Effect of uterine torsion intrapartum on concentrations of placental estrogens and progesterone in cattle

Abstract Objective The current study investigates how uterine torsion influences placental oestrogens and progesterone blood concentrations in intrapartum cows. Our research tests the hypothesis that intrapartum uterine torsion impairs the ability of the placenta to synthesize steroids and may also suppress the release of synthesized steroids into the maternal circulation. Methods The study included a total number of 37 intrapartum dairy cows of various breeds and ages. These animals were transported to our clinic by their owners. Furthermore, general and obstetrical examinations of all these animals were performed in our clinic. The uterine torsion (UT) group consisted of 20 animals. The presence of UT was verified during clinical general examinations by vaginal and transrectal examination. The comparison (C) group included 17 animals whose birth was undisturbed or could be terminated with moderate obstetrical assistance. The clinical examination of group C animals showed no problems with their general health and genital organs. Blood samples were collected immediately after the initial obstetrical examination from 37 cows for radioimmunological measurement of estradiol‐17β (E2), free total estrogen (FTE), conjugated total estrogen (CTE), and progesterone (P4). Results In terms of P4, there was no statistical difference between the two groups. For all estrogen parameters, however, concentrations were significantly lower in the UT group than in the C group. In the correlation analysis, there was a significant correlation between the P4 and the FTE in the C group. Furthermore, the positive correlation between all estrogen parameters in the UT group was significant. In group C, significant positive correlations were found apart from the correlation between E2 and CTE. Conclusions The results are consistent with the hypothesis and suggest that in UT animals processes dependent on estrogens or other placental hormones may be impaired during the peri‐ or postpartum period.


INTRODUCTION
In the cow, the morphological and functional peripartum changes, such as cervical dilation and uterine contractions, are regulated to a significant extent by endocrinological interactions (Burton et al., 1987;Kindahl et al., 2002;Smith et al., 1973;Wehrend, 2003). Steroid hormones are essential in this endocrinological system for controlling pregnancy and delivery. The placenta is an important temporary endocrine organ in addition to its role in transporting substances between the mother and the fetus. As in many ungulate species, the bovine placenta produces significant amounts of estrogens. The site of estrogen synthesis is the fetal portion of the placentome, the cotyledons, where the key enzyme in estrogen synthesis, aromatase (CYP19A1), is localized in the trophoblast giant cells (Schuler et al., 2006;Schuler et al., 2008).
In the period close to parturition, pregnancy-associated estrogens are associated with the softening of the soft birth pathway and stimulation of myometrial activity (Smith et al., 1973;Schams et al., 2003;Schuler, 2000). However, placental estrogen production in cattle is detectable at the early stages of gestation. Placental estrogens are detectable in amniotic fluid as early as around day 50, 3-4 weeks days after the onset of placentation (Eley et al., 1979). As yet, there is little reliable information on the significance of placental estrogens in the early and middle stages of pregnancy. They presumably serve as a placental or uterine growth and differentiation factor (Schuler et al., 2008;Schuler et al., 2018). In favor of a predominantly local function of placental estrogens in the gravid uterus is the fact that in systemic maternal blood, they circulate predominantly as conjugated forms, that is, as sulfates or glucuronides, with oestrone sulfate being the major estrogen from a quantitative point of view (Hoffmann et al., 1997;Schuler, 2021). Sulphation of placental estrogens apparently already occurs predominantly in the trophoblast, where the estrogen-specific sulphotransferase SULT1E1 is localized in placentomes mainly in the mononuclear trophoblast cells (Khatri et al., 2011). Oestrone sulfate is measurable in the maternal blood of gravid cattle from about day 120-150 of gestation and increases steadily until late gravidity. A greater increase in free estrogens, predominantly estrone, is not observed until the last month of gestation. The concentrations of estradiol-17ß, which is biologically much more potent, are comparatively much lower (Hoffmann et al., 1997), with a considerable proportion apparently originating in the udder (Janowski et al., 2002). Possibly, however, the udder is not capable of de novo synthesis of E2 from cholesterol, but may depend in this respect on the supply of precursors from the placenta. The bovine placenta can also produce P4. However, the biological necessity of an auxiliary/placental minor P4 source, which becomes evident after the 240th day of pregnancy is still elusive (Hoffmann et al., 1979;Hoffmann & Schuler, 2002;Schuler et al., 1994;Schuler et al., 2006;Schuler, 2000). In late pregnant or intrapartum cattle, uterine torsion can endanger the life of the offspring and may lead to severe degenerative and inflammatory lesions in the uterus (Sickinger et al., 2020). Impairment of uterine perfusion can develop in the uterus depending on the degree of rotation and the duration of the torsion. Because the veins are primarily affected by vascular twisting and constriction, uterine congestion and cyanosis are com-mon. If therapy is not provided promptly, the vessels will thrombose and the damaged uterus will be cut off from the bloodstream, leading to substantial tissue changes in the uterine wall. The fetus's supply of nutrients and oxygen as well as the elimination of CO 2 and metabolic end products is reduced. In fatal cases, the fetus subsequently dies as a result of intrauterine respiratory and metabolic acidosis (Busch, 1993;Klein & Wehrend, 2006;Rosenberg & Berchtold, 1978). In addition to the uterus and fetus, the placenta may also be affected by

