Method for determining the sex of a fetus in an ungulate by means of hormonal assay

Abstract

The present invention relates to a method for determining the sex of a fetus in an ungulate.

Description

[0001] Method for determining the sex of a fetus in an ungulate by hormonal assay

[0002] The present invention relates to a method for determining the sex of a fetus in an ungulate by hormonal assay.

[0003] For ungulates, particularly horses, fetal sex determination is crucial for breeders to plan their herds' potential and make informed decisions, especially regarding breeding, sales, and future expenses. If the fetus is determined to be of the "optimal" sex for a particular mare or stallion, a higher selling price for the pregnant mare can be applied. Thus, fetal sex determination is a valuable decision-making tool for breeders, enabling them to implement business strategies such as deciding whether to sell a pregnant mare with a fetus of the desired sex. Furthermore, fetal sex determination also allows breeders to manage their herds more effectively in terms of planning breeding seasons.

[0004] Since 1989, transrectal (TR) ultrasound has been the gold standard for determining fetal sex between 60 and 70 days of gestation, while transabdominal (TA) ultrasound becomes necessary at later stages of gestation (120–210 days) due to fetal growth and changes in fetal position within the mare's abdomen. However, ultrasound-based fetal sex determination can be challenging and requires technical skill to obtain accurate results. This technique relies on identifying and locating the migration of the genital tubercle toward the tail in females (F) and toward the umbilical cord in males (M). The anogenital distance increases during the differentiation process in male fetuses (M).

[0005] Doppler ultrasound and detailed three-dimensional imaging are other techniques that can help determine the sex of equine fetuses, but they require sophisticated equipment and specialized technical skills. Analysis of circulating fetal DNA in the maternal bloodstream has emerged as a promising alternative method during the last trimester of gestation. However, this approach still requires improvements in sensitivity and accuracy. The diagnosis of female sex relies on the absence of the Y chromosome, which introduces a risk of false male fetuses if the amount of circulating fetal DNA is insufficient to be detected.

[0006] Unfortunately, as the above indicates, current methods for determining fetal sex are relatively complex and require technical expertise from the veterinarian. Furthermore, current methods lack precision, specificity, and sensitivity.

[0007] Since determining fetal sex has important implications for breeding strategies and stud farm management, there is currently a real need to provide a method for determining the sex of the fetus in an ungulate, particularly in the horse, which is simple while guaranteeing high specificity, high sensitivity and high accuracy.

[0008] To this end, the invention provides a method for determining the sex of a fetus in an ungulate according to a given month of gestation of the latter, comprising:

[0009] - an extraction of steroids from a sample previously taken or collected from said ungulate to obtain an extract containing steroids,

[0010] - a determination of at least one steroid present in said extract by liquid chromatography coupled to mass spectrometry to determine a concentration of said at least one steroid in said extract, said at least one steroid being chosen from the following steroids: 11-Deoxycorticosterone (11 DOC), 17b-Dihydroequiline (17bOHEQ), 17b-Dihydroequilenine (17bOHEQLN), 17a-Hydroxyprogesterone (17OHP4), 17b-Dihydroequiline 3-Sulfate (17bEQS), 21-Deoxycortisol (21 S), 5a-Dihydroprogesterone (5DHP), (3a)-Allopregnanolone (Allo), Androstenedione (A4), Cortisol, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone 3-sulfate (DHEAS), Estrone (E1), Estrone β-D-Glucuronide (E1 G), Estrone 3-Sulfate (E1 S), 17β-Estradiol (E2), 17β-Estradiol 3-β-D-Glucuronide (E2G), 17β-Estradiol 3-O-Sulfate (E2S), Equiline (EQ), Equiline 3-O-Beta-D-Glucuronide (EQG), Equilenine (EQLN), Testosterone (T), 11-Deoxycortisol (S), 20α-

[0011] Dihydroprogesterone (20DHP), 5a-Dihydrotestosterone (5DHT), Corticosterone, Progesterone (P4), Pregnenolone (P5),

[0012] - Determining the sex of the ungulate fetus

[0013] 1) based on said concentration of said at least one steroid present in said extract a) with reference to predetermined threshold values ​​of concentrations of said at least one steroid above or below which the sex of the fetus is determined to be female or male according to a given month of gestation of said ungulate, and / or b) with reference to ranges of predetermined values ​​of concentrations of said at least one steroid, said ranges of predetermined values ​​being associated with the female or male sex of the fetus according to a given month of gestation of said ungulate, and / or

[0014] 2) based on a ratio established between the concentrations of at least two steroids present in said extract a) with reference to predetermined threshold values ​​of concentration ratios established between said at least two steroids, said predetermined threshold values ​​of concentration ratios being associated with the female or male sex of the fetus according to a given month of gestation of said ungulate, and / or b) with reference to predetermined ranges of concentration ratios established between said at least two steroids, said predetermined ranges of concentration ratios being associated with the female or male sex of the fetus according to a given month of gestation of said ungulate.In the context of the present invention, the terms "threshold value" or "reference value" refer to the same thing, namely a value above or below which the sex of the fetus is determined to be female or male and / or a value indicating as such that the sex of the fetus is female or male.

