Hyponatraemia reversibly affects human myometrial contractility. An in vitro pilot study.

BACKGROUND: In a previous study we found a significant correlation between dystocia and hyponatraemia that developed during labour. The present study examined a possible causal relationship. In vitro studies often use area under the curve (AUC) determined by frequency and force of contractions as a measure of myometrial contractility. However, a phase portrait plot of isometric contraction, obtained by plotting the first derivate of contraction against force of contraction, could indicate that bi-or multiphasic contractions might be less effective compared to the smooth contractions.
MATERIAL AND METHODS: Myometrial biopsies were obtained from 17 women undergoing elective caesarean section at term. Each biopsy was divided into 8 strips and mounted isometrically in a force transducer. Seven biopsies were used in the first part of the study when half of the strips were immersed in the hyponatraemic study solution S containing Na+ 120 mmol/L and observed for 1 hour, followed by 1 hour in normonatraemic control solution C containing Na+ 136 mmol/L, then again in S for 1 hour, and finally 1 hour in C. The other half of the strips were studied in reverse order, C-S-C-S. The remaining ten biopsies were included in the second part of the study. Response to increasing doses of oxytocin (OT) in solutions S and C was studied. In the first part of the study we calculated AUC, and created phase portrait plots of two different contractions from the same strip, one smooth and one biphasic. In both parts of the study we registered frequency and force of contractions, and described appearance of the contractions.
RESULTS: First part of the study: Mean (median) contractions per hour in C: 8.7 (7.6), in S 14,3 (13). Mean (SD) difference between groups 5.6 (4.2), p = 0.018. Force of contractions in C: 11.8 (10.2) mN, in S: 10.8 (9.2) mN, p = 0.09, AUC increased in S; p = 0.018. Bi-/multiphasic contractions increased from 8% in C to 18% in S, p = 0.001. All changes were reversible in C. Second part of the study: Frequency after OT 1.65 x 10-9 M in C:3.4 (2.9), in S: 3.8 (3.2), difference between groups: p = 0.48. After OT 1.65 x 10-7 M in C: 7.8 (8.9), increase from previous OT administration: p = 0.09, in S: 8.7 (9.0), p = 0.04, difference between groups, p = 0.32. Only at the highest dose of OT dose was there an increase in force of contraction in S, p = 0.05, difference between groups, p = 0.33. Initial response to OT was more frequently bi/multiphasic in S, reaching significance at the highest dose of OT(1.65 x 10-7 M), p = 0.015. when almost all contractions were bi/multiphasic.
CONCLUSION: Hyponatraemia reversibly increased frequency of contractions and appearance of bi-or multiphasic contractions, that could reduce myometrial contractility. This could explain the correlation of hyponatraemia and instrumental delivery previously observed. Contractions in the hyponatraemic solution more frequently showed initial multiphasic contractions when OT was added in increasing doses. Longer lasting labours carry the risk both of hyponatraemia and OT administration, and their negative interaction could be significant. Further studies should address this possibility.

Introduction

Maternal and neonatal hyponatraemia caused by hypotonic fluid administration during labour is well recognised [1, 2]. The antidiuretic effect of endogenous vasopressin and oxytocin administration will also increase the susceptibility of women to develop hyponatraemia during labour. In a previous observational study including 287 women at term we found that 26% of the women who received 2.5 liters of fluids or more during labour developed hyponatraemia defined as plasma sodium < 130mmol/L [3]. Maternal reduction in plasma sodium was significantly correlated with increased duration of labour and prolonged second stage, as well as instrumental delivery or emergency caesarean section for failure to progress (p<0. 001)[3]. These results do not necessarily indicate a causal relationship, but the possibility is supported also by animal studies that showed reduced contractility when myometrial biopsies were immersed in fluids with low concentration of sodium [4].

Different ions and ion channels are involved in the complex events that precede myometrial contractions [5]. The main extracellular ions are Na+, Ca2+, and Cl-, whereas K+ predominates in the intracellular compartment. A negative membrane potential of the myometrial cell is determined by the unequal charge of the ions on both sides of the cell membrane. The movement of ions is determined by their relative concentrations in the two compartments and by the membrane potential.

Myometrial contraction is determined by influx of Ca2+ that follows an action potential (AP). Extracellular sodium is involved in this process through different ion channels [6, 7]. Influx of ions are believed to reduce the membrane polarisation to a level when the calcium channel opens, causing depolarisation. Sodium is also involved in the termination of contractions through the Na/Ca exchange [8] Thus, there are several possible ways alterations of extracellular sodium concentration may affect myometrial contractility.

We performed the present in vitro study to determine a possible influence of hyponatraemia on myometrial contractility. In the first part of the study we registered frequency and force of contractions in normonatraemic and hyponatraemic solutions, and calculated AUC. We described the shapes of isometric contractions, and created phase portrait plots as described by Shmygol and Gullam [8, 9]. In the second part of the study we observed response to increasing doses of oxytocin, in normonatraemic and hyponatraemic solutions respectively. Our results confirm our hypothesis that hyponatraemia may negatively affect myometrial contractility.

Material and methods

Ethical approval

The study was approved by Regional Ethical Review Board in Stockholm (Dnr 2012/610-3173) on April 25th 2012, and registered 15th October 2012 with ClinTrials. com (NCT01708811, LOF-KSI). All study participants signed informed consent before being enrolled in the study. The first patient enrolled March 8th 2013, the last patient was enrolled June 5th 2015.