Animals
The study included a total number of 37 intrapartum dairy cows of various breeds (German Brown Swiss, German Holsteins, and German Fleckvieh) and ages. All these animals were housed on farms in Germany's Hesse region before being transported to our clinic by their calving was spontaneous or could be terminated with moderate obstetrical assistance (simple fetal extraction). Examination of these animals showed no problems with their general health and genital organs.

Blood sample collection
In
Toluene was used for sample extraction; the antiserum exhibited the following cross-reactivity: E2: 100%, oestrone: 1.30%; androstenedione, cortisol, dehydroepiandrosterone, pregnenolone, P4, testosterone: < 0.01%. The minimum detectable concentration was 2pg/mL. Intra-and interassay CV were 7.1% and 17.6%, respectively. The assay applied for the measurement of free and conjugated total estrogens followed in principle the procedure described in detail by Hoffmann et al. (1996) for the measurement of free and conjugated oestrone.
The identical radioimmunological method was used for the determination of free and conjugated estrogens. Different from the cited work, in which a specific antiserum against oestrone was applied, in this study, an antiserum generated against 17β-estradiol-17-hemisuccinate-BSA was used, which exhibits virtually identical cross-reactivity against oestrone, estradiol-17α and estradiol-17β (Hoffmann, 1976). The separate determination of the free and conjugated forms was achieved by extracting and measuring the free estrogens. Then the sample residue was enzymatically hydrolyzed, extracted again, and the conjugated forms were subsequently determined via the 'detour' of the corresponding free forms. The measurement was performed on an estrone standard curve.

Statistical analyses
In this study, the group's UT and C were compared with respect to the

RESULTS
The results are summarised in Figures 1 and 2 the C (p < 0.01) groups ( Figure 2). Also, there was significant positive correlation between FTE and E2 in the UT (p < 0.01) and C (p < 0.01) groups ( Figure 2). A significant positive correlation between CTE and E2 (p < 0.01) was only found in the UT group but not in C animals. In the C group, there was a significant positive correlation between FTE and P4 (p < 0.05) (Figure 2).