[0015] Preferably, according to the invention, said predetermined threshold values ​​of concentrations of said at least one steroid, said ranges of predetermined values ​​of concentrations of said at least one steroid, said predetermined threshold values ​​of concentration ratios established between said at least two steroids and said predetermined ranges of concentration ratios established between said at least two steroids are determined by a comparative statistical analysis of steroid concentrations measured by said chromatographic method coupled with mass spectrometry between a group of samples of pregnant ungulates carrying a male fetus and a group of samples of pregnant ungulates carrying a female fetus for which a significant difference in concentration of said at least one steroid is observed / measured.

[0016] Preferably, according to the present invention, said at least one steroid is chosen from the following steroids: 17b-Estradiol 3-O-Sulfate (E2S), Estrone 3-Sulfate (E1 S), Dehydroepiandrosterone 3-sulfate (DHEAS), Estrone bD-Glucuronide (E1 G), Equiline 3-O-Beta-D-Glucuronide (EQG), 17b-Estradiol 3-bD-Glucuronide (E2G), 17b-Dihydroequiline 3-Sulfate (17bEQS).

[0017] Of course, any other steroid that can be identified, measured, and monitored during the management of an ungulate falls within the scope of the present invention, such as the following steroids (this list is not exhaustive): Testosterone sulfate, Testosterone glucuronide,

[0018] Dihydrotestosterone glucuronide, Dehydroepiandrosterone glucuronide, Aldosterone, Androstenedione sulfate, Androstenedione glucuronide, 17α-Hydroxypregnenolone (17OHP5), 17α-Estradiol, 17α-Dihydroequiline (17αOHEQ), 17α-Dihydroequilenine (17αOHEQLN), Pregnenolone sulfate (P5S), Allotetrahydrodeoxycortisone, Cortisone, Estriol, Androstanedione, Androsterone sulfate, Androsterone glucuronide, Hydroxyestrone, ... The predetermined threshold values ​​of the concentrations of said at least one steroid above or below which the sex of the fetus is determined to be female or male are determined by a comparative analysis between a group of samples from pregnant ungulates carrying a male fetus and a group of gravid ungulates carrying a female fetus for which a significant difference in concentration of said at least one steroid is observed / measured.

[0019] The predetermined ranges of concentration values ​​associated with the female or male sex of the fetus are determined by a comparative analysis between a group of samples of pregnant ungulates carrying a male fetus and a group of samples of pregnant ungulates carrying a female fetus for which a significant difference in concentration of said at least one steroid is observed / measured.

[0020] Predetermined threshold values ​​of concentration ratios established between at least two steroids and associated with the female or male sex of the fetus are determined by a comparative analysis between a group of samples of pregnant ungulates carrying a male fetus and a group of samples of pregnant ungulates carrying a female fetus for which a significant difference in concentration ratio established between at least two steroids is observed / measured.

[0021] Predetermined ranges of concentration ratios established between at least two steroids and associated with the female or male sex of the fetus are determined by comparative analysis between a group of samples of pregnant ungulates carrying a male fetus and a group of samples of pregnant ungulates carrying a female fetus for which a significant difference in concentration ratio established between at least two steroids is observed / measured.

[0022] Preferably, according to the present invention, said liquid chromatography is of the liquid chromatography-mass spectrometry (LC-MS) type, or of the liquid chromatography-tandem mass spectrometry (LC-MS / MS) type, or of the liquid chromatography-high-resolution mass spectrometry (LC / HR-MS) type. According to the present invention, the determination of at least one steroid present in the extract by liquid chromatography-mass spectrometry to determine the concentration of said at least one steroid in the extract can be a quantitative or semi-quantitative determination.

[0023] According to the present invention, the dosage of a single steroid can make it possible to determine the sex of a fetus in an ungulate according to a given month of gestation of the latter.

[0024] Preferably, according to the method following the invention, several steroids are measured simultaneously, at least one of these steroids being able to determine the sex of a fetus in an ungulate according to a given month of gestation of the latter.

[0025] Within the scope of the present invention, the following steroids can be identified, measured, and monitored during the management of an ungulate (this list is not exhaustive): 11-Deoxycorticosterone (11-DOC) (CAS number 64-85-7), 11-Deoxycortisol (S) (CAS number 152-58-9), 17b-Dihydroequiline (17bOHEQ) (CAS number 3563-27-7), 17b-Dihydroequilenin