Sampling and preparation

We obtained myometrial biopsies from 17 women during planned caesarean sections in spinal anaesthesia at term. None of the women were in spontaneous labour. Indications for caesarean section were breech presentation, previous caesarean sections, or maternal request. The biopsies 15 x 6 mm x full thickness were excised from the midline upper lip of the incision, performed in the lower uterine region after delivery of the baby. The biopsies were immediately immersed in ice cold Tyrodes´s solution that was equilibrated with a gas mixture of 95% O2 and 5% CO2, obtaining pH 7.4, and carried to the laboratory where the biopsy was divided into 8 strips measuring 10 x 2 x 2 mm. The strips were mounted isometrically with one end fixed to a holder and the other end to a MLT0201 force transducer (ADInstruments Ltd, Oxford, UK) and immersed in separate 5 ml chambers at 37◦ C using the ML0186 Panlab Eight Chamber Organ Bath System (Panlab s. l., Barcelona, Spain). All strips were initially immersed in normonatraemic Tyrode´s solution, and were stretched to a passive tension of 19.6 mN. As in previous studies [10], contractions in all strips were stimulated with oxytocin (OT) 8.25 x 10−8 M, and the strips were then allowed to rest for 1 hour, refreshing the organ bath 3 to 4 times with Tyrode´s solution. We studied only one biopsy at a time, and the strips that failed to contract were excluded from further experiment and analysis. All biopsies were treated similarly up to this point. The solutions were at all times equilibrated with a gas mixture of 95% O2 and 5% CO2.

First part of the study: Reversible effect of hyponatraemia

We included biopsies from seven women to study reversible effects of hyponatraemia in the first part of the study. Half of the strips from each biopsy were immersed in the study solution S containing Na+ 120 mmol/L and observed for 1 hour, followed by 1 hour in the control solution C containing Na+ 136 mmol/L, then again immersed in solution S for 1 hour, and finally for 1 hour in solution C. The other half of the strips were studied in reverse order, being immersed for four periods of each 1 hour in the solutions C-S-C-S. The organ baths were refreshed with the actual solutions S or C after 30 minutes of each period. The total observation time was 5 hours.

We registered frequency and peak force (from baseline to peak), and we calculated the AUC. The shape of contractions was also visually examined, and defined as monophasic (smooth) or bi/multiphasic. We created phase portrait plots of two different contractions from the same strip, one smooth and one biphasic. This was done by plotting the first derivate of isometric force against force of contraction, as previously decribed [8, 9]. Data were registered and analysed using LabChart (ADInstruments Ltd, Oxford, UK).

Second part of the study: Combined effect of oxytocin and hyponatraemia

Biopsies from the remaining ten women were included in the second part of the study. Half of the strips from each biopsy were immersed in solution S, the other half in solution C, and remained in the same solution for the whole duration of the experiment. The response to OT was studied by adding OT in increasing doses of 1.65 x 10−9 M, 1.65 x 10−8 M, and 1.65 x 10−7 M (S1 File). Initial response to OT was registered for 20 minutes after each dose of OT. The organ baths were rinsed three times with 20 minutes interval using solution S or solution C respectively before adding the next dose of OT. The total observation time was 4 hours.

We registered frequency and peak force (from baseline to peak). We visually examined the shape of the isometric contractions that were defined as monophasic (smooth) or bi/multiphasic.

Solutions

Tyrode´s solution with Na+ 136 mM used for transport and control (C) contained (in mM) NaCl 114, KCl 4,0. CaCl2 2,0, MgCl2 1,0, NaHCO3 21,4, NaH2PO4 1,4, Glucose 10.

The study solution S with Na+ 120 mM was made hyponatraemic and hypoosmotic by reduction of NaCl to 97,4 mM from 114 mM. The solutions were equilibrated with a gas mixture of 95% O2 and 5% CO2.

Oxytocin 82.5 x 10−8 M (Syntocinon® Novartis Pharma Ag, Basel, Switzerland) was added to all strips before the the experiments, and in increasing doses of 1.65 x 10−9 M, 1.65 x 10−8 M, and 1.65 x 10−7 M in the second part of the study.

Statistics

Power calculation was not performed, as this was a pilot study. Differences of frequency, peak force, AUC, and the frequency of bi-or multiphasic contractions were analysed with Wilcoxon matched pairs test. Mean values for each biopsy were used for calculations. Initial multiphasic contraction rates with increasing doses of OT in solutions S and C were analysed with ANOVA. All tests were two-tailed, and a p-value ≤0.05 was considered significant. The software of Statistica version 12 (Statistica; StatSoft®, Tulsa, OK, USA) was used for all statistics.

Results

Patient demographics are listed in Table 1 (and S1 Table). Due to technical problems we excluded from analysis four of 56 strips from the first part of the study, and one of 80 strips from the second part of the study.

Table 1

Patient demographics.

Parameter	1st part of study	2nd part of study	Total
N	7	10	17
Age (yrs)
Mean (SD)	33.3 (3.1)	36.0 (5.1)	34.9 (4.5)
Median (range)	32 (30–38)	37 (27–43)	36 (27–43)
BMI (kg/m2)
Mean (SD)	24.0 (3.2)	24.8 (7.2)	24.4 (5.7)
Median (range)	22.5 (21.1–30.2)	22.4 (17.8–39.7)	22.5 (17.8–39.7)
Parity
0	2 (28.6)	4 (40.0)	6 (35.3)
1	3 (42.9)	5 (50.0)	8 (47.1)
2	2 (28.6)	1 (10.0)	3 (17.6)
Gestational length (weeks)
Mean (SD)	38.6 (0.4)	38.9 (0.4)	38.8 (0.4)
Median (range)	38.4 (38.0–39.1)	38.9 (38.3–39.6)	38.7 (38.0–39.6)
Previous CS
No	3 (43)	4 (40)	7 (41)
Yes	4 (57)	6 (60)	10 (59)
Indication
Maternal request	4 (57)	10 (100)	14 (82)
Praevia	1 (14)	0 (0)	1 (6)
Previous CS	2 (29)	0 (0)	2 (12)
Infant weight (g)
Mean (SD)	3811 (304)	3529 (413)	3645 (388)
Median (range)	3630 (3530–4280)	3583 (2958–4150)	3620 (2958–4280)
Infant sex
Male	5 (71.4)	2 (20.0)	7 (41.2)
Female	2 (28.6)	8 (80.0)	10 (58.8)

CS: caesarean section, Numbers in () indicate % when not otherwise stated.

