Abstract

We evaluated tumor necrosis factor alpha (TNFα) and uterine artery pulsatility index (UtA-PI) in the triage of patients with suspected preterm delivery (PTD), preeclampsia (PE), fetal growth restriction (FGR), and PE+FGR. The study included 125 pregnant women attending high-risk pregnancy clinics for triage of pregnancy complications. There were 31 pure PE cases, 42 cases of PE combined with FGR, 16 pure FGR cases, 15 PTD cases, and 21 term normal delivery controls. Maternal serum TNFα was determined by immune-diagnostic testing. UtA-PI was measured by Doppler sonography. Demographic, medical and pregnancy history, and mean arterial blood pressure (MAP) were extracted from the hospital medical records. Linear regression coefficients, and Box and Whisker plots were calculated and depicted using non-parametric statistics (Kruskal Wallis and Mann–Whitney). Spearman’s regression coefficient assessed marker accuracy; p<0.05 was considered significant. It was found that high TNFα in cases <34 weeks gestation, when coupled to low UtA-PI and normal blood pressure are found in early PTD most likely linked to maternal inflammation. At term, high TNFα combined with high UtA-PI is associated with any FGR (with/without PE), possibly reflecting inflammation and maternal and fetal hypoxia due to the very long period of altered placental perfusion. Accordingly, TNFα, and Doppler UtA-PI could be used for the differential diagnosis of early PTD, and FGR (with/without PE) near delivery.

Keywords: Fetal growth restriction; Gestational week; Inflammation; Mean arterial blood pressure; Placental hypoxia; Placental perfusion; Preeclampsia; Pregnancy

Abbreviations: FGR: fetal growth restriction; GA: gestational week; MAP: mean arterial blood pressure; PE: preeclampsia; TNFα: Tumor necrosis factor alpha; PTD: preterm delivery; PPROM: premature rupture of the membranes; Endo-PAT: endothelial peripheral arterial tonometry; ISSHP: the International Society for the Study of Hypertension in Pregnancy

Introduction

Tumor necrosis factor alpha (TNFα) is as a major regulator of inflammatory responses in various inflammatory and autoimmune diseases, and is mainly generated by activated macrophages, T-lymphocytes, and natural killer cells Bradley [1], Horiuchi et al. [2]. TNFα binds to two different receptors, triggering signaling pathways involving other cytokines and chemokines, underlying inflammation and cell death Idriss & Naismith [3]. Inappropriate or excessive activation of TNFα signaling is associated with chronic inflammation and can eventually lead to the development of complications such as autoimmune diseases. In pregnancy, TNFα influences hormone synthesis, placental architecture, and embryonic development, while increased TNFα levels in complicated pregnancies draw attention to trophoblast biology Romanowska-Próchnicka et al. [4]. Elevated TNFα may affect maternal-fetal interactions by altering the secretory profile of placental immunomodulatory factors, which in turn affect maternal immune cells Romanowska-Próchnicka et al. [5], Azizieh & Raghupathy [6]. While evidence linked TNFα to early placentation and growth, further evidence has shown increased levels near delivery Romanowska-Próchnicka et al. [5]. Overexpression of TNFα is linked to pro-inflammatory cytokines and the development of fetal growth restriction (FGR) in response to fetal hypoxia, possibly by decreasing the uptake of amino acids by the fetus Bartha et al. [7]. Increased TNFα was reported in preterm delivery (PTD) associated with premature rupture of the membranes (PPROM) and preeclampsia (PE) Gücer et al. [8], Bartha et al. [7]. In first trimester pregnancies, TNFα levels are twice higher in women at high risk of a subsequent development of PE, and at term it could reach five times higher levels in PE cases versus normal term delivery Trisnawati et al. [9].