DISCUSSION
Consistent with our own hypothesis, the UT-induced impairment of placental perfusion and placental function is evident from significantly reduced estrogen concentrations. Here, the difference between the UT and C groups as measured by the p-value from the statistical evaluation is more pronounced for E2 and FTE than for CTE. This is possibly due to the fact that the sulfated steroids have a longer half-life than the free estrogens due to binding to albumin (Schuler, 2021), whereby the degree of impairment of oestrogen production or delivery to the maternal compartment is reflected in a more current form in the case of the free estrogens than by the conjugated estrogens. Decreased availability of placental estrogens or other placental regulatory factors in the maternal compartment could also lead to F I G U R E 1 Blood P4, FTE, CTE, and E2 concentrations in UT and C groups. The box's lower and upper borderlines indicate the first and third quartiles, respectively. The horizontal line crossing the box shows the median of the data. Points below or above the boundary of the vertical line represent possible outliers. In terms of P4, there is no statistical difference between the two groups. All estrogen hormones, however, were significantly lower in the UT group than in the C group. E2: estradiol-17β, FTE: free total oestrogen, CTE: conjugated total oestrogen, P4: progesterone.
impaired uterine function in the subsequent puerperium even in cases of successful obstetric therapy. In addition to impaired production of placental estrogens or their transfer to the maternal compartment due to the disturbance of uterine perfusion, it would also be conceivable that a primary disturbance of placental estrogen and prostaglandin production, via impairment of myometrial activity, could favor the occurrence of uterine torsion.
From a clinical point of view, the differentiation of a prepartum UT from an intrapartum UT is essentially based on the presence of signs of a birth that has already commenced, but which cannot always be clearly assessed by the effects of UT, such as the degree of opening of the cervix or myometrial activity. From an endocrinological point of view, in cattle, the onset of parturition is characterized by the initiation of prepartum luteolysis, which is responsible for the prepartum drop in maternal P4 concentration to basal levels (Schuler et al., 2018).
In two cases, clearly, suprabasal levels were measured in UT animals despite the clinical diagnosis of intrapartum UT, suggesting that luteolysis had not yet commenced or at least had not yet been completed in them. However, in animals with intrapartum UT, a basal progesterone level need not necessarily be due to physiological luteolysis, because in severe cases luteal function could also have been terminated by pathological processes.
In the present work, a significant positive correlation was found between P4 and FTE in group C. This observation is difficult to explain.
Although the bovine placenta exhibits significant P4 concentrations in placental tissue from local production from approximately 6 months of gestation (Tsumagari et al., 1994), by far the majority of P4 measurable in the peripheral maternal circulation is always of ovarian origin throughout gestation (Schuler et al., 2008). In the final phase of gravidity, no contribution of the bovine uterus to maternal progesterone levels was detectable (Comline et al., 1974). Thus, it is unclear how a relationship between the basal or slightly suprabasal P4 concentrations F I G U R E 2 Steroid hormone correlation analyses in the UT and C groups. E2: estradiol-17β, FTE: free total oestrogen, CTE: conjugated total oestrogen, P4: progesterone, ρ: Pearson's correlation coefficient, τ: Kendall's correlation coefficient.
at birth and the highly variable concentrations of free estrogens could be established. In any case, as a precursor of estrogen production, P4 does not play a quantitatively significant role in cattle and other ruminants due to the minimal lyase activity of ruminant CYP17A1 in the Δ4-pathway of steroidogenesis (Schuler et al., 1994;Shet et al., 2007).

CONCLUSION
The current study's findings confirm that intrapartum UT in cows clearly has an adverse influence on the production of placental oestrogens and/or on their release into the maternal circulation, considerably decreasing peripheral levels of estrogen. Significant involvement of the placenta in E2 synthesis is also supported by the fact that in UT cases E2 concentrations are considerably reduced. Further studies are needed on the relationship between the angle of uterine rotation and the reduction in maternal estrogen concentrations. In addition, this study has the following limitations. Blood samples were taken only once during parturition, and there are no data on hormone concentrations before uterine torsion. The animals belonged to different breeds and were of different ages. The degree of uterine torsion varied. In some animals, a retorsion attempt was probably made before transfer to the clinic. Continued studies should clarify the possible effects of each of these factors.