First part of the study: Reversible effect of hyponatraemia

Mean (median) contractions per hour (interquartile range lower to upper [IQR]) in solution C: 8.7 (7.6) contractions per hour (5.6–9.3), in solution S 14,3 (13.0) contractions per hour (10.5–13.8). Mean (SD) increase from C to S was 5.6 (4.2) contractions per hour, p = 0.018.

Bi-or multiphasic contractions increased from 8% in C to 18% in S, p = 0.001. All changes were reversible in solution C. (Figs 1 and 2, and S1 Fig). Mean (median) force of contractions (IQR) in solution C: 11.8 (10.2) mN (8.8–15.3), in solution S: 10.8 (9.2) mN (5.0–9.3) mN, p = 0.09, Fig 2. Three biopsies showed decreased amplitude in solution S, whereas no amplitude decreased in solution C, Fig 2. AUC increased significantly in S, p = 0.018, Fig 2. Phase-plots of one smooth and one biphasic contraction from the same strip are shown in Fig 3 (and S2 Fig).

Fig 1

Isometric contractions from two different biopsies.

Panel A: area of reversible bi- or multiphasic contractions indicated. Bottom trace is excluded from analysis due to technical problems. Panel B: reversible change in frequency of contraction is indicated. C; normonatraemic control solution with sodium136 mM. S; hyponatraemic study solution with sodium 120 mM. 1h; 1 hour observation time in each solution before changing solution.

Fig 2

Frequency, peak force, and area under the curve in hyponatraemic and normonatraemic solutions.

Each line indicates mean values for one biopsy, and shows reversible change of contraction frequency, peak force, and area under the curve in solutions S and C. Left panel: Frequency of contractions per hour. Middle panel: Peak force of contractions. Right panel: Area under the curve. 120: Study solution with sodium 120 mM, 136: Control solution with sodium136 mM, mN: milliNewton.

Fig 3

Phase portrait plots.

Phase portrait plots of two contractions from the strip indicated in Fig 1 panel A Upper panels: Left; monophasic, smooth contraction. Right; biphasic contraction. Lower panels: Left; Phase portrait plot of smooth contraction. Right; Phase portrait plot of biphasic contraction.

Second part of the study: Combined effect of oxytocin and hyponatraemia

Mean (median) frequency of contractions per hour (IQR) after OT 1.65 x 10−9 M in solution C:3.4 (2.9) contractions per hour (2.7–3.5), in solution S: 3.8 (3.2) contractions per hour (2.7–4.5), difference between S and C, p = 0.48. After OT 1.65 x 10−8 M in solution C:6.2 (6.3) contractions per hour (4.0–8.3), increase from previous OT administration, p = 0.003; in solution S: 6.7 (6.6) contractions per hour (4.3–9.3), increase from previous OT administration p = 0.003. Difference between S and C, p = 0.56. After OT 1.65 x 10−7 M in solution C: 7.8 (8.9) contractions per hour (4.6–10.6), increase from previous OT administration, p = 0.09, in solution S: 8.7 (9.0) contractions per hour (6.6–11.8), p = 0.04, difference between S and C, p = 0.32.

Force of contractions mean (median) mN (IQR) after OT 1.65 x 10−9 M in solution C: 16.8 (19.9) mN (9.7–23.1), in solution S: 17.8 (20.2) mN. Difference between C and S, p = 0.5. After OT 1.65 x 10−8 M in solution C: 17.1 (19.5) mN (11.3–22.5), increase from previous OT administration, p = 0.51, in solution S:17.3 (19.3) mN (12.7–21.3). increase from previous OT administration, p = 0.20. Difference between C and S, p = 0.83. After OT 1.65 x 10−7 M in solution C: 16.8 (18.2) mN (13.5–21.3), increase from previous OT administration, p = 0.39, in solution S: 15.7 (16.9) mN (12.6–18.4) increase from previous OT administration, p = 0.05. Difference between C and S, p = 0.83.

Initial multiphasic contraction patterns following OT increased with higher concentrations of OT and were more often observed in solution S. (Table 2, Fig 4, and S2 Table).

Table 2

Initial multiphasic contractions after oxytocin administration.

	Sodium concentration		Differences
	S (120 mmol/L)	C (136 mmol/L)	p-values
Oxytocin (1.65 10−9 M)	0.23 (0.42)	0.25 (0.42)	0.828
Oxytocin (1.65 10−8 M)	0.61 (0.40)**	0.35 (0.41)	0.047
Oxytocin (1.65 10−7 M)	0.94 (0.13)**	0.61 (0.38)*	0.015

Numbers indicate % (SD) of contractions with multiphasic appearance.

* difference from previous OT administration in the same solution p<0.05,

** difference from previous OT administration in the same solution p<0.01.

S:study solution with sodium 120 mmol/L C: control solution with sodium136 mM

Fig 4

Combined effect of hyponatraemia and oxytocin.

Contractions in eight strips from the same biopsy following OT 1.65 x 10−7 M. C; normonatraemic control solution with sodium136 mM. S; hyponatraemic study solution with sodium 120 mM.

Discussion

Main results and interpretation

Our in vitro study showed that hyponatraemia significantly altered contractility of human myometrium. In the first part of the study we found that frequency of contractions increased significantly when the specimens were immersed in the hyponatraemic solution. The increased frequency caused higher AUC, as no increase in peak force was observed. Contractions were also significantly more often biphasic or multiphasic in the hyponatraemic solution. All changes were reversible in the normonatraemic solution, indicating that hyponatraemia caused these changes.

The phase portait plot of the biphasic contraction suggests that bi- and multiphasic contractions could give less effective myometrial contractions [8, 9].