[0026] (17bOHEQLN) (CAS number 1423-97-8), 17a-

[0027] Hydroxyprogesterone (17OHP4) (CAS number 68-96-2), 17b-Dihydroequiline 3-Sulfate (synonym of 17-(3>-dihydroequiline sulfate) (17bEQS) (CAS number 16680-49-2), 20a-Dihydroprogesterone (20DHP) (CAS number 145-14-2), 21-Deoxycortisol (21S) (CAS number 641-77-0), 5a-Dihydroprogesterone (5DHP) (CAS number 566-65-4), 5o-Dihydrotestosterone (5DHT) (CAS number 521-18-6), (3a)-Allopregnanolone (Allo) (CAS number 516-54-1), Androstenedione (A4) (CAS number 63-05-8), Corticosterone (CAS number 50-22-6), Cortisol (CAS number 50-23-7), Dehydroepiandrosterone (DHEA) (CAS number 53-43-0), Dehydroepiandrosterone 3-sulfate (synonym of dehydroepiandrosterone sulfate) (DHEAS) (CAS number 651-48-9), Estrone (E1) (CAS number 53-16-7), Estrone β-Glucuronide (synonym of estrone glucuronide) (E1 G) (CAS number 2479-90-5), Estrone 3-Sulfate (synonym of estrone sulfate) (E1 S) (CAS number 438-67-5), 17β-Estradiol (E2) (CAS number 50-28-2),17b-Estradiol 3-bD-Glucuronide (synonym of 17-(3>-estradiol glucuronide) (E2G) (CAS number 15270-30-1), 17b-Estradiol 3-O-Sulfate (synonym of 17-(3>-estradiol sulfate) (E2S) (CAS number 4999-79-5), Equiline (EQ) (CAS number 474-86-2), Equiline 3-O-Beta-D-Glucuronide (synonym of equiline glucuronide) (EQG) (CAS number 27610-12-4), Equilenin (EQLN) (CAS number 517-09-9), Progesterone (P4) (CAS number 57-83-0), Pregnenolone (P5) (CAS number 145-13-1), Testosterone (T) (CAS number 58-22-0).

[0028] The process according to the present invention is a non-invasive and simple process ensuring high specificity, high sensitivity and high precision, in particular by avoiding cross-reactions.

[0029] Preferably, according to the invention, said sample previously taken or collected from said ungulate is a blood sample, a urine sample, a fecal sample, a milk sample, a saliva sample, a sweat sample, a hair or mane sample, or even a mucus sample.

[0030] Optionally, according to the invention, said blood sample is subjected to treatment in such a way as to obtain a sample in the form of plasma or serum.

[0031] Preferably, according to the invention, the sample previously taken or collected from the ungulate is stored at a temperature below 15°C, preferably below 10°C, for 6 hours following the taking or collection of the sample before steroid extraction. It has been shown, within the scope of the present invention, that such storage prevents the alteration of steroid concentrations (conjugated or not) in the sample observed during storage at room temperature.

[0032] More preferably, according to the invention, the sample previously taken or collected from the ungulate is frozen at a temperature below 0°C, preferably below -10°C, more preferably at a temperature ranging from -20°C to -80°C, and even more preferably at -80°C, within 6 hours of the sample being taken or collected, before the steroid extraction. It has been demonstrated, within the scope of the present invention, that such a freezing step prevents the alteration of steroid concentrations (conjugated or not) in the sample observed during storage at room temperature. Preferably, according to the invention, the steroid extraction from a sample previously taken or collected from the ungulate is carried out using an organic solvent, for example, acetonitrile.

[0033] Preferably, according to the invention, said predetermined threshold values ​​of concentrations of said at least one steroid are between 1 pg / mL and 9999 ng / mL.

[0034] The present invention also relates to the use of a kit comprising the following elements for determining the sex of a fetus in an ungulate according to the method of the invention, said kit comprising:

[0035] - an extraction buffer for extracting steroids from a sample taken or collected from said ungulate,

[0036] - a reconstitution buffer to reconstitute the extracted steroids in a precise volume of solution compatible with a separation method coupled to mass spectrometry,

[0037] - a separation method, for example a chromatographic column, to separate steroids using the separation method coupled with mass spectrometry,

[0038] - at least one mobile phase to transport the extracted steroids through said separation means, for example through the chromatographic column,

[0039] - an internal standard mixture being a mixture of isotopically labeled compounds chemically similar to the steroids of interest, said internal standard mixture comprising at least one steroid being selected from the following: 11-Isotopicly labeled deoxycorticosterone (11-DOC), 17b-Isotopicly labeled dihydroequiline (17bOHEQ), 17b-Isotopicly labeled dihydroequilenine (17bOHEQLN), 17a-Isotopicly labeled hydroxyprogesterone (17OHP4), 17b-

[0040] Isotopically labeled dihydroequiline 3-sulfate (17bEQS), isotopicly labeled 21-deoxycortisol (21S), isotopicly labeled 5a-dihydroprogesterone (5DHP), isotopicly labeled (3a)-allopregnanolone (Allo), isotopicly labeled androstenedione (A4), isotopicly labeled cortisol, isotopicly labeled dehydroepiandrosterone (DHEA), isotopicly labeled dehydroepiandrosterone 3-sulfate (DHEAS), isotopicly labeled estrone (E1), isotopicly labeled cestron β-D-glucuronide (E1G), isotopicly labeled estrone 3-sulfate (E1S), isotopicly labeled 17b-estradiol (E2), 17b-estradiol Isotopicly labeled 3-β-D-Glucuronide (E2G), Isotopicly labeled 17β-C Estradiol 3-O-Sulfate (E2S), Isotopicly labeled Equiline (EQ), Isotopicly labeled Equiline 3-O-Beta-D-Glucuronide (EQG), Isotopicly labeled Equilenine (EQLN), Isotopicly labeled Testosterone (T), Isotopicly labeled 11-Deoxycortisol (S), Isotopicly labeled 20α-Dihydroprogesterone (20DHP), 5α-

[0041] Isotopically labeled dihydrotestosterone (5DHT), Isotopicly labeled corticosterone, Isotopicly labeled progesterone (P4), Isotopicly labeled pregnenolone (P5)

