**4. Hormonal regulation of pregnancy in normal mares**

#### **4.1 Progesterone**

"Maternal recognition of gestation-MGR" it is essential to establish a complete and uninterrupted interaction between the uterus and the conceptus to prevent the regression of primary CL as a result of the blocking of luteolysis. The mobility of the conceptus within the uterine lumen between days 11 and 15 (or "first luteal response of pregnancy"); [27] seem to compensate for the reduced contact surface due to the relatively small size of the equine trophoblast, demonstrating that restriction of movement only partially leads to early embryo loss [105]. The PGs synthesized and secreted by the concept itself stimulate myometrial contractions that promote their migration through the uterus, avoiding premature regression of CL. Additionally, the longitudinal direction of the uterine folds, as well as the spherical shape of the embryo due to the persistence of the glycoprotein capsule, contribute to facilitating this movement [106, 107]. During the mobility phase and its subsequent fixation uterine high amounts of estrogen, mainly oestrone sulfate (E1S) by the equine conceptus are synthesized, related to the development of the embryonic and endometrial vasculature and local effects on myometrial activity, uterine mobility and endometrial gland secretion [108, 109].

Embryo implantation begins around day 36 post-ovulation and involves the development of the chorionic band from the trophoblast, whose cells invade the maternal endometrium giving rise to endometrial cups [110]. Ginther [28] reported that the embryonic cup cells produce a hormone called equine chorionic gonadotropin (eCG), formerly known as pregnant mare's serum gonadotropin. This hormone is first detectable systemically between days 35 and 40 of pregnancy. The cups are mature and robustly secreting eCG at approximately days 50–60, but they will subsequently undergo sloughing by days 100–150 in most mares This resurgence phase of P4 secretion by the primary CL is termed the "secondary luteal phase or output 2," whereas the production by supplementary CL is termed the "third luteal phase" or "output 3". These accessory CLs formed, respectively, causing an increase in P4 secretion around the 75th day of gestation [27, 28, 111]. Thus, during this period, two secretion peaks of P4 are described, which gradually decreasing to undetectable levels at the 200 days of gestation [112, 113].

Ovarian P4 is necessary for the early maintenance of gestation in the mare until 150 days of pregnancy. After the regression of CLs, the placenta is then the organ in charge of maintaining gestation [114]. Several studies describe maximum levels of P4 during the second and third months of gestation, followed by a significant decrease to minimum values (<1 ng/ml) from mid-gestation to term [115]. Additionally, the presence of eCG causes a change in luteal steroidogenesis. In this case, CL changes from synthesizing only P4 to secreting also estrogens and androgens, increasing plasma levels rapidly and tripling the basal values [116]. However, it is not until approximately day 35 that systemic estrogen rises. The source of this estrogen is the ovary, more specifically, the CL and possibly follicles. The stimulation of the ovaries by eCG is responsible for the timing of this increase in estrogen. It appears that estrogen is not actually necessary for pregnancy

**15**

**4.2 Progestagens**

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy…*

maintenance, because ovariectomized mares administered only exogenous progestins will maintain pregnancy without the administration of estrogens [28]. The origin of both steroids is found in the primary CL, since their increase takes place before the formation of the secondary CLs and is absent in mares without functional CL. Although the mechanism by which gonadotropin exerts this activity is unknown, an increase in the expression of the enzyme 17α-hydroxylase in charge of the conversion of P5 into dehydroepiandrosterone (DHEA) and P4 into A4 has been described. Both events coincide with the secretion of eCG, they seem to be limited to the first period since they are not detected towards the middle of gestation [116]. The increase in P4 responds primarily to the growth of primary CL and the develop-

During the period of endometrial cups activity, secretion peaks are described for testosterone (T) and A4 [118, 119], whose activity may be decisive in uterine processes related to cell transformation associated with decidualization [120]. In addition, estrogen production depends on the increased synthesis and availability of androgens that are subsequently metabolized by the enzyme aromatase, present in luteal tissue even before eCG secretion. Thus, total estrogen levels are like right-handed during the first 35 days of gestation and increase around day 40 due to follicular development before the formation of CL [121]. Additionally, primary gestational CL produces E1S in response to eCG stimulation [113, 115, 118]. The regression of the endometrial cups to 100–120 days of gestation causes the cessation of eCG secretion and luteal development, observing a progressive decrease in plasma levels of P4 to reach basal values around 200 days of gestation [115]. Currently, all the luteal structures present in the ovary have completely involuted [27]. From this moment onwards, various metabolites derived from P4 (progestins) increase in the systemic circulation, that exceed 500 ng/ml during the last weeks of gestation, which subsequently fall in the 24–48 h prior to birth [122].