In the second part of the study OT induced equal increase in frequency of contractions in both groups. Only at the highest dose of OT was there a slight increase in force of contraction in the hyponatraemic group, but no significant difference between the groups. The most significant difference was the increase of multiphasic contractions in the hyponatraemic solution when OT was added in increasing doses. Indeed, at the highest OT dose almost all contractions in the hyponatraemic solution were bi-or multiphasic.

Longer lasting labours carry the risk both of hyponatraemia and OT administration. However, studies in this field rarely, if ever, consider maternal plasma electrolytes, even when addressing issues regarding fluid administration [11, 12]. In physiologic conditions OT is secreted in a pulsative manner, and several studies have addressed the hypothesis that pulsative administration of OT for induction or augmentation of labour could improve the effect of oxytocin [13, 14]. However, a recent large trial showed that pulsative administration of OT for augmentation of labour resulted in longer lasting labours and higher frequency of instrumental delivery when compared to continuous infusion [15].

Our results indicate that the concomitant exposure to pulsative OT and hyponatraemia could further hamper myometrial contractility as the initial response to OT was more often multiphasic in the hyponatraemic solution, resulting in potentially ineffective contractions. Oxytocin stimulates myometrial contraction by elevating intracellular calcium, and sodium is involved both in contraction as well as and the termination of contraction[16], possibly explaining a negative interaction between OT and sodium. Further studies should address longer lasting exposure to OT and hyponatraemia.

Strengths and limitations

Our studies on excised human myometrium may more cosely represent the physiology of human labour than the rodent model frequently used. To maintain other variables as close to human physiology as possible, we studied the effect of hyponatraemia without maintaining osmolality, as hyponatraemia in vivo causes a corresponding reduction in osmolality. For the same reason both study and control solutions contained Mg in physiologic concentration.

The strategy of changing and reversing the solutions S and C showed reproducible and reversible effects of hyponatraemia in the first part of our study. We found no difference in the force of contraction between the groups, but the small number of biopsies and the weak p value could indicate a type II error. Our ambition was to study spontaneous contractions, but we used OT to initiate contractions. However, the effect of OT would have disappeared due to repeated change of Tyrode´s solution before starting the experiments. Only initial effects of OT was studied in the second part of the study.

Clinical aspects

No electrolyte free solutions should ever be administered intravenously, particularly when vasopressin levels could be increased, as this will significantly increase the risk of acute hyponatraemia[17]. However, some hospitals and centers still use glucose solutions without electrolytes for intravenous energy supply as well as for dilution of OT used to augment labour. Together with maternal overdrinking, this practice may lead to hyponatraemia.

Registration of fluid consumption is an easy preventive measure against hyponatraemia, without medicalisation of the physiological process of childbirth [18]. Blood sampling for plasma analysis may be indicated when hyponatraemia is suspected, as in long lasting labours with unknown maternal hydration status. When diagnosed, fluid limitation will resolve most cases of dilutional hyponatraemia. Treatment with hypertonic saline is indicated only in the rare occurrence of hyponatraemic encephalopathy causing neurological symptoms [19].

During intrauterine resuscitation Hartmann´s solution or Ringer´s acetate are administered in bolus of 0.5–1 L intravenously, usually without analysing the labouring woman´s plasma electrolytes. Both these solutions contain 130mmol/L of sodium, and will therefore not aggravate any hyponatraemia, and will also compensate any hypernatraemia due to dehydration. In both instances, the bolus will increase maternal blood volume, favouring placental perfusion.

Conclusion

In a previous clinical observational study we found an association between hyponatraemia and instrumental delivery for failure to progress. The present study indicates that there could be a causal relationship. Dystocia is likely to be multifactorial, and we propose that hyponatraemia could be one of several factors. Hyponatraemia should be considered in long lasting labours with slow progress.

Oxytocin added in second part of the study.

(DOCX)

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Frequency, peak force, and area under the curve.

(XLSX)

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(XLSM)

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(XLSX)

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Initial multiphasic contractions.

(XLSX)

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17 Sep 2019

PONE-D-19-18851

Hyponatraemia reversibly affects human myometrial contractility. An in vitro pilot study

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Reviewer #1: uploaded in file Moen Bruden 2019.

Prior paper 2009 looked at oxytocin in multvar regression, but using 5 U total dose and bimodal analysis, showed .072 p value, although CI - .9 – 7.4. Thus, probably should not strongly say that oxytocin is not related to hyponatremia, as noted in this abstract. Not to review a 2009 publication, but excessive oxytocin may cause low Na in some patients.

Also no mention of inappropriate ADH due to labor in either paper

Phase plot reference 8 should be 10, page 14. The phase plot analysis proposed by the Warwick group actually was not intended primarily as a measure of effectiveness of contractions (especially in vivo), rather normalizing results among different samples.

While customary in the field of uterine contractility, the area under the curve (AUC) is actually a force-time integration, which is a mechanical impulse. This unfortunately was also chosen decades ago as the word to describe an action potential, hence the potential (pun intended) confusion in a field where the tissue emits both an impulse in the form of an action potential and an impulse in the form of an integrated force. Just a comment, not a recommendation for change, since that boat left dock a while ago. If would be nice to occasionally see the correct terms used, however.

Intro –

• Should mention oxytocin at least as a possibility for causing hyponatremia

• Cl- is largely distributed by passive electrical properties – donnan/Nernst

• Intro describing the action potential is a bit long and the main point is only that these ions create electrical activity that “opens” voltage gated calcium channels which initiates the contraction. If you want to go into detail, you should also mention the Na/Ca exchanger which is probably more important in Na metabolism than the Na channel, esp regarding duration of contractions

Sampling

• 4 years to write manuscript?

• Omit humanitarian or define it. Given the routine nature of this biopsy, we really don’t even need to know other than “clinical indications” and implying that none were done for research purposes alone.

• Typo labouratory – “were” to where

• Putting normonatraemic Tyrode’s once (earlier) is enough, redundant is redundant. Tyrode’s is normal Na. Also, you realize that Mg2+ is a potential tocolytic, probably should comment at some point. Most groups avoid Mg, although for these studies it probably makes no difference. For storage/transport on ice, you should mention pH, though since real Tyrode’s is ~ 6.5 unless outgassed with CO2

• Did you stabilized the 100 mg (9.8 mN) tension after tissue creep (i.e. rest period)?

• You studied one biopsy at a time, although up to 8 strips simultaneously, correct?

First part of the study

• No need to repeat studying one biopsy at a time

• Phase plot reference is Gullum – 10. My guess is that you need to use endnote or similar program, since it looks like several of your references are incorrectly numbered

Second part of the study

• No need to repeat studying one biopsy at a time

• This description of oxytocin concentrations brings into question if the 82.5 nM oxytocin was used

Solutions

• While this is a complete description, it would be good to not repeat all the unchanged chemicals, but simply state the that the S solution was made hyponatremic and hypoosmotic by reduction of NaCl to 97.4 mM from 114 mM. Also, if oxytocin was added to the rest period (which I am not sure), you could place oxytocin here as well so that it is clear all tissues were exposed to oxytocin. On the other hand, if all tissues were not exposed to oxytocin, please correct your description on page 7 (top)

Statistics

• Do you mean “mean values for each biopsy..? – or each tissue strip?

Results

• Exclusion due to technical problems, or simply the tissue strip failed to contract (which can be up to 20% and still be OK)

• Now you have to define humanitarian. I think you mean “elective”

• Earlier you probably should note that none of your subjects were in spontaneous labor

First part

• First sentence is not a sentence

• Needs to be really clear – peak force is the force change from “resting” to maximum force generated. (or however you measured)

• Fig 1 is confusing. The 1 h is not clear. How many strips is this? Upper panel is noted, but not lower panel. Needs much better labeling

• Fig 2. As above, amplitude is probably peak force, should note if measured from onset to peak or simply peak from absolute 0.

• Fig 3 phase plots of the contractions above?

• the really interesting finding is the oscillatory behavior in the lower 4 tracings in the lower panel – if only I could figure out the history of the tissues.

Second part

• This is confusing to read – could this just be a table?

• All the figure legends should be put together at the end, near the figures.

Discussion

• This is where the concept of peak force rather than just force helps keep confusion with AUC (which to some is a force) low.

• All changes were reversible in the…. Indicating that hyponatremia or hypoosmolarity …

• Probably could mention Parkington’s work, but it is a bit overboard to attribute your results to spike-like rather than plateau potentials.

• The paragraph that starts with tachysystole doesn’t hang together well. You never really got close to 5 contractions in 10 min, so I would suggest you avoid this topic

• You really short-changed the discussion on the 2nd part of the study. What do you think caused multiphasic appearance? What is the physiological underpinning, and why would OT bring that out?

• Pulsatile OT is neither here nor there -suggest you either justify or omit

• What about multiphasic contractions in vivo – do your multiphasic relate to them (doubling, tripling, etc)

• Given the effort on phase plots – they are also a way to quantify multiphasic behavior – that is the areas of the closed circles of the plots return a measure of the impulse attributable to each phase of the contraction. I actually don’t think this must be done because it is a lot of calculating for very little information, but just to point out that it could be done for the 2nd part oxytocin effects.

Streghts (sic-type) and limitations

• Our studies on excised human tissue may more closely represent the physiology of human labor better than rodent model. Or similar phrasing

• OT was used to initiate contractions and establish a constant starting point to investigate the specific effects of hyponatremia/ hypoosmolarity on uterine contractions.

• You already mention type II error, no need to have the last two lines as a limitation

• The key limitation was that you studied hyponatremia without maintaining osmolarity constant, so the effects of Na are mixed with hypoosmolarity. However, since this is the clinical condition you wished to mimic, that may be what you intended to do.

Conclusion

• Some of the other factors for dystocia could be …. For example Sue Wray’s group showed that pH is a key factor as well.

• Fluid intake was a conclusion of your prior paper, so you probably should merely say that low Na is a modifiable clinical parameter

In summary

This is a very good paper with very good data. While the philosophy of PLoS One is to publish data regardless of perceived relevance, this paper has both good data and relevance. As pointed out above, there are a few areas of potential confusion and a few missed opportunities. The authors should consider most of this review as suggestions, with questions requiring answers are written in bold.

This manuscript satisfies PLoS One criteria for publication

Reviewer #2: The present paper reports about the effect of hyponatremia on myometrial contractility, evaluated “in vitro” on myometrial biopsies obtained from women undergoing elective caesarean section at term. The authors conclude that hyponatremia present a reversible effect on myometrial contractility, since it may determine an increased frequency of contractions and of bi- or multiphasic contractions.

The work is well written, and the reported results are indeed interesting, since it could explain

the previously observed correlation between hyponatremia and instrumental delivery; however, I believe that the following points need to be addressed:

• The authors should at least comment about the intrauterine resuscitation techniques and how they could affect the incidence of hyponatremia, in particular those who provide intravenous fluid bolus; indeed, in several countries, these tecniques are often used for the management of non-reassuring fetal heart monitoring.

• Sometimes “hyponatremia” is written “hyponatraemia”, please correct.

• Figure 1 and Figure 2 are hard to understand, please review to make them clearer

• In the Discussion section more space must be given to the potential clinical implications of these findings: what could be the best method for hyponatremia correction? In an asymptomatic patient, when the electrolytes should be dosed? Only when there is a suspect of dystocic labor? Every three-four hours?

**********

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Submitted filename: review Moen Brudin 2019.pdf

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19 Nov 2019

Dear Reviewers,

Thank you for your effort in reviewing our manuscript. We have followed your advice to the best of our knowledge, and hope you will appreciate this renewed version of our manuscript.

Below you will find our answers and comments.

Vibeke Moen

Reviewer #1: uploaded in file Moen Bruden 2019.