[0042] - a calibration solution for calibrating the separation method coupled with mass spectrometry, said calibration solution comprising at least one steroid selected from the following: 11-Deoxycorticosterone (11 DOC), 17b-Dihydroequiline (17bOHEQ), 17b-Dihydroequilenine (17bOHEQLN), 17a-Hydroxyprogesterone (17OHP4), 17b-Dihydroequiline 3-Sulfate (17bEQS), 21-Deoxycortisol (21 S), 5a-Dihydroprogesterone (5DHP), (3a)-Allopregnanolone (Allo), Androstenedione (A4), Cortisol, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone 3-Sulfate (DHEAS), Estrone (E1), Estrone bD-Glucuronide (E1 G), Estrone 3-Sulfate (E1 S), 17b-Estradiol (E2), 17b-Estradiol 3-bD-Glucuronide (E2G), 17b-Estradiol 3-O-Sulfate (E2S), Equiline (EQ), Equiline 3-O-Beta-D-Glucuronide (EQG), Equilenine (EQLN), Testosterone (T), 11-Deoxycortisol (S), 20a-Dihydroprogesterone (20DHP), 5o-Dihydrotestosterone (5DHT), Corticosterone, Progesterone (P4), Pregnenolone (P5).

[0043] Advantageously, the kit for use according to the invention further includes a pre-column.

[0044] Preferably, the kit for use according to the invention further includes at least one control solution for monitoring the accuracy and precision of the separation method coupled with mass spectrometry.

[0045] Advantageously, the kit for use according to the invention further includes a rinsing solution for rinsing the separation medium and the system used for the mass spectrometry-coupled separation method. Typically, such a rinsing solution is used to clean the separation medium (e.g., the chromatographic column) and to clean the system used for the mass spectrometry-coupled separation method (e.g., the LC-MS or LC-MS / MS system) between analyses to prevent cross-contamination between samples.

[0046] Preferably, a kit for use according to the present invention further comprises a well plate, for example a multi-well plate for holding samples ready to be analyzed by the separation method coupled with mass spectrometry. Such a plate is placed in an autosampler which injects the samples into the separation medium, in particular into the chromatographic column.

[0047] Typically, the internal standard mixture is added to the samples before analysis using the separation method coupled with mass spectrometry. Isotopicly labeled compounds, chemically similar to the steroids of interest, allow for correction of analytical variations and ensure accurate quantification of the steroids.

[0048] Typically, reconstitution buffer is used after evaporation of extraction buffer to resuspend samples.

[0049] According to the invention, the kit for use according to the present invention may include several control solutions, preferably a level 1 control solution (low concentration solution), a level 2 control solution (medium concentration solution) and a level 3 control solution (high concentration solution) to control the accuracy and precision of the separation method coupled with mass spectrometry, particularly in clinical diagnostics, for the quantitative determination of steroids in the sample.

[0050] According to the invention, the standard solution is a multi-level standard solution with varying concentrations, including a blank and a double blank. Such a standard solution is intended to calibrate the measurement method, particularly in clinical diagnostics, for the quantitative determination of steroids in the sample.

[0051] Other features, details, and advantages of the invention will become apparent from the example given below, which is not intended to be limiting. Example

[0052] To determine fetal sex, various steroid levels were measured for each month of gestation in ungulates to determine if significant differences in steroid concentrations were observed for a given month of gestation when the fetus was male or female. Based on steroid concentration data obtained from a group of samples of pregnant ungulates carrying a male fetus and from a group of samples of pregnant ungulates carrying a female fetus, a comparative statistical analysis was performed to define, in particular, threshold steroid concentration values ​​above or below which the fetal sex is determined to be male or female for each month of gestation.

[0053] Animals and sampling

[0054] Between November 2020 and June 2024, a cohort of pregnant mares was selected. The mares were housed and managed under standardized conditions in terms of nutrition, breeding techniques, and exercise levels. No mares intended as embryo recipients or suffering from pregnancy-related pathologies were selected.

[0055] A total of 82 pregnant mares were considered and inseminated once or several times between November 2020 and June 2024, resulting in 147 pregnancies. For each mare considered, age, breed, parity, and gestational age were recorded, and fetal sex was confirmed by direct clinical examination after birth.

[0056] Once a month, from 4 months of gestation until parturition, a transrectal ultrasound (TR) was performed and the combined thickness of the uterus and placenta (CTUP) was measured using the EXAPad Mini® with a 7.5 to 10 MHz linear probe (IMV Imaging, Angoulême, France) according to the technique described by Renaudin et al. (Ultrasonographic evaluation of the equine placenta by transrectal and transabdominal approach in the normal pregnant mare. Theriogenology, 1997, 47:559-573) to diagnose possible placentitis.

[0057] Exclusion criteria include signs of placental pathology, including an increased mean uterine prolapse (UTP) (Bucca S. Diagnosis of the compromised equine pregnancy. Vet. Clin. North Am. Equine Pract, 2006, 22(3)749-761), heterogeneous UTP or allantoic fluid, placental abruption, vulvar discharge, premature mammary development, abortion, and a weak foal at birth, as well as histological examination of the placenta. If the cervical portion of the allantochorion appears thickened and / or congested after postnatal macroscopic examination, the mare is also excluded from the study. After applying exclusion criteria (not pregnant, placental pathology, missing data, euthanasia), 71 mares were selected, including 31 Purebred Spanish (PRE) mares, 33 show jumping (SJ) type mares, 2 Icelandic breed mares and 5 Trotter breed mares.