In previous studies of this Slovenian cohort, we evaluated the uterine artery pulsatility index (UtA-PI) in differentiating PTD, PE and FGR. Using a UtA PI cutoff = 0.85, all cases of term controls and PTD <37 weeks gestation were significantly lower from all cases of FGR and PE+FGR Sharabi-Nov et al. [10]. Previous studies indicated that differential diagnosis of PE and PE+FGR are assisted by pro- and-anti-angiogenesis markers, inhibin A and the use of Endo-PAT (endothelial peripheral arterial tonometry) Sharabi-Nov et al. [11], Kumer et al. [12]. Here, we conducted a secondary analysis to explore whether the maternal serum level of TNFα, an inflammation markers could serve for the differential diagnosis of the complications alone, and whether vascular physiology that can be assessed by the blood flow through the uterine arteries measured by the Doppler pulsatility index (UtA-PI) and blood pressure measured by mean arterial pressure (MAP) could assist in the differential diagnosis of these complications from mid-gestation till term delivery.

Materials and methods

Samples and patients

Our dataset for this secondary analysis is based on patient records collected between 2012 and 2015 Sharabi-Nov et al. [11], Kumer et al. [12]. The National Medical Ethics Committee of the Republic of Slovenia approved the study (No. 104/04/12). The cohort database included women who signed their informed consent. Medical and delivery records were extracted from the outpatient clinics of the Department of Obstetrics and Gynecology of the University Medical Center of Ljubljana, Slovenia, attended for suspected PE, FGR, PE+FGR and PTD. Control term delivery (TD) cases were admitted due to a history of these complications in previous pregnancies. Excluded were women at gestational week <24, those in labor at enrolment, younger than 18 years, those with multifetal pregnancies, and major fetal malformation or chromosomal/genetic anomalies. We also excluded patients with pre-existing renal, hematological, autoimmune conditions, or chorioamnionitis along with mental disorders jeopardizing informed consent reliabillity.

TNFα measurements

TNFα was measured by immune-diagnostics. Ten ml blood were drawn into a vacutainer at enrolment, left for 1.5 h to clot at room temperature, then centrifuged at 1,500 x g for 15 min. Serum was collected and aliquots were kept at -70°C, and thawed once for testing in a LAB microplate analyzer (Adaltis, Italy). TNFα kits (International GMBH, Hamburg, Germany) were used according to the manufacturer’s instructions. The minimal detectable concentration was 5 pg/ml with inter- and intra-assay coefficients of variation of 8.1% and 7.7%.

Blood pressure measurements

Data of the blood pressure were extracted from the medical records from measurements performed according to the FMF guidelines using arm-adjusted cuffs, and a pre-calibrated automated device (OMRON M6 Comfort, Omron Healthcare Co., Ltd., Kyoto, Japan) Poon et al. [13]. Mean arterial blood pressure (MAP) was calculated according to (systolic+diastolic*2)/3 Poon et al. [13].

Uterine artery pulsatility index

Data were extracted from the medical records from records made with trans-abdominal sonography with a GE Voluson U6 and GE Voluson 8Expert and a 2-7 MHz GE RAB6-D probe (GE Healthcare GmbH, Solingen, Germany). The A pulsed Doppler sampling gate of 2 mm was used to cover each vessel, and an angle of insonation <30° with peak systolic velocity of >60 cm/sec was used to obtain the necessary waveforms before calculating the average of the pulsatility index in the left and right uterine arteries Oros et al. [14], Sharabi-Nov et al. [10].

Definitions of the clinical complications

Preeclampsia (PE) was diagnosed according to the American College of Obstetricians and Gynecologists, and the International Society for the Study of Hypertension in Pregnancy (ISSHP) guidelines at the time of admission (American College of Obstetricians and Gynecologists 2020, Magee et al. [15]). Diagnosis of PE required the presence of new-onset hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 90 mmHg) at ≥ 20 weeks’ gestation or chronic hypertension and either proteinuria (≥ 300 mg/24 h or protein-to-creatinine ratio ≥ 30 mg/mmol or ≥ 2+ on dipstick testing) or evidence of renal dysfunction (serum creatinine > 97 μmol/L), hepatic dysfunction (transaminases ≥ 65 IU/L) or hematological dysfunction (platelet count <100x103/mL). Fetal growth restriction (FGR) was defined according to ISUOG criteria Salomon et al. [16]. Preterm delivery (PTD) was defined as delivery <37 weeks’ gestation Goldenberg et al. [17].