Prior paper 2009 looked at oxytocin in multvar regression, but using 5 U total dose and bimodal analysis, showed .072 p value, although CI - .9 – 7.4. Thus, probably should not strongly say that oxytocin is not related to hyponatremia, as noted in this abstract. Not to review a 2009 publication, but excessive oxytocin may cause low Na in some patients.

Also no mention of inappropriate ADH due to labor in either paper

Answer:

In our previous paper we addressed the antidiuretic effects of oxytocin, and commented that the women who received/ingested most fluids ”received oxytocin at rates necessary for oxytocin to express antidiuretic effect, but probably for a period of time too short for significant antidiuretic effect to develop”

We addressed the probable increased (physiological and appropriate) secretion of ADH, and suggested that hyponatraemia was partly caused by increased ADH secretion, as the hourly fluid volumes were well below maximum renal excretion at rest, and “therefore the development of hyponatraemia indicates increased vasopressin activity during labour” We also wrote that “Labour itself is however not a situation causing inappropriate vasopressin secretion” with an appropriate reference. Our conclusion was that “fluid volume is the major determinant of hyponatraemia, but the antidiuretic effects of endogenous vasopressin and oxytocin administration increase the susceptibility of women to develop hyponatraemia during labour”.

The problem with the term” inappropriate ADH secretion” is that the term often includes all non-osmotic stimuli, even if non-osmotic stimuli for ADH secretion are highly physiologic.

In our statistical analysis we separated oxytocin dose from fluid volume used for oxytocin administration, but pointed out that electrolyte free glucose was used.

We have now in the present paper included: “The antidiuretic effect of endogenous vasopressin and oxytocin administration will also increase the susceptibility of women to develop hyponatraemia during labour.”

Phase plot reference 8 should be 10, page 14. The phase plot analysis proposed by the Warwick group actually was not intended primarily as a measure of effectiveness of contractions (especially in vivo), rather normalizing results among different samples.

Answer: The ref 10, now ref 8 ( Schmygol) was published in 2007, and was to our knowledge the first to introduce the concept of phase portrait plot. In this paper they interpret the phase plot of the biphasic contraction: “Obviously, the type of contraction shown in FIG. 2B would be thermodynamically less efficient”

We therefore think it correct to use this article as a reference.

In the study by Gullam, published in 2009, a sophisticated analysis of the phase portait plot is described. Our analysis of the phase portrait plot does not match this method, but we now also use Gullam as reference[9].

While customary in the field of uterine contractility, the area under the curve (AUC) is actually a force-time integration, which is a mechanical impulse. This unfortunately was also chosen decades ago as the word to describe an action potential, hence the potential (pun intended) confusion in a field where the tissue emits both an impulse in the form of an action potential and an impulse in the form of an integrated force. Just a comment, not a recommendation for change, since that boat left dock a while ago. If would be nice to occasionally see the correct terms used, however.

Intro –

• Should mention oxytocin at least as a possibility for causing hyponatremia

We have now added:” The antidiuretic effects of endogenous vasopressin and oxytocin administration will increase the susceptibility of women to develop hyponatraemia during labour”.

Cl- is largely distributed by passive electrical properties – donnan/Nernst

Answer: We think our description covers this aspect without being too detailed: “A negative membrane potential of the myometrial cell is determined by the unequal charge of the ions on both sides of the cell membrane. The movement of ions is determined by their relative concentrations in the two compartments and by the membrane potential.”

• Intro describing the action potential is a bit long and the main point is only that these ions create electrical activity that “opens” voltage gated calcium channels which initiates the contraction. If you want to go into detail, you should also mention the Na/Ca exchanger which is probably more important in Na metabolism than the Na channel, esp regarding duration of contractions

Answer: All aspects of myometrial contraction are extremely complicated, and usually far from the every-day clinician´s knowledge. Our intention was to very briefly outline basic elements. We have now deleted the speculations regarding plateau and spike-like Aps in the Discussion, but included your suggestion regarding Na/Ca exchanger in this section.

Sampling

• 4 years to write manuscript?

• Omit humanitarian or define it. Given the routine nature of this biopsy, we really don’t even need to know other than “clinical indications” and implying that none were done for research purposes alone.

Answer: We have now changed “humanitarian”in text and Table 1 to “maternal request”. We hope clinicians will read our paper, and omitting the indications for caesarean section will appear strange to most clinicians, we therefore maintain the indications in Table 1. Caesarean section in a woman in active labour is by definition an “emergency“caesarean”. Elective caesarean will imply a woman who is not in labour, but for the benefit of a reader without obstetric knowledge we included in “Methods” this clarification: “None of the women were in spontaneous labour.”

• Typo labouratory – “were” to where

Corrected, thank you

• Putting normonatraemic Tyrode’s once (earlier) is enough, redundant is redundant. Tyrode’s is normal Na.

Answer; We have deleted accordingly

Also, you realize that Mg2+ is a potential tocolytic, probably should comment at some point. Most groups avoid Mg, although for these studies it probably makes no difference.

The content of Mg corresponds to physiologic values, and we aimed at altering only plasma sodium (and consequently osmolality)

For storage/transport on ice, you should mention pH, though since real Tyrode’s is ~ 6.5 unless outgassed with CO2

We have completed: The biopsies were immediately immersed in ice cold Tyrodes´s solution that was equilibrated with CO2 5%/O2 95%, obtaining pH 7.4

• Did you stabilized the 100 mg (9.8 mN) tension after tissue creep (i.e. rest period)?

We realise we used 19.6 mN and have corrected accordingly.

• In all experiments, the baseline tension was induced by a sequential increase of tension to get the basal tension of (2g) After this, the tissue was left to rest and equilibrate before exposing the tissue for OT. During the experiments the resting baseline was stable over several hours and was not needed to be adjusted.

You studied one biopsy at a time, although up to 8 strips simultaneously, correct?

Yes, that is correct

First part of the study

• No need to repeat studying one biopsy at a time

Deleted as suggested

• Phase plot reference is Gullam – 10. My guess is that you need to use endnote or similar program, since it looks like several of your references are incorrectly numbered

We used Endnote, and have now checked again our references. To our astonishment, Endnote apparently did not follow the previous changes in manuscript. We have no recreated the whole reference list.

As previously mentioned, we maintain the ref 10 ( now 8) as this was published in 2007, and was to our knowledge the first to introduce the concept of Phase portrait plot.

Second part of the study

• No need to repeat studying one biopsy at a time

Adjusted, as suggested

• This description of oxytocin concentrations brings into question if the 82.5 nM oxytocin was used

Answer: In the Methods/Sampling and preparation we include: “As in previous studies [10], contractions in all strips were stimulated with oxytocin (OT) 8.25 x 10 -8 M, and the strips were then allowed to rest for 1 hour,”

Solutions

• While this is a complete description, it would be good to not repeat all the unchanged chemicals, but simply state the that the S solution was made hyponatremic and hypoosmotic by reduction of NaCl to 97.4 mM from 114 mM.

Thank you, we have followed this suggestion, and also moved this section and Statistics so that they precede “First” and “Second” part of the study

Also, if oxytocin was added to the rest period (which I am not sure), you could place oxytocin here as well so that it is clear all tissues were exposed to oxytocin.

We have followed this suggestion, and included “……Oxytocin 82.5 nM (Syntocinon® Novartis Pharma Ag, Basel, Switzerland) was added to all strips before the the experiments, and ……..in the second part of the study”.

On the other hand, if all tissues were not exposed to oxytocin, please correct your description on page 7 (top)

All biopsies were exposed to oxytocin. To make this quite clear, contractions in all strips

Also, after describing OT addition, we write “All biopsies were treated similarly up to this point.”

Statistics

• Do you mean “mean values for each biopsy..? – or each tissue strip?

Yes, mean values for each biopsy, for statistical correctness

Results

• Exclusion due to technical problems, or simply the tissue strip failed to contract (which can be up to 20% and still be OK)

We have followed this suggestion : “…and the strips that failed to contract were excluded”

• Now you have to define humanitarian. I think you mean “elective”

All caesareans were elective, and we have now changed to ”maternal request”, to specify that there were no medical reasons for the elective caesarean section.

• Earlier you probably should note that none of your subjects were in spontaneous labor

For the obstetrician “elective caesarean” will imply caesarean section in a woman who is not in active labour”. For the benefit of all readers we include the clarification in the Methods: “None of the women were in spontaneous labour”.

First part

• First sentence is not a sentence

We have corrected and it now reads “We included biopsies from seven women…”

• Needs to be really clear – peak force is the force change from “resting” to maximum force generated. (or however you measured)

Answer: We have altered to peak force, and peak force is measured from onset to peak. We have now included this specification in Statistics and Fist and Second part of the study.

• Fig 1 is confusing. The 1 h is not clear. How many strips is this? Upper panel is noted, but not lower panel. Needs much better labeling

Answer: The figure shows contractions in two biopsies, eight strips from each biopsy. We have now altered their placement and description, in an effort to increase understanding. • Fig 2. As above, amplitude is probably peak force, should note if measured from onset to peak or simply peak from absolute 0.

During the experiments the resting baseline was stable over several hours and was not needed to be adjusted. Thus, the different peaks of force during the experiments were calculated from the similar levels of basal tension. We have now specified this in Methods, First and Second Part, by writing peak force (from baseline to peak).

• Fig 3 phase plots of the contractions above?

The portrait plots are obtained from two contractions shown in Fig 1 panel A, and this is now mentioned in text to Fig 1

• the really interesting finding is the oscillatory behavior in the lower 4 tracings in the lower panel – if only I could figure out the history of the tissues.

Comment: These were four of the strips from the same biopsy as the strips above.. I am not sure I understand what you mean by “oscillatory behaviour” but we point out that appearance and frequency reversibly changes from C to S.

Second part

• This is confusing to read – could this just be a table?

Fig 4 Is now a table

• All the figure legends should be put together at the end, near the figures.

We have followed the ”instruction to authors”in the PLOS guidelines, and also checked this with the editorial office, and adhered to these guidelines inserting Figure text where the figure first is mentioned in the manuscript.

Discussion

• This is where the concept of peak force rather than just force helps keep confusion with AUC (which to some is a force) low.

• All changes were reversible in the…. Indicating that hyponatremia or hypoosmolarity …

• Probably could mention Parkington’s work, but it is a bit overboard to attribute your results to spike-like rather than plateau potentials.

We have now deleted this part

• The paragraph that starts with tachysystole doesn’t hang together well. You never really got close to 5 contractions in 10 min, so I would suggest you avoid this topic

Answer: Please see our answer below, following question regarding pulsatile OT.

We are aware that studies often ignore this initial response to oxytocin, and the effect studied in a “stabilised” situation, but we consider also the initial effect to be important, as the clinical use of intermittent oxytocin administration was (is?) common.

• You really short-changed the discussion on the 2nd part of the study. What do you think caused multiphasic appearance? What is the physiological underpinning, and why would OT bring that out?

Answer: All aspects of myometrial contraction are complicated, and any explanation to the interaction between hyponatraemia and oxytocin would be very speculative from our part. We therefore limit ourselves to adding: Oxytocin stimulates myometrial contraction by calciumsensitisation, and as sodium is involved both in calcium influx and the termination of contraction, this could explain a possible potentiation by oxytocin.

• Pulsatile OT is neither here nor there -suggest you either justify or omit

Answer: The practice of pulsative administration of oxytocin justified the second part of our study, we therefore maintain and justify by including Fig 4. This figure shows increased frequency and multiphasic contractions after OT, and illustrates the possible negative interaction between OT and hyponatraemia.:

• What about multiphasic contractions in vivo – do your multiphasic relate to them (doubling, tripling, etc)

The answer to this is beyond our knowledge and study results

• Given the effort on phase plots – they are also a way to quantify multiphasic behavior – that is the areas of the closed circles of the plots return a measure of the impulse attributable to each phase of the contraction. I actually don’t think this must be done because it is a lot of calculating for very little information, but just to point out that it could be done for the 2nd part oxytocin effects.