[0058] In parallel, blood samples were collected via the jugular vein into dry tubes (Vacuette® CAT serum sep clot activator tubes). The samples were collected at the same time of day, and the blood tubes were centrifuged (1500 x g for 15 min) within two hours of collection (Hettich zentrifugen EBA 200TM, Tuttlingen, Germany). The serum was divided into three 1 mL aliquots, and the samples were then stored in a freezer at -80°C until analysis. For each blood sample, the time in days between the date of insemination and the date of blood collection was calculated. Blocks of 4 weeks, or 28 days, were defined as follows and correspond to months of gestation (M) (Table 1):

[0059] Table 1

[0060] Steroid dosages

[0061] The concentrations of the following steroids were determined in maternal serum samples for each month of gestation using a validated LC-MS / MS method according to current recommendations: 11-Deoxycorticosterone (11 DOC), 17b-Dihydroequiline (17bOHEQ), 17b-Dihydroequilenine (17bOHEQLN), 17a-Hydroxyprogesterone (17OHP4), 17b-Dihydroequiline 3-Sulfate (17bEQS), 21-Deoxycortisol (21 S), 5a-Dihydroprogesterone (5DHP), (3a)-Allopregnanolone (Allo),

[0062] Androstenedione (A4), Cortisol, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone 3-sulfate (DHEAS), Estrone (E1), Estrone βD-Glucuronide (E1 G), Estrone 3-Sulfate (E1 S), 17β-Estradiol (E2), 17β-Estradiol 3-βD-Glucuronide (E2G), 17β-Estradiol 3-O-Sulfate (E2S), Equiline (EQ), Equiline 3-O-Beta-D-Glucuronide (EQG), Equilenine (EQLN), Testosterone (T), 11-Deoxycortisol (S), 20α-Dihydroprogesterone (20DHP), 5α-

[0063] Dihydrotestosterone (5DHT), Corticosterone, Progesterone (P4), Pregnenolone (P5).

[0064] This method involves the extraction of 400 pL of serum with 1200 pL of acetonitrile and 20 pL of an internal standard mixture [11-deoxycorticosterone-d7 (1 mg / mL) (CAS number 64-85-7), 11-deoxycortisol-d5 (1 mg / mL) (CAS number 1258063-56-7), 17b-Dihydroequiline-16, 16, 17-d3 (10 pg / mL) (CAS number 350820-03-0), 17a-Hydroxyprogesterone-D8 (100 pg / mL) (CAS number 850023-80-2), 20o-Dihydroprogesterone-d4 (10 pg / mL) (CAS number not available), 5o-Dihydroprogesterone-d3 (100 pg / mL) (CAS number 79037-34-6), (3a)-Allopregnanolone-d5 (1 mg / mL) (CAS number 516-54-1), 4-Androstene-3,17-dione-2,3,4-13C3 (1 mg / mL) (CAS number 327048-86-2), Cortisol-d4 (10 pg / mL) (CAS number 73565-87-4), Dehydroepiandrosterone-d5 (1 mg / mL) (CAS number 97453-25-3), Dehydroepiandrosterone-d5-3-sulfate (10 pg / mL) (CAS number not available), Estrone-d4 (10 pg / mL) (CAS number 53866-34-5), D4-Estrone 3-Sulfate (1 mg / mL) (CAS number 285979-80-8), 17beta-Estradiol-2,4,16,16,17-d5 (1 mg / ml) (CAS number 221093-45-4),Sodium 17betaC Estradiol-16,16,17-d3 3-glucuronide (1 mg / mL) (CAS number not available), 17beta-C Estradiol 3-O- Sulfate-d3 (10 pg / mL) (CAS number 4999-79-5), Equiline-d4 (10 pg / mL) (CAS number 285979-79-5), Progesterone-d9 (10 pg / mL) (CAS number 15775-74-3), Pregnenolone-d4 (1 mg / mL) (CAS number 61574-54-7), Testosterone-2,3,4-13C3 (10 pg / mL) (CAS number 327048-83-9)], followed by centrifugation at 13600 rpm for 10 minutes at 4°C.

[0065] A volume of 1500 pL of supernatant is collected, transferred to a 96-well plate, and evaporated to dryness at 45°C. Then, 45 pL of dansyl chloride and 30 pL of carbonate / bicarbonate buffer are added to enhance the ionization of at least one given steroid, and the mixture is incubated in an oven at 60°C for 15 minutes. Finally, 50 pL of water is added to stop the reaction, before centrifugation at 3600 rpm for 10 minutes at 4°C. Next, the samples obtained are injected with a volume of 7 pL for analysis using the ExionLC Series UHPLC system (ABSciex, Framingham, Massachusetts, USA) equipped with an ACQUITY UPLC BEH C18 column (2.1 mm x 150 mm, 1.7 pm; Waters, Milford, USA) and a linear combination of triple quadrupole and QTrap 6500+ mass analyzer (ABSciex, Framingham, Massachusetts, USA) operating in triple-quadrupole mode.