Progestins can be subclassified as pregnenes and 5α-pregnenes. The pregnenes includes P5, P4 and 5-pregnene-3β,20β-diol (P5ββ), while 5α-pregnenes includes 5α-pregnane-3,20-dione (5αDHP), 3β-hydroxy-5α-pregnan-3-one (3β5P), 20α-hydroxy-5α-pregnan-3-one (20α5P), 5α-pregnane-3β,20β-diol (ββ-diol) and 5α-pregnane-3β,20α-diol (βα-diol). Of them, the most important ones in maternal plasma during this period are the 5αDHP and its derivatives, 20α5P, and βα-diol. The origin of all of them is found in P5, synthesized mainly in the fetal adrenal gland, with a production rate exceeding 10 μmol/min. In the placenta, P5 is converted to P4 and this is transformed into 5αDHP in the endometrium [123]. The pattern of secretion of 5αDHP at beginning of gestation runs parallel to that of P4, while around 90 days the onset of P4 decline gives way to fetoplacental synthesis of the different progestogens whose concentrations continue to increase during the second half of gestation. Thus, 20α5P, which is initially at 5 ng/ml, reaches 69 ng/ ml at 200 days of gestation and 300 ng/ml at term. In addition, the concentrations of βα-diol increase to 484 ng/ml [112], while 3β5P, P5ββ and ββ-diol reach values of

100, 10 and 100 ng/ml, respectively, towards the end of gestation [124].

The 5αDHP is found primarily at the uterine level during midgestation, but as labor approaches, its distribution changes and is predominantly in fetal circulation. This metabolite is an immediate precursor of allopregnanolone, a potent gamma-aminobutyric acid (GABA) receptor agonist with activity on myometrial relaxation in other species [125–127]. Serum allopregnanolone increases similarly to its precursor, reaching maximum values at the middle of gestation and a term [112]. However, both P4 and 5αDHP prevent weakly myometrial contractions induced by

*DOI: http://dx.doi.org/10.5772/intechopen.90387*

ment of secondary and accessory CLs [4, 117].

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy… DOI: http://dx.doi.org/10.5772/intechopen.90387*

maintenance, because ovariectomized mares administered only exogenous progestins will maintain pregnancy without the administration of estrogens [28]. The origin of both steroids is found in the primary CL, since their increase takes place before the formation of the secondary CLs and is absent in mares without functional CL. Although the mechanism by which gonadotropin exerts this activity is unknown, an increase in the expression of the enzyme 17α-hydroxylase in charge of the conversion of P5 into dehydroepiandrosterone (DHEA) and P4 into A4 has been described. Both events coincide with the secretion of eCG, they seem to be limited to the first period since they are not detected towards the middle of gestation [116]. The increase in P4 responds primarily to the growth of primary CL and the development of secondary and accessory CLs [4, 117].

During the period of endometrial cups activity, secretion peaks are described for testosterone (T) and A4 [118, 119], whose activity may be decisive in uterine processes related to cell transformation associated with decidualization [120]. In addition, estrogen production depends on the increased synthesis and availability of androgens that are subsequently metabolized by the enzyme aromatase, present in luteal tissue even before eCG secretion. Thus, total estrogen levels are like right-handed during the first 35 days of gestation and increase around day 40 due to follicular development before the formation of CL [121]. Additionally, primary gestational CL produces E1S in response to eCG stimulation [113, 115, 118].

The regression of the endometrial cups to 100–120 days of gestation causes the cessation of eCG secretion and luteal development, observing a progressive decrease in plasma levels of P4 to reach basal values around 200 days of gestation [115]. Currently, all the luteal structures present in the ovary have completely involuted [27]. From this moment onwards, various metabolites derived from P4 (progestins) increase in the systemic circulation, that exceed 500 ng/ml during the last weeks of gestation, which subsequently fall in the 24–48 h prior to birth [122].