Results

Cohort Flow Chart and Characterization: This is a secondary analysis included 125 pregnant women who had blood samples drawn at the time of enrolment. There were 21 cases of TD, 15 PTD cases (6 delivered <34 weeks gestation, and 9 between 34 to<37 weeks). There were 31 PE cases (3 delivered <34 weeks, 8 between 34 to <37 weeks, and 10 at term). There were 16 FGR cases (12 early who delivered <34 weeks, and 4 at term). The group of FGR+PE (not counted in the former groups) included 42 cases (28 delivered <34 weeks, 10 between 34 to <37 weeks, and 4 at term) (Table 1). Among the groups who delivered <34 weeks, most parameters were similar. MAP was significantly higher at enrolment and at delivery in the PE and FGR+PE groups (Tables 1 & 2, A). Delivery by Caesarean section had a higher incidence in FGR+PE, while any of the FGR cases delivered smaller (lower birth weight) newborns (Table 1). Similar differences were recorded among women who delivered between 34 to <37 weeks (Tables 1&2, B), although patients with a higher BMI were found in the FGR+PE group. MAP was higher in any PE patient at enrolment and at delivery. At term delivery, FGR cases had lower birthweight. MAP was higher in any case of PE and FGR+PE at enrolment and at delivery (Tables 1&2, C).

Uterine artery pulsatility index (UtA-PI): Among the groups who delivered <34 weeks (Table 2, A), UtA-PI at enrolment was higher in any of the PE, FGR and PE+FGR groups but no difference was found between TD and PTD, who were both significantly lower from any of the above. Among women who delivered between 34 to <37 weeks (Table 2, B), UtA-PI at enrolment was only higher in FGR+PE (no pure FGR cases in this time frame). Lower UtA-PI was measured in the TD and PTD cases who were indistinguishable. At term (Table 2, C), UtA-PI was higher in FGR+PE cases compared to TD.

Maternal serum TNFα: Figure 1 depicts the Box and Wiskers Plot of maternal blood levels of TNFα in the different groups divided according to the time of delivery. In the PTD group delivered <34 weeks, the group median was 321 pg/ml (95% Confidence Interval (95% CI): 145-715). This value was significantly higher compared to the level at GA between 34 to <37 weeks, which was 53 pg/ml (95 CI: 20-269), and also higher compared to TD cases, which was 22 pg/ml (95% CI: 14-145) (Figure 1A). Among the PE cases, the values of TNFα between 34 to <37 weeks were the highest for the group, 122 pg/ml (95% CI: 35-405), whereas TNFα values in cases <34 weeks and at term were lower, corresponding to 29 and 19 pg/ml, respectively (Figure 1B). However, the differences did not reach significance. FGR cases <34 weeks showed TNFα values of 50 pg/ml (95% CI: 12-312), which were ten times lower from 572 pg/ml (95% CI: 18-1349) in term FGR cases (Figure 1C). The group of combined FGR+PE also showed a reversed trend to the PTD group with 36 pg/ml (95% CI: 21-74) in cases delivered <34 weeks, reaching 42 pg/ml (95% CI: 17-366) at 34 to <37 weeks and ten times higher at term, 485 pg/ml (95% CI: 64-848) (Figure 1D).