[0066] A skilled professional is perfectly capable of determining and adjusting the usual analytical parameters of LC-MS or LC-MS / MS, whether it be mass transitions for the quantification and qualification of steroids or instrumental conditions (e.g., source, fragmentation, chromatography and ionization parameters).

[0067] The composition of mobile phase A is 0.1% formic acid in water, and mobile phase B is 0.1% formic acid in acetonitrile. The injection rate is 0.5 mL / min, and the separation is performed using a gradient. The initial mobile phase composition is 25% B, increasing to 45% over 14 minutes. Between 14 and 40 minutes, the percentage of B is increased linearly to 69%. Between 40 and 40.1 minutes, the percentage of B reaches 95%, a proportion maintained for 2 minutes before returning to the initial gradient conditions. A person skilled in the art is perfectly capable of determining the appropriate calibration curve for each steroid, particularly by choosing the suitable regression model with or without weighting.

[0068] The limits of quantification are established as follows: 11 DOC (4 pg / mL), S (9.9 pg / mL), 17bOHEQ (9.9 pg / mL), 17bOHEQLN (2.07 pg / mL), 17OHP4 (9.9 pg / mL), 17bEQS (51.75 pg / mL), 20DHP (9.9 pg / mL), 21 S (2000 pg / mL), 5DHP (207 pg / mL), 5DHT (9.9 pg / mL), Allo (2250 pg / mL), A4 (9.9 pg / mL), Corticosterone (2.07 pg / mL), Cortisol (207 pg / mL), DHEA (247.5 pg / mL), DHEAS (51 .75pg / mL)„ E1 (9.9pg / mL), E1 G (247.5pg / mL), E1 S (2000pg / mL), E2 (2.07pg / mL), E2G (2.07pg / mL), E2S (51.75pg / mL), EQ (2.07pg / mL), EQG (247.5pg / mL), EQLN (2.07pg / mL), P4 (51.75pg / mL), P5 (100pg / mL), T (2.07pg / mL).

[0069] Statistical analyses and determination of threshold values ​​for steroid concentrations

[0070] All statistical analyses were performed using SAS software (version 9.4). The threshold for statistical significance was set at p < 0.05.

[0071] To determine the threshold values ​​for steroid concentrations, a comparative statistical analysis as described below can be carried out.

[0072] The Shapiro-Wilk test is used to assess the normality in the distribution of steroid concentration data obtained from a group of samples from pregnant ungulates carrying a male fetus and from a group of samples from pregnant ungulates carrying a female fetus.

[0073] Due to the non-normal distribution of some data, logarithmic or square root transformations may be performed. A skilled professional is perfectly capable of determining which type of transformation can be applied when the distribution is non-normal for certain data.

[0074] It should be noted that it has been determined that there is no influence of the breed, age and parity of the mares.

[0075] If a univariate approach is used in the comparative statistical analysis, a univariate logistic regression model is used to assess the association, for a given month of gestation, between steroid concentration (or a variable such as age, race, or parity) and fetal sex. The following equation was used: L = logit(p) = ln(p / (1 - p)) = [30 + P1X with p = 1 / (1 + e A (-L)) = 1 / (1 + e A (- J3O+|31X)), p determining the probability that the fetus is male. The optimal threshold values ​​were determined using Youden's index.

[0076] If a multivariate approach is used in the comparative statistical analysis, a multivariate logistic regression model is used to assess the association, for a given month of gestation, between the concentration of at least one steroid and fetal sex, possibly taking into account the age, breed, and / or parity of the mares. The following equation was used: L = logit(p) = ln(p / (1 - p)) = [30 + [31X1 + (32X2 + ... + [3kXk] with p = 1 / (1 + ... + e A (-L)) = 1 / (1 + e A (- p0 + piX1 + p2X2 + - + pkXk)), where p determines the probability that the fetus is male. Optimal threshold values ​​were determined using Youden's index.

[0077] Optionally, the performance evaluation of the univariate or multivariate approach is assessed by ROC curves with a confidence interval set at 95%.

[0078] If several concentration results for different steroids are available for a given month of gestation of the ungulate, the determination of fetal sex will be carried out from the steroid with the highest discriminating power between males and females, the discriminating power being based on the p-value determined for a given steroid.

[0079] Such a comparative statistical analysis for determining threshold / reference values ​​is common practice for those skilled in the art, who may modify it without departing from the scope of the present invention. It is understood that any other method or statistical analysis for determining threshold / reference values ​​falls within the scope of the present invention. It is also understood that the comparative statistical analysis as presented above could take into account other additional data / variables.

[0080] The predetermined threshold values ​​of the concentrations of said at least one steroid are linked to the assay method and the parameters applied during the implementation of this assay method, but also to the comparative statistical analysis implemented and the variables considered for this comparative statistical analysis.

[0081] Results

[0082] Table 2 shows, for each month of gestation, the threshold values ​​for steroid concentrations (± 10%) above or below which the fetus is determined to be male. The significance levels (corresponding to the difference in concentration between mares carrying male and female fetuses) are indicated for each steroid (p-value). The threshold values ​​shown in Table 2 were determined by a comparative statistical analysis using a univariate approach as described above.