#### **4.2 Progestagens**

*Animal Reproduction in Veterinary Medicine*

**4.1 Progesterone**

triggering the onset of puberty [102, 103]. A study in pony mares demonstrated the anticipated ovulation when treated with 10 mg of kisspeptin. Another report identified that the administration of 500 μg and 1.0 mg of kisspeptin induces indistinguishable LH and FSH responses to 25 μg GnRH. However, a single injection of 1.0 mg of kisspeptin (iv) was insufficient to induce ovulation in the mare in heat [104].

"Maternal recognition of gestation-MGR" it is essential to establish a complete and uninterrupted interaction between the uterus and the conceptus to prevent the regression of primary CL as a result of the blocking of luteolysis. The mobility of the conceptus within the uterine lumen between days 11 and 15 (or "first luteal response of pregnancy"); [27] seem to compensate for the reduced contact surface due to the relatively small size of the equine trophoblast, demonstrating that restriction of movement only partially leads to early embryo loss [105]. The PGs synthesized and secreted by the concept itself stimulate myometrial contractions that promote their migration through the uterus, avoiding premature regression of CL. Additionally, the longitudinal direction of the uterine folds, as well as the spherical shape of the embryo due to the persistence of the glycoprotein capsule, contribute to facilitating this movement [106, 107]. During the mobility phase and its subsequent fixation uterine high amounts of estrogen, mainly oestrone sulfate (E1S) by the equine conceptus are synthesized, related to the development of the embryonic and endometrial vasculature and local effects on myometrial activity,

Embryo implantation begins around day 36 post-ovulation and involves the development of the chorionic band from the trophoblast, whose cells invade the maternal endometrium giving rise to endometrial cups [110]. Ginther [28] reported that the embryonic cup cells produce a hormone called equine chorionic gonadotropin (eCG), formerly known as pregnant mare's serum gonadotropin. This hormone is first detectable systemically between days 35 and 40 of pregnancy. The cups are mature and robustly secreting eCG at approximately days 50–60, but they will subsequently undergo sloughing by days 100–150 in most mares This resurgence phase of P4 secretion by the primary CL is termed the "secondary luteal phase or output 2," whereas the production by supplementary CL is termed the "third luteal phase" or "output 3". These accessory CLs formed, respectively, causing an increase in P4 secretion around the 75th day of gestation [27, 28, 111]. Thus, during this period, two secretion peaks of P4 are described, which gradually decreasing to undetectable

Ovarian P4 is necessary for the early maintenance of gestation in the mare until 150 days of pregnancy. After the regression of CLs, the placenta is then the organ in charge of maintaining gestation [114]. Several studies describe maximum levels of P4 during the second and third months of gestation, followed by a significant decrease to minimum values (<1 ng/ml) from mid-gestation to term [115]. Additionally, the presence of eCG causes a change in luteal steroidogenesis. In this case, CL changes from synthesizing only P4 to secreting also estrogens and androgens, increasing plasma levels rapidly and tripling the basal values [116]. However, it is not until approximately day 35 that systemic estrogen rises. The source of this estrogen is the ovary, more specifically, the CL and possibly follicles. The stimulation of the ovaries by eCG is responsible for the timing of this increase in estrogen. It appears that estrogen is not actually necessary for pregnancy

**4. Hormonal regulation of pregnancy in normal mares**

uterine mobility and endometrial gland secretion [108, 109].

levels at the 200 days of gestation [112, 113].

**14**

Progestins can be subclassified as pregnenes and 5α-pregnenes. The pregnenes includes P5, P4 and 5-pregnene-3β,20β-diol (P5ββ), while 5α-pregnenes includes 5α-pregnane-3,20-dione (5αDHP), 3β-hydroxy-5α-pregnan-3-one (3β5P), 20α-hydroxy-5α-pregnan-3-one (20α5P), 5α-pregnane-3β,20β-diol (ββ-diol) and 5α-pregnane-3β,20α-diol (βα-diol). Of them, the most important ones in maternal plasma during this period are the 5αDHP and its derivatives, 20α5P, and βα-diol. The origin of all of them is found in P5, synthesized mainly in the fetal adrenal gland, with a production rate exceeding 10 μmol/min. In the placenta, P5 is converted to P4 and this is transformed into 5αDHP in the endometrium [123]. The pattern of secretion of 5αDHP at beginning of gestation runs parallel to that of P4, while around 90 days the onset of P4 decline gives way to fetoplacental synthesis of the different progestogens whose concentrations continue to increase during the second half of gestation. Thus, 20α5P, which is initially at 5 ng/ml, reaches 69 ng/ ml at 200 days of gestation and 300 ng/ml at term. In addition, the concentrations of βα-diol increase to 484 ng/ml [112], while 3β5P, P5ββ and ββ-diol reach values of 100, 10 and 100 ng/ml, respectively, towards the end of gestation [124].

