferred (
In a recent study, we performed a benchmarking experiment to assess discrimination capability of a variety of classification algorithms on prediction of embryonic sex using CM spectra (submitted). Among the classifiers tested, k-nearest neighbor (k-NN) provided the highest prediction accuracy. Therefore, we applied k-NN method for viability prediction in this study.
In the distance based local k-NN model the class label of a test sample is decided to be the same as the most frequent class among its
Specifically, a weighted k-NN approach was applied where the contributions of neighbors to the class choice were weighted by the inverse of distances to the test sample.
The study dataset is a typical example of high dimension low sample size (HDLSS) problem with 49 samples of CM, 96 samples from plasma, and 1704 features, obtained from spectroscopy analysis. We utilized principal component analysis (PCA) to spectra data for dimensionality reduction.
We applied 10-fold cross validation training-testing strategy in the classification experiments. The entire dataset was randomly divided into 10 bins. The predictive model was developed on the 9 bins (training samples) and the performance of prediction was assessed on the remaining bin (test samples). In order to overcome sampling bias, the training-testing procedure was repeated 10 times replacing the test samples with a bin from the training samples. The average results obtained from the repeated tests are presented.
The discrimination capability of the classifiers was assessed in terms of accuracy, sensitivity and specificity (i.e., the proportion of correctly detected positive and negative pregnancy outcomes, resp.), and receiver operating characteristics curve (ROC) analysis [
In Experiment 1, we obtained the metabolomic profile of individually cultured embryos, upon FTIR analysis of frozen/thawed CM samples of 24 h in vitro-cultured, in vivo-derived fresh embryos. Values were normalized versus those of blank samples cultured without embryos. Predictive models compared pregnant versus nonpregnant animals on Day 60 and at birth within all categories of embryos analyzed.
In Experiment 2, we predicted pregnancy success from recipient plasma metabolomic analysis. For normalization purposes, in the absence of blank controls, we included two days of plasma sampling. Our aim was that subtraction of Day 0 and Day 7 plasma values could be an appropriate tool to normalize recipient data between laboratories. Therefore, plasma spectral values were analyzed on Day 0 and Day 7, each being an independent prediction day, and normalized (i.e., Day 7 and Day 0).
All spectral model development, data preprocessing steps, and postprediction statistical analysis were performed using Matlab (R2011b; The MathWorks, Natick, MA). Classifier benchmarking tasks were conducted using Weka (Waikato Environment for Knowledge Analysis), an open-source data mining system [
Day 6 in vivo recovered early morulae (
Day 60 pregnancies in each individual laboratory involved and within cumulative data were higher than predictions at birth (Table
Day 60 pregnancy and birth predictions on PCA transformed spectra data derived from metabolomic analysis of spent culture medium (CM) of Day 6 in vivo embryos cultured in vitro for 24 h and transferred fresh to recipients on Day 7 in two laboratories (UNCEIA, France, and UTE-Bos, Spain).
Laboratory | Sample analyzed | Pregnancy endpoint |
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PCA | Accuracy | Sensitivity | Specificity | AUC |
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France | Embryo CM | Day 60 | 26 | 16 | 10 | 3 | + |
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Birth | 26 | 14 | 12 | 3 | + |
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Spain | Embryo CM | Day 60 | 23 | 13 | 10 | 3 | + |
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Birth | 23 | 12 | 11 | 3 | + |
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France + Spain | Embryo CM | Day 60 | 49 | 29 | 20 | 3 | − |
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Birth | 49 | 26 | 23 | 3 | − | <0.6 |
PCA: principal component analysis (+: applied; −: did not improve the results when applied).
In contrast, cumulative analysis improved predictions with plasma (Table
Day 60 pregnancy and birth predictions on PCA transformed spectra data derived from metabolomic analysis of plasma recovered on Day 0 and Day 7 from recipients prior to transfer on Day 7 of embryos that had been cultured in vitro for 24 h in two laboratories (UNCEIA, France, and UTE-Bos, Spain).
Laboratory | Plasma analyzed | Pregnancy endpoint |
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Positive | Negative |
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PCA | Accuracy | Sensitivity | Specificity | AUC |
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France | Day 0 | Day 60 | 25 | 15 | 10 | 3 | − |
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Birth | 25 | 13 | 12 | 3 | − |
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France | Day 7–Day 0 | Day 60 | 25 | 15 | 10 | 3 | − | <0.6 | |||
Birth | 25 | 13 | 12 | 3 | − |
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Spain | Day 0 | Day 60 | 23 | 13 | 10 | 1 | − |
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Birth | 23 | 12 | 11 | 1 | − | <0.55 | |||||
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Spain | Day 7–Day 0 | Day 60 | 23 | 13 | 10 | 3 | + |
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Birth | 23 | 12 | 11 | 3 | + |
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France + Spain | Day 0 | Day 60 | 48 | 28 | 20 | 1 | + |
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Birth | 48 | 25 | 23 | 1 | + |
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France + Spain | Day 7–Day 0 | Day 60 | 48 | 28 | 20 | 3 | + | <0.6 | |||
Birth | 48 | 25 | 23 | 3 | + |
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PCA: principal component analysis (+: applied; −: did not improve the results when applied).
Day 7 plasma alone (not represented) yields <0.6 AUC.
In vitro development and pregnancy rates of Day 6 in vivo embryos that recovered from superovulated, artificially inseminated cows in UNCEIA (France) and UTE-Bos (Spain) followed by a 24 h individual culture step in SOF + 6 g BSA/L prior to embryo transfer.
Development rates | |||||
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Day 60 pregnancy | Calving |
France | 27 | 26 | 26 | 17 (65%) | 16 (61%) |
Spain | 24 | 23 | 23 | 13 (56%) | 12 (52%) |
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Total | 51 | 49 | 49 | 30 (59%) | 28 (55%) |
France: all embryos were transferred in a single herd (
Spain: embryos transferred in 2 herds (
Principal component analysis (PCA) of spectra obtained from Day 0 plasma. The percentages of variability represented by the first three principal components are displayed across PC nos. 1, 2, and 3 on
Plasma Day 0 gave AUC and accuracy cumulative values higher than those from Day 0 to Day 7 normalized.
We show here that CM metabolome reflects viability of in vivo embryos and that the metabolic fingerprint of recipient plasma provides robust information on the likelihood of pregnancy and birth. The embryo produced in vitro shows capacity to modify its environment early in development. Thus, in the cow uterus, IVP early embryos trigger detectable maternal responses on Day 8 in the uterine fluid [
The present findings suggest that the predictive value of CM is limited with in vivo embryos, and future work should consider gaining insight into normalization procedures. Normalization should include classification by embryonic stages with higher sample numbers, and probably culture conditions and time periods other than 24 h.
We did not estimate the effects of 24 h in vitro culture on in vivo embryos after transfer. However, Grade 3 morulae that recovered from superovulated animals yield pregnancy rates similar to noncultured Grade 1 morulae after 24 h in vitro culture [
CM from in vivo embryos was less predictive for birth than for Day 60 pregnancy rates. This is surprising, as recipients carrying in vivo-derived embryos usually show less than 5% embryonic losses from the second month of pregnancy to term [
The viability profiles of CM with in vivo embryos were not more efficient than conventional selection of embryos. Interestingly, the plasma recipient was generally more predictive of pregnancy success than the CM profile. Within cumulative results, plasma Day 0 gave AUC and accuracy values higher than those from Day 0 to Day 7 normalized, indicating that (combinations of) factors exist in single Day 0 samples able to act as internal controls. Information from recipient (plasma) was generally more predictive of pregnancy success than the embryo CM. Recipient plasma can be affected by different feeding, management, and environmental conditions. However, Day 0 plasma enabled identification of common predictive profiles between recipient herds with an AUC higher than those from each laboratory analyzed apart. These data suggest that two-day sampling could not be necessary, once single plasma samples on Day 0 may provide endogenous normalization.
The ability of a recipient to reach birth has been suggested to be a source of variation higher than the ability of the embryo to survive to term [
To our knowledge, the association of embryos with specific recipients has been not yet studied, and it could help optimize the use of recipient herds. Promising research lines can investigate recipients that are usually discarded for ET using conventional selection criteria. In addition, as recipients and embryos can enter into an early dialogue into which immunological concerns are prominent [
Selection of in vivo embryos by FTIR analysis of CM to increase pregnancy rates may be performed. Although this approach did not improve the results obtained with conventional selection of in vivo embryos, it is likely that establishing more homogeneous procedures may allow normalization between laboratories leading to improved prediction rates. In contrast, cumulative analysis of recipient plasma from both laboratories identified pregnancy predictive profiles with an AUC higher than those from each laboratory analyzed apart, suggesting that FTIR can be an interesting, simple tool to select recipients on field in conventional MOET programs.
FTIR analysis of CM provides a noninvasive, rapid, and inexpensive method compatible with the highest sanitary standards and international exchanges of embryos. Using embryos and recipients with improved viability indexes will significantly increase pregnancy rates and economic benefit in the cattle breeding industry.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Marta Muñoz and Asli Uyar contributed equally to this paper.
The authors thank F. Goyache for valuable scientific comments. Project support was provided by the Spanish Ministry of Economy and Competitiveness, Project AGL2012-37772 and FEDER. Marta Muñoz, Eva Correia, and Beatriz Trigal are supported by MICINN-RYC08-03454, MEC-FPU-AP2009-5265, and Cajastur, respectively. Emre Seli is supported by Award R01HD059909 from the National Institute of Health (NIH), USA. The authors are members of the COST Action FA1201 Epiconcept: Epigenetics and Periconception Environment.