[0083] Table 3 shows, for each month of gestation, the threshold values ​​for concentration ratios between the steroids E2S and 17bEQS (± 10%) above or below which the sex of the fetus is determined to be male. Significance levels are indicated for each steroid (p-value). The threshold values ​​for concentration ratios between the steroids E2S and 17bEQS, as shown in Table 3, were determined by a comparative statistical analysis using a univariate approach as described above.

[0084] For a given month of gestation, based on the threshold values ​​of steroid concentrations as presented in Table 2, it is possible to determine whether the sex of the fetus is male or female by carrying out an assay of at least one steroid from a sample previously taken or collected from an ungulate and by comparing the concentration measured during this assay for said at least one steroid to predetermined threshold values ​​of concentrations of said at least one steroid above or below which the sex of the fetus is determined to be female or male according to a given month of gestation of said ungulate.

[0085] For a given month of gestation, based on the threshold values ​​of concentration ratios established between at least two steroids as presented in Table 3, it is possible to determine whether the sex of the fetus is male or female by carrying out a assay of at least two steroids from a sample previously taken or collected from an ungulate, by establishing a concentration ratio between said at least two steroids and by comparing this concentration ratio to predetermined threshold values ​​of concentration ratios established between said at least two steroids, said predetermined threshold values ​​of concentration ratios being associated with the female or male sex of the fetus according to a given month of gestation of said ungulate.

[0086] The present invention has been described in relation to specific embodiments, which are purely illustrative and should not be considered limiting. Generally, it will be obvious to those skilled in the art that the present invention is not limited to the examples illustrated and / or described above.

[0087] The use of the verbs "comprendre", "includer", "comporter", or any other variant, as well as their conjugations, cannot in any way exclude the presence of elements other than those mentioned.

[0088] The use of the indefinite article "un", "une", or the definite article "le", "la" or "I'", to introduce an element does not exclude the presence of a plurality of these elements.

[0089] Table 2 Table 3

Claims

1. Demands 1. A method for determining the sex of a fetus in an ungulate according to a given month of gestation of the latter, comprising: - an extraction of steroids from a sample previously taken or collected from said ungulate to obtain an extract containing steroids, - a determination of at least one steroid present in said extract by liquid chromatography coupled with mass spectrometry to determine a concentration of said at least one steroid in said extract, said at least one steroid being chosen from the following steroids: 11-Deoxycorticosterone (11 DOC), 17b-Dihydroequiline (17bOHEQ), 17b-Dihydroequilenine (17bOHEQLN), 17a-Hydroxyprogesterone (17OHP4), 17b-Dihydroequiline 3-Sulfate (17bEQS), 21-Deoxycortisol (21 S), 5a-Dihydroprogesterone (5DHP), (3a)-Allopregnanolone (Allo), Androstenedione (A4), Cortisol, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone 3-sulfate (DHEAS), Estrone (E1), Estrone β-Glucuronide (E1 G), Estrone 3-Sulfate (E1 S), 17β-Estradiol (E2), 17β-Estradiol 3-β-Glucuronide (E2G), 17β-Estradiol 3-O-Sulfate (E2S), Equiline (EQ), Equiline 3-O-Beta-D-Glucuronide (EQG), Equilenine (EQLN), Testosterone (T), 11-Deoxycortisol (S), 20α- Dihydroprogesterone (20DHP), 5a-Dihydrotestosterone (5DHT), Corticosterone, Progesterone (P4), Pregnenolone (P5), - Determining the sex of the ungulate fetus 1) based on said concentration of said at least one steroid present in said extract a) with reference to predetermined threshold values ​​of concentrations of said at least one steroid above or below which the sex of the fetus is determined to be female or male according to a given month of gestation of said ungulate, and / or b) with reference to ranges of predetermined values ​​of concentrations of said at least one steroid, said ranges of predetermined values ​​being associated with the female or male sex male sex of the fetus according to a given month of gestation of said ungulate, and / or 2) based on a ratio established between the concentrations of at least two steroids present in said extract a) with reference to predetermined threshold values ​​of concentration ratios established between said at least two steroids, said predetermined threshold values ​​of concentration ratios being associated with the female or male sex of the fetus according to a given month of gestation of said ungulate, and / or b) with reference to predetermined ranges of concentration ratios established between said at least two steroids, said predetermined ranges of concentration ratios being associated with the female or male sex of the fetus according to a given month of gestation of said ungulate.

2. A method according to claim 1, characterized in that said predetermined threshold values ​​of concentrations of said at least one steroid, said ranges of predetermined values ​​of concentrations of said at least one steroid, said predetermined threshold values ​​of concentration ratios established between said at least two steroids and said predetermined ranges of concentration ratios established between said at least two steroids are determined by a comparative statistical analysis of steroid concentrations measured by said chromatographic method coupled with mass spectrometry between a group of samples of pregnant ungulates carrying a male fetus and a group of samples of pregnant ungulates carrying a female fetus for which a significant difference in concentration of said at least one steroid is observed / measured.

3. A method according to claim 1 or 2, characterized in that said liquid chromatography is of the liquid chromatography coupled with mass spectrometry (LC-MS) type or of the liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS) type or of the type liquid chromatography coupled with high-resolution mass spectrometry (LC / HR-MS)...