Comment: We consider Shmygol´s conclusion regarding the phase portrait plot of a multiphasic contraction indicates the relevance and importance of the portrait plots.:

“Obviously,the type of contraction shown ….would be thermodynamically less efficient”

To the clinician, we believe that the phase plot best illustrates the changes caused by hyponatraemia, as well as their possible implication.We have therefore maintained the plots.

Streghts (sic-type) and limitations

• Our studies on excised human tissue may more closely represent the physiology of human labor better than rodent model. Or similar phrasing

• OT was used to initiate contractions and establish a constant starting point to investigate the specific effects of hyponatremia/ hypoosmolarity on uterine contractions.

Thank you for these suggestions.

We altered: Our studies on excised human myometrium may more closely represent the physiology of human labour than the rodent model frequently used. To maintain other variables as close to human physiology as possible, we studied the effect of hyponatraemia without maintaining osmolality, as hyponatraemia in vivo causes a corresponding reduction in osmolality. For the same reason both study and control solutions contained Mg in physiologic concentration.

• You already mention type II error, no need to have the last two lines as a limitation

• The key limitation was that you studied hyponatremia without maintaining osmolarity constant, so the effects of Na are mixed with hypoosmolarity. However, since this is the clinical condition you wished to mimic, that may be what you intended to do.

Indeed, this is exactly what we did. We studied the effect of hyponatraemia without maintaining osmolality, as the physiological effect hyponatraemia in vivo necessarily implies hypoosmolality. Plasma sodium is the main responsible for extracellular osmolality, and hyponatraemia predictably reduced osmolality in the study participants in our previous study.

We performed this in vitro study to test the hypothesis that the observed dystocia was caused by hyponatraemia (and therefore hypoosmolality)

Conclusion

• Some of the other factors for dystocia could be …. For example Sue Wray’s group showed that pH is a key factor as well.

Comment: Hyponatraemia will cause a metabolic acidosis, and the reduction of pH will depend on the respiratory compensation, and we did observe a slight decrease in pH in our labouring and hyponatraemic women. However, if we understand Sue Wray correctly, decrease in systemic pH does not have negative effect on contractility, it is only local pH that has this influence.

• Fluid intake was a conclusion of your prior paper, so you probably should merely say that low Na is a modifiable clinical parameter

We have modified accordingly.

In summary

This is a very good paper with very good data. While the philosophy of PLoS One is to publish data regardless of perceived relevance, this paper has both good data and relevance. As pointed out above, there are a few areas of potential confusion and a few missed opportunities. The authors should consider most of this review as suggestions, with questions requiring answers are written in bold.

This manuscript satisfies PLoS One criteria for publication

Reviewer #2: The present paper reports about the effect of hyponatremia on myometrial contractility, evaluated “in vitro” on myometrial biopsies obtained from women undergoing elective caesarean section at term. The authors conclude that hyponatremia present a reversible effect on myometrial contractility, since it may determine an increased frequency of contractions and of bi- or multiphasic contractions.

The work is well written, and the reported results are indeed interesting, since it could explain

the previously observed correlation between hyponatremia and instrumental delivery; however, I believe that the following points need to be addressed:

• The authors should at least comment about the intrauterine resuscitation techniques and how they could affect the incidence of hyponatremia, in particular those who provide intravenous fluid bolus; indeed, in several countries, these tecniques are often used for the management of non-reassuring fetal heart monitoring.

Answer: We have included a new paragraph “Clinical aspects” with comments on fluid administration during labour.

• Sometimes “hyponatremia” is written “hyponatraemia”, please correct.

Comment. Done

• Figure 1 and Figure 2 are hard to understand, please review to make them clearer

CommenT. We have modified, hoping to be more understandable.

• In the Discussion section more space must be given to the potential clinical implications of these findings: what could be the best method for hyponatremia correction?

In an asymptomatic patient, when the electrolytes should be dosed? Only when there is a suspect of dystocic labor? Every three-four hours?

Answer: we address these issues in the paragraph “Clinical implications”

Registration of fluid consumption is an easy preventive measure against hyponatraemia, without medicalisation of the physiological process of childbirth [18]. Blood sampling for plasma analysis may be indicated when hyponatraemia is suspected, as in long lasting labours with unknown maternal hydration status, When diagnosed, fluid limitation will resolve most cases of dilutional hyponatraemia. Treatment with hypertonic saline is indicated only in the rare occurrence of hyponatraemic encephalopathy causing neurological symptoms [19].

We believe “prevention is better than cure”, and information to mothers and widwives as well as obstetricians is crucial. Frequent blood sampling for analysis of electrolyte would lead to medicalisation of the natural process of childbirth, and also unnecessarily increase the economic burden for the health care system.

________________________________________

Submitted filename: Response to Reviewers.docx

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16 Dec 2019

Hyponatraemia reversibly affects human myometrial contractility. An in vitro pilot study

PONE-D-19-18851R1

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Reviewer #1: Yes

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Reviewer #1: All comments in inititial review are addressed. This is an interesting paper, with interesting approaches and analyses. There remain a few quibble points, but nothing that is clinically or scientifically important, hence will suggest accept in current form.

Minor typo - page 9 could use a comma or something between 82.5 nM and 8.25. The the. Several more of these, but I think the copy editors will catch most.

I find it interesting that you note that some hospitals administer D5W. I don't think many in the US do that (none in my experience). This would be an interesting study to compare outcomes in that group with matched D5LR group.

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Reviewer #1: Yes: Roger C. Young, MD, PhD

14 Jan 2020

PONE-D-19-18851R1

Hyponatraemia reversibly affects human myometrial contractility. An in vitro pilot study

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