Figure 2 shows the individual values of TNFα at the time of enrolment versus the outcome groups. It shows that for any of the FGR groups (pure FGR and FGR+PE), the regression line has a coefficient “r” with a clearly positive slope increasing with GA, with the respective values of r = 0.53 for pure FGR, and r = 0.37 for FGR+PE. Any of these is significantly different compared to the flat regression curves of the pure PE cases (r = 0.06) and the TD control cases (r = 0.04) (Fig. 2). By contrast, the regression line of the PTD cases shows a negative r-coefficient (r = - 0.19), reflecting that in these cases, TNFα values are decreasing over the second half of gestation. This regression coefficient of PTD is significantly different from the regression coefficients of the FGR and the FGR+PE groups.

TNFα and uterine artery pulsatility index in the second half of pregnancy: Figure 2 shows how different the two markers, TNFα (A) and UtA-PI (B), change with gestational age in the different groups. For the term delivery group, the values do not change during the evaluated period, with a slope of r = 0.04 for TNFαand a small increase (r=0.13) for UtA-PI. For the PTD group, TNFαdecreases from a higher value <34 weeks to a lower value at <37 weeks, with a moderate negative slope (r = -19). UtA-PI for this group is moderately increasing (r=0.23). For the PE group, TNF has almost a horizontal curve over GA, whereas the slope for UtA-PI sharply decreases with r = -067. For the FGR+PE group, TNFα significantly increases between GA <34 to GA >37 weeks with an r = 0.37, whereas UtA-PI decreases with an r = -0.44. Pure FGR shows the clearest changes with TNFα increasing with an r = 0.59 and UtA- PI decreasing with an r = -0.81.

Slope of UtA-PI / TNFα by gestational age: We plotted the ratio of UtA-PI (x 100)/TNFα) against gestational age at enrolment (Fig. 3). For the PTD and TD groups the ratio remains flat at the evaluated gestational ages (r = 0). For the pure PE group, the slope slightly decreased (r = -0.20). By contrast, the slope for FGR and FGR+PE groups showed a steep decrease with increasing gestational age. Accordingly, a differential diagnosis can be offered, indicating high TNF and low UtA-PI for early PTD cases (<34 weeks gestation) could mark these cases as unique groups compared to the patients with high UtA-PI who have low TNFα who subsequently developed pure FGR and FGR+PE. At term, when the PTD group already delivered, term FGR and to a lower extent term FGR+PE demonstrates higher TNFα compared to UtA-PI due to a continuous increase of TNFα in any FGR from the start of the third trimester to delivery at term. This was different from the level of TNFαin TD cases which was flat all through

Discussion

Principal findings

The main finding is the presence of high TNFα in PTD cases <34 weeks gestation with low UtA-PI and without hypertension. This creates an avenue for differential diagnosis of early PTD versus pure FGR, pure PE or PE+FGR. At GA>37 (term delivery), compared with PE group, the pure FGR group presents significantly higher TNFα values combined with high UtA-PI. There were no significant trends in values of TNFα in PE cases.

Results in the context of what is already known in the literature

TNFα is an inflammatory marker Idriss & Naismith [3], Bradley [1], Horiuchi et al. [2], and it is known that PE is associated with significant inflammation Guan et al. [18]. In fact, pregnant women with documented inflammation due to the COVID-19 pandemic developed symptoms similar to PE Lai et al. [19]. Impaired TNFα in PE cases were previously found to have gene polymorphisms at position −308 G/A of the TNFα promotor associated with the mother's reduced tolerance to the growing fetus Chen et al. [20], Lin et al. [21]. Such TNFα polymorphism was so far identified in patients of Chinese or African ethnic origin Chen et al. [20], Lin et al. [21], Raguema et al. [22], while our cohort was of a pure Caucasian ethnicity. This may explain why we did not see any significant differences in TNFα levels in pure PE cases. Molvarec et al. [23] have found that the SNP – 308 G/A of TNFα has a role in the risk of developing severe FGR complicated by PE. Hence, there is a need to explore this polymorphism in our population.

Clinical and research implications

The bimodal changes in TNFα between PTD (high <34 weeks, low >34 weeks) and FGR (low <34 weeks, high >37 weeks) with high UtA-PI <34 weeks in early FGR but not in early PTD without hypertension, may well be used to identify and differentiate between early FGR and PTD.

The graphical representation of our findings (Figure 4) indicates that in PTD cases, early alterations of the tissues in the reproductive organs (such as microfractures of the fetal membranes) Mikkelsen et al. [24] may result in an increased maternal inflammatory response, and thus lead to an early increase of TNFα levels (red arrow in Fig. 4).

The high level of TNFα decreases during the third trimester of pregnancy and thus allows the segregation between the very early PTD cases <34 weeks gestation to later PTD cases. This may reflect unaltered placental perfusion with maternal blood, and a placental unstressed status Huppertz [25], Huppertz et al. [26].

By contrast, in FGR cases (with and without PE), the long-lasting alterations of placental perfusion with maternal blood, due to an impaired transformation of uterine spiral arteries, may result in an increasing placental stress Bradley [1], Huppertz [25], Huppertz et al. [26]. Hence, a late increase of TNFα levels in such cases appears to be initiated by the placenta (blue arrow, Fig. 4). In FGR cases, high velocities of maternal blood flowing into the placenta induce damage to villous tissues and increased peripheral resistance in the placental vasculature Huppertz [25], Huppertz et al. [26]. This leads to a reduced transfer of oxygen and nutrients to the fetus, which in turn results in increased oxygen levels of the maternal blood in the intervillous space (hyperoxia) and at the same time hypoxia of fetal blood and the fetus (Figure 4) Huppertz [25], Huppertz et al. [26].

Strengths and limitations:

A strength of the study is coverage of the pregnancy epidemiology of Slovenia. The prevalence of PE in Slovenia (pure PE and FGR+PE combined) accounts for 2-3% of all deliveries. Having a cohort of 73 cases of PE (combining 31 cases of pure PE and 42 cases of PE+FGR) accounts for an approximate sample size of 2,433-3,650 pregnant women, corresponding to 15-20% of all deliveries in Slovenia, and around 40% of the deliveries in the medical center of Ljubljana. In this respect, our study has a power of 0.85-0.90. This appears reasonable, given we also included a group of preterm deliveries that were unrelated to PE and FGR (mainly spontaneous PTD cases, and had also a control group of unaffected cases at term. Of course, larger studies are needed.

A limitation of this study is the small cohort size. Although powered for the analysis, TNFα value standardization and their conversion to medians of the mean was not possible. Hence, our model was based on regression analysis and used the trend of changes for each group against gestational age and the ratio of the two markers (each of them behaved very differently) as a way to extract the values for the differential diagnosis of the use of TNFαand UtA-PI as aiding tools for differential diagnosis.

We measured the marker value at admission with a window between 24-41 weeks, and not in fixed gestational age. However, this mimics the real-life situation. Another limitation was the lack of repeated testing, which in principle could have helped to increase marker accuracy [27,28], (supplementary table),(Figure3,5).

Conclusions

This study showed how combining TNFα with UtA-PI could add to the differential clinical diagnosis of pregnancy complications from the 24th week of gestation towards delivery. High TNFα in cases <34 weeks gestation, when coupled to low UtA-PI and normal blood pressure reflects early PTD linked to maternal inflammation. At term, high TNFα combined with high UtA-PI is associated with any FGR (with/without PE), possibly reflecting inflammation and maternal and fetal hypoxia due to the very long period of altered placental perfusion. More and larger studies are warranted to verify the findings.

References

1. Bradley JR (2008) TNF-Mediated inflammatory disease. J Pathol 214(2): 149-160.
2. Horiuchi T, Mitoma H, Harashima S, Tsukamoto H, Shimoda T, et al. (2010) Transmembrane TNF-alpha: Structure, function and interaction with anti-TNF agents. Rheumatology 49(7): 1215-1228.
3. Idriss HT, Naismith JH (2000) TNF alpha and the TNF receptor superfamily: structure-function relationships. Microsc Res Tech 50(3): 184-195.
4. Romanowska PK, Felis GA, Olesi´nska M, Wojdasiewicz P, Paradowska GA et al. (2021) The Role of TNF-α and Anti-TNF-α Agents during Preconception, Pregnancy, and Breastfeeding. Int J Mol Sci 22(6): 2922.
5. Azizieh FY, Raghupathy RJ (2015) Tumor Necrosis Factor-α and Pregnancy Complications: A Prospective Study. Med Princ Pract 24(2): 165-170.
6. Bartha JL, Romero CR, Comino DR (2003) Inflammatory cytokines in intrauterine growth retardation. Acta Obstet Gynecol Scand 82(12): 1099-1102.
7. Gücer F, Balkanli KP, Yüksel M, Yüce MA, Türe M, et al. (2001) Maternal serum tumor necrosis factor-alpha in patients with preterm labor. J Reprod Med 46: 232-236.
8. Trisnawati E, Nontji W, Nurasni St (2020) Tumour necrosis factor-α (TNF-α) serum levels in preeclampsia pregnant women and pregnant women at risk with preeclampsia. Enfermería Clínica 30(S2): 27-30.
9. Sharabi NA, Tul N, Kumer K, Premru ST, Fabjan VV et al. (2022) Biophysical Markers of Suspected Preeclampsia, Fetal Growth Restriction and The Two Combined-How Accurate They Are? Reprod Med 3(2): 62-84.
10. Sharabi NA, Premru ST, Kumer K, Fabjan VV, Fabjan T, et al. (2021) Maternal Serum Inhibin-A Augments the Value of Maternal Serum PlGF and of sFlt-1/PlGF Ratio in the Prediction of Preeclampsia and/or FGR Near Delivery-A Secondary Analysis. Reprod Med 2(1): 35-49.
11. Kumer K, Sharabi NA, Fabjan VV, Premru ST, Tul N, et al. (2021) Pro- and Anti-Angiogenic Markers as Clinical Tools for Suspected Preeclampsia with and without FGR near Delivery-A Secondary Analysis. Reprod Med 2: 12-25.
12. Poon LC, Kametas NA, Chelemen T, Leal A, Nicolaides KH, et al. (2010) Maternal risk factors for hypertensive disorders in pregnancy: A multivariate approach. J Hum Hypertens 24(2): 104-110.
13. Oros D, Figueras F, Cruz MR, Meler E, Munmany M, et al. (2011) Longitudinal changes in uterine, umbilical and fetal cerebral Doppler indices in late-onset small-for-gestational age fetuses. Ultrasound Obstet Gynecol 37(2): 191-195.
14. Sharabi NA, Kumer K, Fabjan VV, Premru ST, Tul N, et al. (2019) Establishing a differential marker profile for pregnancy complications near delivery. Fetal Diagn Ther 47(6): 471-484.
15. Magee LA, Brown MA, Hall DR, Gupte S, Hennessy A, et al. (2021) The 2021 International Society for the Study of Hypertension in Pregnancy classification, diagnosis & management recommendations for international practice. Pregnancy Hypertens 27: 148-169.
16. Salomon LJ, Alfirevic Z, Da Silva CF, Deter RL, Figueras F, et al. (2019) ISUOG practice guidelines: Ultrasound assessment of fetal biometry and growth. Ultrasound Obstet Gynecol 53(6): 715-723.
17. Goldenberg RL, Culhane JF, Iams JD, Romero R (2008) Epidemiology and causes of preterm birth. Lancet 371(9606): 75-84.
18. Guan X, Fu Y, Liu Y, Cui M, Zhang C et al. (2023) The role of inflammatory biomarkers in the development and progression of pre-eclampsia: a systematic review and meta-analysis. Front Immunol 14: 1156039.
19. Lai J, Romero R, Tarca AL, