Natural and Synthetic Oxytocin—Structurally Same but Differ in Effects—A Pilot Study

Katarzyna Wszołek (  katarzyna.wszolek@ump.edu.pl ) Poznan University of Medical Sciences: Uniwersytet Medyczny imienia Karola Marcinkowskiego w Poznaniu https://orcid.org/0000-0001-9336-7060 Karolina Chmaj-Wierzchowska Poznan University of Medical Sciences: Uniwersytet Medyczny imienia Karola Marcinkowskiego w Poznaniu Małgorzata Pięt Poznan University of Medical Sciences: Uniwersytet Medyczny imienia Karola Marcinkowskiego w Poznaniu Agata Tarka Poznan University of Medical Sciences: Uniwersytet Medyczny imienia Karola Marcinkowskiego w Poznaniu Maciej Wilczak Poznan University of Medical Sciences: Uniwersytet Medyczny imienia Karola Marcinkowskiego w Poznaniu


Introduction
Oxytocin was rst described to stimulate uterine muscle by Dale [1]. The term "oxytocin" is related to the Greek term "ωκνξ" and "τoκoxξ," meaning "quick delivery" [2]. It is synthesized, similar to vasopressin, by the large cellular neurons located in the neurons of the supraoptic and paraventricular hypothalamic nuclei, transported in combination with the neurophysins in axons in the form of neurogenic granules to the posterior pituitary lobe, and stored there. In the hypothalamus, it is present as an inactive precursor. The inactive precursor gradually hydrolyzes into smaller fragments and relocates along axons to the posterior pituitary lobe leading to its activation [3]. It is released by the stimulation of receptors located in the nipples, vagina, and cervix [4].
The concentration of oxytocin during pregnancy gradually increases, reaching its maximum during delivery and maintaining its high levels in the third and fourth stages of labor. Oxytocin is naturally secreted in the cerebrospinal uid in high quantities, thereby directly affecting the female brain [5]. In this way, the stress level in the mother decreases, her mood improves, and speci c behavioral patterns develop, which is closely related to the feeling of motherhood and the formation of an emotional bond with the newborn baby [5,6]. These effects are possible due to the presence of oxytocin receptors in the following structures of the brain: the dorsomedial hypothalamic nucleus, amygdala, septum pellucidum, bed nucleus of the stria terminalis, anterior olfactory nucleus, preoptic and ventral tegmental area, and hippocampus [2]. Although oxytocin is secreted continuously in the central nervous system, it is released into the bloodstream by pulsation. The pulsating secretion depends on the depolarization of the cell membrane of the posterior pituitary lobe neurons, which occurs as a result of speci c stimuli [2,[5][6][7][8].
Synthetic oxytocin was rst obtained in 1953 by Vincent du Vigneaud [2]. It is currently used as an integral part of clinical practice in obstetrics to increase the contraction of the uterine muscles-both to induce labor and strengthen the contractile function in the rst or second stage of labor. It is also used therapeutically and prophylactically in the third stage of labor for accelerating the involution of the uterus and preventing postpartum bleeding [6][7][8][9][10]. The structure of synthetic oxytocin is identical to that of the natural hormone-it is a cyclic peptide containing nine amino acids. Increasing the permeability of the cell membrane to Ca 2+ ions leads to a decrease in the membrane tension and an increase in excitability. This causes an increase in the amplitude, frequency, and duration of the contraction of uterine smooth muscle cells and nipple myoepithelial cells [2,5,6]. The oxytocin receptors, which belong to the group of protein Gbinding receptors, are highly present in the limbic system, spinal cord, heart, intestine, immune system, uterus, and breast tissues [3,8].
According to some researchers, the key difference in the action of synthetic oxytocin is the fact that it does not penetrate the blood-brain barrier in the parturient. This is theoretically impossible due to the particle size and its hydrophilic nature [5][6][7]11], but according to some studies, intravenous or nasal administration allows oxytocin to penetrate into the intracerebral structures, which is bene cial to the selected patient groups [12][13][14][15]. However, this data does not apply to parturients.
In the context of childbirth, high-dose, continuous infusions of oxytocin usually result in regular uterine contraction, but the absence or average permeability of the blood-brain barrier and the continuity of infusions, unlike the pulsed release of natural oxytocin, may result in a different effect from the physiological hormone. The contractions are more painful according to the mothers giving birth, the concentration of stress hormones in the serum increases [12,13] Aim of the study In this study, we aimed to correlate the dose and duration of synthetic oxytocin infusions used during induction of labor, augmentation of labor in the rst and second stage of labor, and during active management of labor in the third stage of labor to the level of prolactin and cortisol in the serum of the parturient blood during the course of full-term pregnancy: during admission to labor, during the third stage of labor (before the infusion with oxytocin is started), on the second day postpartum. To evaluate the newborn baby: prolactin and cortisol levels were measured and gasometry from the umbilical cord vein just cutting was performed.

Page 4/18
The study comprised of patients who were either admitted to the K. Marcinkowski Gynecological and Obstetrical Clinical Hospital in Poznan for the induction of labor and/or those in the active phase of the rst stage of labor. This is a prospective cohort study. At this stage, after decontamination of the puncture site, blood was collected from a venous vessel using a closed aspiration and vacuum set SARSTEDT S-MONOVETTE 9 mL, containing a clotting activator (silicate). The sample collected was labeled with the date and time of collection, along with the term "Oxytocin 1" and was transferred to the laboratory, where the level of cortisol and prolactin was evaluated by electrochemiluminescence (ECLIA) using Cobas 6000 apparatus. Taking daily uctuations in serum cortisol levels into account, the rst sampling was performed in the morning. Before starting with the cortisol analysis, the hourly range, corresponding to the hours of sampling, was marked in relation to the cortisol test (6-10 and 16-20). In the case of high concentrations of hormone, reassessment was performed after sample dilution.
Seventy-eight patients provided the rst blood sample. Of them, 66 women gave birth naturally, out of which, the blood sample was collected from 65 patients immediately after the birth in order to reassess the levels of prolactin and cortisol. This sample was labeled as "Oxytocin 2." After the cessation of umbilical cord pulsation and after the sterile clamping and cutting, the blood sample was collected from the umbilical cord vein in a group of 61 women. This sample was labeled with the date and time of collection along with the terms "Umbilical cord oxytocin." The blood sample was also collected from the umbilical cord vein and artery in order to separate heparinized capillaries and assess the pH, base de cit (BD), pO 2 , and CO 2 concentration. The analysis was performed immediately after the blood collection using Cobas B 123 apparatus.
In the case of the remaining patients, 9 underwent labor with the use of a vacuum extractor and 6 underwent a Cesarean section. These patients were excluded from the subsequent stages of the study, which they were informed at the stage of quali cation.
Out of 60 patients who gave birth naturally, we collected the blood sample from a venous vessel on postpartum day 2 in order to determine the levels of prolactin and cortisol. This sample was labeled "Oxytocin 3." The reasons for the loss of patients, apart from the fact that the birth was terminated during the surgical procedure, were as follows: sudden indications for Cesarean sections (no sampling "Oxytocin 1" -3 patients), heavy postpartum bleeding after obtaining written consent (no sampling "Oxytocin 2" -1 patient), insu cient blood in the umbilical cord vessels (no "Umbilical cord oxytocin" sampling, 5 patients), earlier discharge of the patient from our hospital (no sampling "Oxytocin 3" -6 patients). Ultimately, 59 patients with 1, 2, 3, and umbilical cord blood samplings were nalized (Scheme 1). We were able to collect umbilical cord blood samples from all patients to determine pH, BD, pO 2 , and CO 2 , but in some cases, it was not possible as there was insu cient blood in the umbilical cord vessels or due to rapid clotting of the sample, which made analysis impossible (Scheme 2).

Results
A total of 81 women agreed to participate in this study. The participants were recruited based on the following inclusion criteria: singleton pregnancy, not complicated by severe maternal or fetal disease, gestational age from 37+0 to 42+0 weeks, and no existing contraindications to natural labor at the time of the interview. The women were admitted to the hospital either for delivery (starting uterine contraction) and/or for the induction of the labor with synthetic oxytocin.

Characteristics of the study group
In this study, participants were in the age group of 21 to 41 years. Of the 66 participants whose labor was natural, 45 were multiparous (68%), and for the remaining 21 participants, it was the rst labor (32%). Natural labor without an incision of the perineum took place in 24 women (30%); natural labor with an incision of the perineum took place in 42 parturients (52%); in 9 respondents (11%), it was necessary to complete the labor by a surgical procedure using a vacuum extractor, and in 6 (7%) patients, a Cesarean section was performed (Table 1). In the study group, 25 (38%) were female and 37 (56%) were male infants born. In 4 cases (6%), there was no data related to the child's gender. Out of 66 participants, 14 (21%) participants had gestational diabetes (G1 and G2), 25 (38%) had hypothyroidism, and 18 (27%) had other diseases. Cesarean section 6 81 7 Course of labor and postpartum period The duration of the rst stage of labor varied greatly (range = 40 min to 17 h 30 min). The condition of newborns, evaluated according to the V. Apgar score, was good or medium. In their rst minute of life, 56 newborns (90.3%) were given 10 points, 4 newborns (6.5%) were given 9 points, and 2 newborns (1.6%) were given 8 and 6 points each. In their fth minute of life, only one newborn child was given 9 points (1.6%), and the remaining 61 newborns were given 10 points (98.4%) ( Table 2). The following analgesics, nalbuphine and paracetamol via intravenous infusions and nitrous-oxide via inhalation, were administered during the labor. None of the parturients received pethidine or epidural anesthesia. Individual patients also received "other" drugs (such as levothyroxinum natricum, ursodeoxycholic acid or insulin at different doses). After the analysis, the in uence of individual analgesics and "other" drugs on the concentration of prolactin and cortisol collected during the rst stage of labor, immediately after delivery, and from the umbilical cord blood and on day 2 after delivery was not found.
Among the group of patients who gave birth naturally, 14 patients (21%) had to undergo labor induction, 49 patients (74%) spontaneously developed systolic function, and there was no data available for 3 patients (5%) with regard to the necessity of labor induction.
In relation to systolic enhancement in the rst stage of labor, oxytocin was administered to 7 parturients (11%), 55 women (83%) did not require oxytocin, and there was no data available for 4 patients (6%) in the rst stage of labor are available.
In the second stage of labor, oxytocin was administered to 12 parturients (18%) to enhance contraction. Fifty women (76%) gave birth without additional stimulation, and there was no data available for 4 patients (6%) with regard to the enhancement of the systolic function in the second stage of labor.
The active management of labor by administering oxytocin after cutting of the umbilical cord was applied in 39 patients (59%); in 23 patients (35%), there was no infusion with oxytocin; and there was no data available for 4 patients (6%) with the regard to the active management of the third stage.
Among the 66 patients who gave birth naturally, the color of amniotic uid was clear in 59 cases (89%), green in 3 cases (5%), and there was no data available for 4 cases (6%) with regard to the color of the amniotic uid. Umbilical cord collision (umbilical cord wrapped around the neck, trunk, or limbs, or a true node on the umbilical cord) was found in 15 newborns (23%). In 4 cases (6%), there was no data with regard to the occurrence of umbilical cord collision, and there was no collision recorded for 47 children (71%).
During the rst 2 h after the labor, 45 women (68%) breastfed the baby. For various reasons, 14 women (21%) were not able to breastfeed their baby, and there was no data was available for 7 women (11%).
Among the 78 women who provided their rst blood sample, the minimum concentration of prolactin 1, collected before or during the rst stage of labor, was 67.24 ng/mL and the maximum was 639.60 ng/mL. Of the 65 patients in whom blood was collected immediately after the labor, the lowest level of prolactin 2 was 40.75 ng/mL and the highest was 597.20 ng/mL. The level of prolactin determined from the umbilical cord vein (n=61) was between 125.90 nmol/L and 854.40 nmol/L. The concentration of prolactin 3 collected on day 2 after the labor (n=60) ranged from 187.80 nmol/L to 688.30 nmol/L.

Statistical analysis of the results
Our analysis shows that the level of prolactin 1, collected during the rst stage of labor or before the infusion of synthetic oxytocin, was higher the older the woman was (p=0.049). A similar relationship was not observed in the case of sampling immediately after the labor and from the umbilical cord blood, as well as on day 2 after the labor.
A reduction in the levels of prolactin 2, in the sample collected immediately after the labor, depending on the total dosage of synthetic oxytocin used during the labor was also signi cant (p=0.019).
