Equine Pregnancy Endocrinology

Abstract

There is a complicated interplay between a number of hormones produced during the establishment and maintenance of the equine pregnancy and subsequent expulsion of the fetus at term. Any clinician involved in equine reproduction is required to have a thorough understanding of the endocrinology of the equine pregnancy. This allows the clinician the ability to monitor the viability of the pregnancy and intervene when problems occur. This chapter will review the main hormones produced during various phases of the equine pregnancy, which hormones can be utilized to monitor pregnancy viability and how and when to initiate parturition utilizing hormonal therapy.

1. Introduction

This chapter will review the main hormones produced during the establishment and maintenance of pregnancy in our equine patients, as well as how to initiate parturition utilizing hormonal therapy. It is imperative that equine clinicians have a thorough understanding of the hormonal analysis during the pregnancy in mares; this will allow clinicians to monitor pregnancy establishment and to assess its viability. The chapter will examine which hormones are important during the different phases of pregnancy and the use of exogenous supplementation of progesterone, when it is required, how to monitor and when it would be safe to discontinue such treatment. Induction of parturition in a clinical setting is advocated under certain clinical situations that lead to compromise of the pregnancy or indeed the welfare of the mare herself. This chapter will review the protocols used in initiating parturition in the mare, and under what clinical parameters should these protocols be used.

2. Equine pregnancy endocrinology

There is a complicated interplay between the mare’s uterus, ovaries, and subsequent feto-placental unit, to establish and maintain pregnancy. A thorough understanding of the physiology allows attending clinicians to establish whether the pregnancy is viable, and in cases of compromise to the pregnancy, to assist in its maintenance to term.

When a mare ovulates a dominant follicle(s) a corpus hemorrhagicum is formed over the following 24 hours; formed by blood filling the vacuated cavity post ovulation. Over the preceding 24 hours, the luteinization of these cells form a structure known as a corpus luteum (CL). Within this structure specialized cells start to produce progesterone. For the first several days after ovulation, there is a linear increase in the production of progesterone, rising to around 4 ng/mL systemically on day 5 post ovulation [1]. In the non-pregnant mare prostaglandins are released from the endometrium which causes luteolysis of the CL, and returns the mare into estrus. In the pregnant mare, the fertilized embryo enters the uterus around day 5 post ovulation [2]. Between this day and around day 15 maternal recognition takes place. While this mechanism in the mare is not fully understood, embryonic migration throughout the uterus is paramount [3]. On day 16 post ovulation, embryo fixation occurs, typically at the base of one uterine horn [3]. During this time, embryonic estrogens are produced, increasing uterine tone [3]. Initially over this period the source of progesterone is the corpus luteum. This is termed output D progesterone production [4]. Around day 30 post ovulation, progesterone production by the corpus luteum declines [3]. At day 35, endometrial cups begin to form. They are formed by specialized cells of the trophoblast chorionic girdle of the developing fetus, which invade the endometrium [3]. Their job is to produce equine chorionic gonadotropin (eCG) which has luteinizing properties, which cause ovarian follicles to either ovulate and produce a corpus luteum or for follicles to luteinize without ovulation [3]. These secondary, or supplementary, corpora lutea cause further production of progesterone [4]. Equine chorionic gonadotropin (eCG) is first detected in serum of pregnant mares between days 35–40, and peak around day 60 post ovulation [3]. Progesterone production by these corpora lutea is termed progesterone output three [4]. Around day 45 post ovulation, reliance on ovarian sources of progesterone shifts to feto-placental production, but the supplementary corpora lutea continue to play a crucial role, peaking their production of progesterone between days 60 and 120, to around 10–15 ng/mL [5]. Regression of these supplementary corpora lutea occurs around days 150–180 [3]. After which the reliance is entirely focused on local progesterone-like substances by the feto-placental unit. A number of important progestins, including 5-ɑ-pregnanes, are detectable in serum from day 60 [3]. The sources of these progestins are maternally derived cholesterol [3]. Production occurs within the utero-placental unit, the fetal gonads, and fetal adrenal glands [3]. The production of these progestins, occurs until near term and are the source for maintaining pregnancy. These progestins peak around the last few days of gestation, and precipitously fall on the day of parturition [6, 7].

Around day 80 post ovulation, feto-placental derived estrogen production starts to increase [3]. These include estrone, estradiol-17β, estradiol-17ɑ, along with the equine specific estrogens, equilin and equilenin, all increase [3]. The source for the production of these estrogen compounds are the fetal gonads [3]. There is a continued decline in production of these compounds over the last two months of gestation. It has been postulated that these estrogens increase blood flow to the feto-placental unit, promoting the tone of the uterus and increased viability of the pregnancy [7].