The 5αDHP is found primarily at the uterine level during midgestation, but as labor approaches, its distribution changes and is predominantly in fetal circulation. This metabolite is an immediate precursor of allopregnanolone, a potent gamma-aminobutyric acid (GABA) receptor agonist with activity on myometrial relaxation in other species [125–127]. Serum allopregnanolone increases similarly to its precursor, reaching maximum values at the middle of gestation and a term [112]. However, both P4 and 5αDHP prevent weakly myometrial contractions induced by

oxytocin *in vitro*, suggesting the intervention of the other hormones in the maintenance of uterine quiescence [128]. On the other hand, an umbilical increase of P4 after 300 days of gestation related to a greater expression in the trophoblast of the enzyme necessary for the conversion of P5 into P4 has been described [129].

Simultaneously with the production of progestagens, the feto-placental unit (FPU) synthesizes phenolic estrogens, E1S and E2 17β and 17α, through the aromatization of dihydroandrosterone (DHA), DHEA and its precursors (3β-hydroxyl C-19). The estrogens β unsaturated, equilin and echinelin, specific to the equine species, derive from farnesyl pyrophosphate, through a noncholesterol-dependent pathway. In general, the pattern of estrogen secretion during gestation is characterized by the first peak of secretion around day 40 in relation to follicular development before the formation of secondary and accessory CLs and a subsequent increase from day 80, reaching maximum levels around 210 days of gestation [130–132]. Thus, the initial plasma concentrations of E1S, corresponding to ovarian synthesis and are affected by ovariectomy. On the contrary, the subsequent peak of liberation comes only from fetoplacental synthesis, descending drastically after fetal death [108, 113, 115, 133].

This increase in estrogens temporarily coincides with the hypertrophy of fetal gonads, which together with local expression of the enzyme 17α-hydroxylase, lead to elevated umbilical levels of P5, T and DHEA [134]. At the same time, maternal plasma concentrations of T and DHEA increase after 100 days of gestation, reaching maximum values at 6 months [116, 135] to promote greater perfusion in the fetal compartment and the uterine tonicity [27, 136]. Legacki et al. [112] describe DHEA values that increase since the first 2 months of gestation to at 6–8 months, decreasing afterward.

The mitochondrial cytochrome P450 side-chain cleavage *enzyme* (P450scc), necessary for the conversion of cholesterol into P5 is present in the glomerulosa and reticularis zone of the fetal adrenals from 150 days of gestation. However, its expression increases noticeably at the end of gestation, is also found in the fasciculata zone, in the placenta, and the utero-placental tissues. At the same time, fetal plasma levels of P5 and its uteroplacental diffusion are doubled and tripled between 200 and 300 days of gestation and that subsequently descend in the days prior to birth [132, 137]. One of the main metabolites of P4, the 5α-DHP, returns to umbilical circulation after synthesis in the endometrium, excreting only 30% of its production to the maternal circulation. Thus, it has been suggested that it could play a relevant role within fetoplacental tissues [137].

#### **4.3 Estrogens**

Estrogen production can likewise be determined in serum obtained from the mare and used as an indicator of feto-placental health [136]. Although total estrogen levels decrease in term gestation, E2 increases dramatically hours before parturition with accentuated myoelectric activity at the uterine level, suggesting the involvement of E2 in myometrial activation [132, 138]. In fact, estrogens promote PGs synthesis and increase endometrial sensitivity to oxytocin, stimulating myometrial contractile activity during delivery [137].

#### **4.4 Cortisol**

A few days before parturition, fetal adrenals change from mainly synthesizing P5 to producing cortisol in response to the stimulation of adrenocorticotropic hormone (ACTH). The increase of fetal cortisol is related to preparing the fetus for extra-uterine life by stimulating different processes necessary for the maturation of

**17**

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy…*

organs such as the liver, thyroid gland, lungs, digestive system, bone marrow and cardiovascular system [137]. In addition, cortisol activates the enzymes responsible for the synthesis of PGs which, without the presence of progestogens, increase continuously stimulating the onset of myometrial contractions. In addition, E2 favors

PGF2α play an important role during delivery by promoting myometrial contractibility, along with oxytocin, and cervical ripening and relaxation (PGE2). Utero-placental tissues are capable of synthesizing PGs and can be found in maternal plasma, fetal plasma and allantoic fluid [140]. However, its bioactivity is controlled by the enzyme 15-hydroxyprostaglandin dehydrogenase (PGDH), which converts the PGs into inactive metabolites, present in the maternal endometrium since approximately 150 days of pregnancy. Since the labile nature of PGs makes it difficult to measure one of these metabolites, 13,14-dihydro-15-keto-prostaglandin F-2α (PGFM) remained at low levels until day 200, then increased to peak pregnancy levels by day 300 and remained at this value until parturition. PGFM uses one of its metabolites as an indicator of its circulating levels, with a term increasingly being described, although it is during the second labor stage when its value

Relaxin is produced by the trophoblastic cells of the placenta and its activity is related to myometrial [137] as well as of the cervix and pelvic ligaments relaxation [142]. Maternal plasma levels increase at the end of gestation and during the second labor stage. After the expulsion of the placenta, it returns to basal values below the detection limit at 36 h, remaining elevated in cases of placental retention [143].

P4 concentrations above 4.0 ng/ml are considered adequate to support early pregnancy. However, when levels are <2.0 ng/ml, P4 supplementation is considered [137]. Several types of P4 products have been used to maintain pregnancies in mares. After oral administration altrenogest is readily absorbed, reaching peak levels after 3–6 h [144]. Altrenogest acts by binding to the P4 receptors but has little effect on endogenous plasma total progestagen concentrations. Specifically, altrenogest is not metabolized to 5α-pregnanes in the horse [128]. For this reason, the only scientific evidence that altrenogest prevents loss pregnancy in mares is during the first trimester, when it prevented abortion induced by repeated administration of PGF2α (cloprostenol) [145]. P4 may exert its effects by interfering with PG production stimulated by proinflammatory cytokines. Daels et al. [146] demonstrated that the rise in endogenous PGF2α concentrations was inhibited by altrenogest treatment. Indeed, when early pregnant mares (21–35 days post-ovulation) were exposed to *Salmonella typhimurium* endotoxin all mares supplemented with

altrenogest until day 70 remained pregnant, whereas 6 out of 7 mares aborted when

Mares with suspected luteal insufficiency can be supplemented with altrenogest (0.044 mg/kg per os once or twice daily) or P4 (150 mg/day IM) starting on day

**5. High-risk mares and hormone supplementation**

altrenogest therapy was discontinued on day 50 [147].

the uterine response to PGs and may also promote their synthesis [139].

*DOI: http://dx.doi.org/10.5772/intechopen.90387*

**4.5 Prostaglandins**

increases up to 50 times [141].

**4.6 Relaxin**

**5.1 Progesterone**

*Physiological and Clinical Aspects of the Endocrinology of the Estrous Cycle and Pregnancy… DOI: http://dx.doi.org/10.5772/intechopen.90387*

organs such as the liver, thyroid gland, lungs, digestive system, bone marrow and cardiovascular system [137]. In addition, cortisol activates the enzymes responsible for the synthesis of PGs which, without the presence of progestogens, increase continuously stimulating the onset of myometrial contractions. In addition, E2 favors the uterine response to PGs and may also promote their synthesis [139].

### **4.5 Prostaglandins**

*Animal Reproduction in Veterinary Medicine*

fetal death [108, 113, 115, 133].

relevant role within fetoplacental tissues [137].

metrial contractile activity during delivery [137].

ing afterward.

**4.3 Estrogens**

**4.4 Cortisol**

oxytocin *in vitro*, suggesting the intervention of the other hormones in the maintenance of uterine quiescence [128]. On the other hand, an umbilical increase of P4 after 300 days of gestation related to a greater expression in the trophoblast of the enzyme necessary for the conversion of P5 into P4 has been described [129].

Simultaneously with the production of progestagens, the feto-placental unit (FPU) synthesizes phenolic estrogens, E1S and E2 17β and 17α, through the aromatization of dihydroandrosterone (DHA), DHEA and its precursors (3β-hydroxyl C-19). The estrogens β unsaturated, equilin and echinelin, specific to the equine species, derive from farnesyl pyrophosphate, through a noncholesterol-dependent pathway. In general, the pattern of estrogen secretion during gestation is characterized by the first peak of secretion around day 40 in relation to follicular development before the formation of secondary and accessory CLs and a subsequent increase from day 80, reaching maximum levels around 210 days of gestation [130–132]. Thus, the initial plasma concentrations of E1S, corresponding to ovarian synthesis and are affected by ovariectomy. On the contrary, the subsequent peak of liberation comes only from fetoplacental synthesis, descending drastically after

This increase in estrogens temporarily coincides with the hypertrophy of fetal gonads, which together with local expression of the enzyme 17α-hydroxylase, lead to elevated umbilical levels of P5, T and DHEA [134]. At the same time, maternal plasma concentrations of T and DHEA increase after 100 days of gestation, reaching maximum values at 6 months [116, 135] to promote greater perfusion in the fetal compartment and the uterine tonicity [27, 136]. Legacki et al. [112] describe DHEA values that increase since the first 2 months of gestation to at 6–8 months, decreas-

The mitochondrial cytochrome P450 side-chain cleavage *enzyme* (P450scc), necessary for the conversion of cholesterol into P5 is present in the glomerulosa and reticularis zone of the fetal adrenals from 150 days of gestation. However, its expression increases noticeably at the end of gestation, is also found in the fasciculata zone, in the placenta, and the utero-placental tissues. At the same time, fetal plasma levels of P5 and its uteroplacental diffusion are doubled and tripled between 200 and 300 days of gestation and that subsequently descend in the days prior to birth [132, 137]. One of the main metabolites of P4, the 5α-DHP, returns to umbilical circulation after synthesis in the endometrium, excreting only 30% of its production to the maternal circulation. Thus, it has been suggested that it could play a

Estrogen production can likewise be determined in serum obtained from the mare and used as an indicator of feto-placental health [136]. Although total estrogen levels decrease in term gestation, E2 increases dramatically hours before parturition with accentuated myoelectric activity at the uterine level, suggesting the involvement of E2 in myometrial activation [132, 138]. In fact, estrogens promote PGs synthesis and increase endometrial sensitivity to oxytocin, stimulating myo-

A few days before parturition, fetal adrenals change from mainly synthesizing P5 to producing cortisol in response to the stimulation of adrenocorticotropic hormone (ACTH). The increase of fetal cortisol is related to preparing the fetus for extra-uterine life by stimulating different processes necessary for the maturation of

**16**

PGF2α play an important role during delivery by promoting myometrial contractibility, along with oxytocin, and cervical ripening and relaxation (PGE2). Utero-placental tissues are capable of synthesizing PGs and can be found in maternal plasma, fetal plasma and allantoic fluid [140]. However, its bioactivity is controlled by the enzyme 15-hydroxyprostaglandin dehydrogenase (PGDH), which converts the PGs into inactive metabolites, present in the maternal endometrium since approximately 150 days of pregnancy. Since the labile nature of PGs makes it difficult to measure one of these metabolites, 13,14-dihydro-15-keto-prostaglandin F-2α (PGFM) remained at low levels until day 200, then increased to peak pregnancy levels by day 300 and remained at this value until parturition. PGFM uses one of its metabolites as an indicator of its circulating levels, with a term increasingly being described, although it is during the second labor stage when its value increases up to 50 times [141].

#### **4.6 Relaxin**

Relaxin is produced by the trophoblastic cells of the placenta and its activity is related to myometrial [137] as well as of the cervix and pelvic ligaments relaxation [142]. Maternal plasma levels increase at the end of gestation and during the second labor stage. After the expulsion of the placenta, it returns to basal values below the detection limit at 36 h, remaining elevated in cases of placental retention [143].