The higher the concentration of prolactin 1 before the labor, the lower was the level of cortisol 1 before the labor (p=0.000008) and the level of cortisol 2 immediately after the labor (p=0.012685). The higher the level of prolactin 1, the higher was the level of prolactin 2 immediately after the labor (p=0.002913) and prolactin 3, collected on day 2 postpartum (p=0.005651). We did not observe similar relationships with respect to the level of cortisol evaluated from the blood collected from the umbilical cord vein (p=0.062867), and cortisol 3 evaluated on day 2 after the labor (p=0.326436), as well as the concentration of prolactin collected from the umbilical cord vein (p=0.205800). The analysis of the relationship between prolactin 1 before the labor and gasometry values assessed immediately after the labor from the umbilical cord blood showed that an increase in prolactin 1 level is accompanied by a decrease in the pH and BD values (p=0.018081 and p=0.017754, respectively).
The level of cortisol 1 signi cantly correlated with the level of prolactin 1-the higher the values of cortisol 1, the lower was the concentration of prolactin (p=0.000008). An increase in the levels of cortisol 1 also positively correlated with an increase in the concentration of cortisol 2 (p=0.000000). Similarly, there was an increase in the levels of cortisol in the sample collected from the umbilical cord vein (p=0.000000) and serum levels on day 2 after the labor (p=0.001237). There were no signi cant correlations between the level of cortisol and prolactin 2 in the sample collected before the labor (p=0.262137) and between prolactin levels in the sample collected from the umbilical cord vein (p=0.276800) and prolactin on postpartum day 2 (p=0.428186).
Considering the relationship between the level of prolactin 2 immediately after the labor, we obtained a positive correlation between the level of prolactin 2 with prolactin 1 (p=0.002913), and prolactin 2 with the value of prolactin 3 on postpartum day 2 (p=0.000033). There was no correlation between the levels of prolactin 2 and cortisol 1 before the labor (p=0.262137), the levels of cortisol 2 immediately after the labor (p=0.124322), the levels of prolactin and cortisol in umbilical cord blood (p=0.158318 and p=0.264867, respectively), and levels of cortisol on postpartum day 2 (p=0.946579).
Higher levels of cortisol 2 immediately after the labor correlated with lower levels of prolactin 1 and higher levels of cortisol 1 before the labor (p=0.012685 and p=0.000000, respectively). Higher levels of cortisol 2 positively correlated with the levels measured in umbilical cord blood and on postpartum day 2 (p=0.000000 and p=0.002897, respectively). There were no signi cant correlations between the level of cortisol 2 and prolactin 2 (p=0.124322) and between the levels of prolactin in umbilical cord blood (p=0.378587) and the level of prolactin on postpartum day 2 (p=0.329469).
With respect to the level of prolactin in umbilical cord blood, we did not obtain any signi cant relationships between the levels of prolactin 1 (p=0. The fact that a newborn baby rst latched within 2 h after birth, which 45 women (68%) from the study group did (n of signi cant in this case =60), turned out to be important in the context of the concentration of the tested hormone and in relation to some of the obtained results. Among them, the level of prolactin 1 (p=0.009939) and lower levels of cortisol 1 during the rst stage of the labor (p=0.010453), cortisol 2 immediately after the labor (p=0.010837), and from the umbilical cord blood (p=0.027262) were more frequently statistically signi cant.
At the end of pregnancy, the values of body mass index were not signi cantly related to the level of prolactin and cortisol during the rst stage of labor, immediately after the labor, and from the umbilical cord blood, as well as on postpartum day 2. Table 3 and Figures 1-4 show a statistically signi cant relationship between the level of individual prolactin levels on the analyzed factors.  Based on the information collected from 12 countries, including Poland in 2020 [7], it is clear that the amount of the initial dose, the maximum dose, the time of increasing the ow of the infusion with oxytocin, as well as the sources of recommendations according to which in a given country the chosen procedure is adopted to induce labor through the use of the infusion with synthetic oxytocin are very divergent. Poland was among the countries where the guidelines concerning the analyzed parameters were regulated by institutions-hospitals where the infusion with synthetic oxytocin is turned on. The dose of oxytocin added to 50 mL of the solvent (0.9% saline or 5% glucose solution) in Poland was 5 IU, the initial dose ranged from 0.06 to 1.2 IU/h, the ow was increased every 30 min by 1.2 mL/h until 3 contractions lasting about 60 seconds within 10 min, the maximum ow was 18 mL/h [7]. It was similar in our study group of patients-1 mL of oxytocin preparation containing 5 IU/mL of solution was added to 49 mL of 0.9% saline or 5% glucose solution. The initial ow rate in all patients was 3 mL/h, which corresponds to a dose of 0.3 IU/h. Most often, the maximum dose needed to achieve satisfactory contraction was 6 mL/h; in a single case, it was 8 mL/h, and in another case, it was 10 mL/h. The frequency of contractions and fetal heart rate were monitored continuously. The total dose of oxytocin, administered during 8 h, was determined between 7.65 and 8.10 IU, whereas data from Germany indicate the administration of up to 27 IU of synthetic oxytocin during 8 h of labor induction. In Germany, the manufacturers provide instructions on doses as part of the hospital guidelines [7]. The total dose of synthetic oxytocin we evaluated was related to the duration of the entire labor, which was as follows: 5 IU -43 patients, 10 IU-13 parturients, and 20 IU-1 patient with uterine atony and postpartum hemorrhage. Interestingly, in the case of the patient with uterine atony, the total duration of the infusion was 9 h, and the maximum ow was 10 mL/h. It was necessary to turn on an additional 5 IU of oxytocin (in 500 mL of 5% glucose solution) in order to strengthen the contraction during the second stage of the labor. It was her second induction of labor. The rst 10-hour induction attempt was made the day before the labor, also with the use of synthetic oxytocin, obtaining a maximum ow of 6.5 mL/h, but without the effect of regular uterine contraction.
This information may be extremely important. It may lead to the de nition of uniform norms and principles with regard to the dosage of synthetic oxytocin for labor induction [17][18][19]. It is also important in the context of the effect of the synthetic hormone on the reduction of the natural secretion of oxytocin in response to the "skin-to-skin" contact between the mother and the newborn [20]. According to a previous study on the in uence of drugs that may affect the newborn taking of breast sucking during the rst hour after the labor, it was demonstrated that the probability of the newborn baby taking breast sucking during the rst 60 min after delivery decreases with an increase in the total dosage of synthetic oxytocin administered to the mother during the labor [9,[21][22][23][24]. In the case of the group of patients we examined, we did not observe such a relationship. However, we observed signi cant correlations with regard to hormone levels. Mothers with higher levels of prolactin and lower levels of cortisol before the labor were more likely to breastfeed the newborn within 2 h after the labor. Among these women, the concentration of cortisol collected from the umbilical cord blood and immediately after the labor was also lower. These relationships seem to be interesting and worth further observation.
Jonas et al. [20] observed a signi cantly higher level of prolactin on postpartum day 2 in the group of women who received an infusion of synthetic oxytocin during the labor, with a simultaneous decrease in the level of endogenous oxytocin, especially in women who received epidural analgesia during the labor. This is a very interesting observation, because in our study group, there was a marked decrease in the level of prolactin immediately after the labor depending on the total dose of synthetic oxytocin used (p=0.019). We did not observe any relationship between the level of prolactin on postpartum day 2 on the dose of administered hormone or the fact of labor induction. However, it should be emphasized that none of the patients we examined had an epidural during the labor.
The condition of newborns assessed according to the V. Apgar scale at the end of their rst minute of life was good or average and in the fth minute of life, the condition of all the newborns was good. Similar results have been reported by Karaçor et al. [17], regardless of the fact of synthetic oxytocin administration. Similarly, the parameters of pH, BD, PO 2 , and CO 2 were within the range of standards; however, due to the size of the study group in the pilot study, a wider evaluation is planned.

Declarations
Funding: This study was nanced from the National Science Center scheme, Miniatura 3, No 2019/03/X/NZ7/00296. Con icts of interest/Competing interests: The authors report no con icts of interest.
Availability of data and material: data are avalable for you if needed.
Code availability: Not applicable.
Ethics approval: Patient enrollment methods, methods of obtaining the research material, and its storage were previously approved by the Poznan University of Medical Sciences Bioethics Committee, No. 869/19 (speci cally approved only for this study on 12 th September, 2019). The patients provided written informed consent for this study.  Relationship between the concentration of cortisol 1 and the fact of newborn latching within 2 hours after the birth.

Figure 3
Relationship between the concentration of cortisol 2 and the fact of newborn latching within 2 hours after the birth.