4. A method according to any one of the preceding claims, characterized in that said sample previously taken or collected from said ungulate is a blood sample, a urine sample, a fecal sample, a milk sample, a saliva sample, a sweat sample, a hair or mane sample, or a mucus sample.

5. A method according to any one of the preceding claims, characterized in that said sample previously taken or collected from said ungulate is kept at a temperature below 15°C, preferably below 10°C, within 6 hours following the taking or collection of the sample before the extraction of the steroids.

6. A method according to any one of the preceding claims, characterized in that said sample previously taken or collected from said ungulate is frozen at a temperature below 0°C, preferably below -10°C, more preferably at a temperature ranging from -20°C to -80°C, even more preferably at a temperature of -80°C, within 6 hours of taking or collecting the sample before extracting the steroids.

7. A method according to any one of the preceding claims, characterized in that said extraction of steroids from a sample previously taken or collected from said ungulate is carried out using an organic solvent, for example using acetonitrile.

8. A method according to any one of the preceding claims, characterized in that said predetermined threshold values ​​of concentrations of said at least one steroid are between 1 pg / mL and 9999 ng / mL.

9. Use of a kit comprising the following elements for determining the sex of a fetus in an ungulate according to the method described in any one of claims 1 to 8, said kit comprising: - an extraction buffer for extracting steroids from a sample taken or collected from said ungulate, - a reconstitution buffer to reconstitute the extracted steroids in a precise volume of solution compatible with a separation method coupled to mass spectrometry, - a separation method, for example a chromatographic column, to separate steroids using the separation method coupled with mass spectrometry, - at least one mobile phase to transport the extracted steroids through said separation means, for example through the chromatographic column, - an internal standard mixture being a mixture of isotopically labeled compounds chemically similar to the steroids of interest, said internal standard mixture comprising at least one steroid being selected from the following: 11-Isotopicly labeled deoxycorticosterone (11-DOC), 17b-Isotopicly labeled dihydroequiline (17bOHEQ), 17b-Isotopicly labeled dihydroequilenine (17bOHEQLN), 17a-Isotopicly labeled hydroxyprogesterone (17OHP4), 17b- Isotopically labeled dihydroequiline 3-sulfate (17bEQS), isotopicly labeled 21-deoxycortisol (21S), isotopicly labeled 5a-dihydroprogesterone (5DHP), isotopicly labeled (3a)-allopregnanolone (Allo), isotopicly labeled androstenedione (A4), isotopicly labeled cortisol, isotopicly labeled dehydroepiandrosterone (DHEA), isotopicly labeled dehydroepiandrosterone 3-sulfate (DHEAS), isotopicly labeled estrone (E1), isotopicly labeled estrone β-D-glucuronide (E1G), isotopicly labeled estrone 3-sulfate (E1S), isotopicly labeled 17b-estradiol (E2), 17b-estradiol Isotopicly labeled 3-bD-Glucuronide (E2G), isotopicly labeled 17b-Estradiol 3-O-Sulfate (E2S), isotopicly labeled Equiline (EQ), Equiline 3-O-Beta-D-Glucuronide isotopically labeled (EQG), Equilenine isotopically labeled (EQLN), Testosterone isotopically labeled (T), Deoxycortisol isotopically labeled (S), Dihydroprogesterone isotopically labeled (20DHP), 5a- Isotopically labeled dihydrotestosterone (5DHT), Isotopicly labeled corticosterone, Isotopicly labeled progesterone (P4), Isotopicly labeled pregnenolone (P5) - a calibration solution for calibrating the separation method coupled to mass spectrometry, said calibration solution comprising at least one steroid being selected from the following: 11-Deoxycorticosterone (11 DOC), 17b-Dihydroequiline (17bOHEQ), 17b-Dihydroequilenine (17bOHEQLN), 17a-Hydroxyprogesterone (17OHP4), 17b-Dihydroequiline 3-Sulfate (17bEQS), 21-Deoxycortisol (21 S), 5a-Dihydroprogesterone (5DHP), (3a)-Allopregnanolone (Allo), Androstenedione (A4), Cortisol, Dehydroepiandrosterone (DHEA), Dehydroepiandrosterone 3-Sulfate (DHEAS), Estrone (E1), Estrone β-D-Glucuronide (E1 G), Estrone 3-Sulfate (E1 S), 17β-Estradiol (E2), 17β-Estradiol 3-βD-Glucuronide (E2G), 17β-Estradiol 3-O-Sulfate (E2S), Equiline (EQ), Equiline 3-O-β-D-Glucuronide (EQG), Equilenine (EQLN), Testosterone (T), 11-Deoxycortisol (S), 20α-Dihydroprogesterone (20DHP), 5a-Dihydrotestosterone (5DHT), Corticosterone, Progesterone (P4), Pregnenolone (P5).

10. Use of a kit according to claim 9, said kit further comprising a pre-column.

11. Use of a kit according to claim 9 or 10, said kit further comprising at least one control solution for monitoring the accuracy and precision of the separation method coupled with mass spectrometry.

12. Use of a kit according to any one of claims 9 to 11, said kit further comprising a rinsing solution for rinsing the means of separation and the system used for the separation method coupled with mass spectrometry.

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