As the pregnancy continues the developing fetus stretches the myometrium. This stretch effect stimulates the production of progestins via the feto-placental unit [3]. This is thought to aid in the quiescence of the uterus, which is essential for the maintenance of the pregnancy [3]. These progestins inactivate the production of endometrial derived prostaglandins, thereby blocking their ecbolic effect [8], thus continuing the quiescence of the uterus, so essential for maintenance of the pregnancy.

During the last few days of pregnancy there are drastic alterations in the hormonal milieu; there is a decrease in progestin production, as the fetal hypothalamus-pituitary–adrenal axis matures. As fetal adrenocortical hormone (ACTH) production is ramped up, there is a negative feedback loop to decrease progestin production, resulting in increased production and secretion of cortisol [9]. This production of cortisol leads to an upregulation of prostaglandin, and eventually myometrial contractions and expulsion of the fetus [3]. During the last week, there is considerable elevation in circulating estradiol-17β, via nocturnal secretion, increasing the responsiveness of the myometrium to prostaglandins [3].

There may be many times where clinicians may wish to establish whether a mare is pregnant without having to do a transrectal ultrasound. This may be due to the mare being too small to safely perform a transrectal ultrasound (such as in the Falabella breed) or indeed for safety reasons. As previously discussed progesterone can be monitored in early pregnancy and low circulating serum levels, would indicate the need to supplement with exogenous progesterone. Progesterone testing in the mid to late gestation in the mare should be avoided, as other progestins from the feto-placental unit are produced to maintain pregnancy, as false negatives would be produced. Pregnant mare serum gonadotropin (also known as eCG) is elevated between days 45–100. It is a positive marker for pregnancy as only those fetal cells invading the endometrium produce this hormone. Testing of estrogens, namely estrone sulfate is a very useful hormone to examine for pregnancy diagnosis. This hormone is produced by the feto-placental unit after day 90 through term, and is also a useful indicator of fetal well being. Low levels may indicate fetal stress or placentitis, and appropriate therapy can be initiated.

3. When should mares be treated with exogenous progesterone

The importance of progesterone is illustrated in a number of studies, where mares were ovariectomized during progesterone output D phase; mares that were supplemented with exogenous progesterone maintained their pregnancy [10]. These studies also show that mares that had circulating blood serum progesterone of above 4 ng/mL all maintained the pregnancies to term, while the minimum circulating serum progesterone levels of 2 ng/mL are required for survivability of the pregnancy [10]. Any mare that has a history of repeated pregnancy loss, serum progesterone levels of 4 ng/mL or less, or mares demonstrating uterine edema on transrectal ultrasonography, should be started on exogenous progesterone. Products include oral altrenogest administered at a dose of 0.044 mg/kg given daily. Most mares tolerate this well. Alternatively a number of injectable altrenogest products are now available, which are administered I.M. weekly. It is recommended that treatment is continued to at least 120 days post ovulation, after which the feto-placental unit will be producing sufficient progestins to maintain pregnancy. To discontinue, a blood sample can be obtained, as the vast majority of commercial laboratory bases progesterone assays do not cross react with these exogenous progesterone products.

Although not the basis of this chapter, ascending placentitis in the mare is a serious condition and a leading cause of pregnancy loss. Diagnosis is based on clinical signs, including premature lactation, vaginal discharge and abortion. On transrectal ultrasonography, the combined thickness of the uterus and placenta (CTUP) can be measured and will allow the attending clinician to evaluate placental thickness and health. The clinician may also identify edema with the placenta, and/or placental separation via transrectal ultrasonography. Any mare suspected to ascending placentitis should immediately be started on progesterone supplementation (altrenogest, administered at a double dose of 0.088 mg/kg), along with systemic broad spectrum antibiotics (such as trimethoprim-sulfamethoxazole), and flunixin meglumine (NSAIDs at 1.0 mg/kg IV) and/or pentoxifylline (8.4 mg/kg PO q 6–8 h).

In studies involving embryo transfer mares, those that are supplemented with exogenous progesterone, had lower circulating endogenous progesterone serum levels [11]. This is likely through the negative feedback of progesterone on the pituitary leading to down regulation of luteinizing hormone. Although other studies have suggested no difference between those mares supplemented with exogenous progesterone and those that were not, had no discernable differences in circulating serum progesterone levels [12]. However it is advisable to wean mares off oral or intramuscular progesterone supplementation over a course of a week.

4. Induction of parturition

Induction of parturition is often called for in a number of clinical cases, such as fetal hydrops and rupture of the prepubic tendon. A number of protocols have been investigated to induce parturition in the mare, including dexamethasone, prostaglandin and/or oxytocin administration. Naturally given that prostaglandins play a role in parturition, researchers have investigated their use to induce parturition. Nonetheless they are unreliable in the duration from time of administration to induction of parturition. This could/would have serious clinical implications especially if the induction is warranted for fetal wellbeing.

Dexamethasone has been investigated for a number of decades, in its ability to induce parturition in the mare. In the 1970s large doses administered to healthy, late term mares lead routinely to parturition. In one study [13] (n = 12), 100 mg/horse administered daily I.M. between days 321 and 324 resulted in the shortening parturition to a mean of day 328 for treated mares versus 340 in the control group. However, variation to day of parturition from last day of administration of dexamethasone between 3 and 11 days occurred. In a similar study [14] (n = 5) administered dexamethasone between days 315 and 317, reduced mean gestation to 322 days versus 335 for controls. Dexamethasone, a glucocorticoid accelerates fetal maturation (including lung maturation, essential for capability with life) and mimics the fetal signal that starts the parturition pathway [15]. In studies examining dexamethasone administration in the late term pregnant mares, has shown that fetal death and dystocia occurs, when administered between days 331 and 347 of gestation [16]. The studies which have investigated the use of dexamethasone of induction of parturition, have utilized healthy mares. The use of this medication in compromised pregnancies, and where the fetal hypothalamic–pituitary axis is not yet functional (as in the previous studies) have not yet been examined.

The ecbolic oxytocin, naturally shown to be elevated during the parturition phase of the pregnancy in the mare, has been shown to be the most reliable in causing parturition. Initially higher doses were administered, but higher rates of premature placental separation (40%) and dystocia (25%) were observed [17]. Lower doses of 5 units (20 units/mL) followed by 10 units of oxytocin, 15 minutes later, routinely and reliably induced parturition [18]. Typically the chorioallantoic membrane ruptures 5–15 minutes after the second dose [18]. This is of particular clinical use should the fetus wellbeing be compromised, and specialized staff can be on hand at delivery. It has been shown that route of administration and degree of cervical relaxation before oxytocin treatment had no effect on fetal viability.

A number of clinicians, the author included, advocates that mares selected for induction of parturition should meet the following criteria: there should be mammary gland development, gestation of at least 330 days, waxy teat ends, and milk calcium carbonate levels of at least 200 ppm. Gestational length is unreliable (equine pregnancy gestation ranges between 320 and 360 days, but even after 360 days, induction of parturition can result in dysmature foals). The clinician should be aware that mares induced into parturition have higher incidence rates of retaining their placenta.

5. Conclusion

In the early phase of pregnancy establishment serum progesterone can be analyzed. It is routine in cases where serum progesterone levels are lower than 4 ng/mL to be supplemented with exogenous progesterone. From day 35 of pregnancy a useful hormone to analyze is eCG. Only fetally derived cells are producing this hormone, meaning such that detection is a positive marker for pregnancy. Towards mid pregnancy from around day 90, the feto-placental unit is taking over production of a number of progestins to maintain the pregnancy. During this time estrone sulfate is a useful hormone to analyze. It is a marker not only of pregnancy but also of fetal wellbeing. Lower levels would indicate a compromise to the pregnancy as only the feto-placental unit is producing this hormone. Induction of parturition in the mare is seldom required, yet it can be a lifesaving procedure in rare clinical cases, such as fetal hydrops or maternal rupture of the prepubic tendon. A number of protocols have been investigated, but it is now established that the oxytocin protocol is most reliable. A number of parameters must be met before induction of parturition is performed, such as establishing milk calcium levels, mammary gland development, and appropriate gestational length. It is advised that induction of parturition should ideally be performed in a hospital setting to ensure adequate staffing to ensure neonatal and maternal wellbeing. Table 1 summarizes the main hormones which can be routinely analyzed during which phase of pregnancy in the mare, and their significance in terms of pregnancy viability.

Days post ovulation	Hormone	Source	Notes
5–35	Progesterone	Corpus luteum	Produced by the corpus luteum in both pregnant and nonpregnant mares between ovulation and 14 days. After which in pregnant mares progesterone will continue to be produced. In non-pregnant mares around day 14 progesterone levels will decline and the mare will return to estrus.; Average serum concentration of 4 ng/mL. In pregnant mares with serum levels under this, clinicians should consider exogenous supplementation.
35–40 (peak 60)	Equine chorionic gonadotropin (eCG)	Endometrial cups (of fetal origin)	Only pregnant mares will produce eCG. This is not a marker of fetal viability. Fetal death after day 35, mares will continue to exhibit eCG in their serum.
90	Estrone sulfate	Feto-placental unit	Useful marker for pregnancy and fetal wellbeing. If levels are on the lower side of normal, or falling, this likely indicates fetal distress, and the clinician should examine the cause and treat accordingly.

Table 1.

Days post ovulation and which hormones can be analyzed to determine pregnancy and fetal wellbeing, their sources and what clinical significant each hormone tested pertains.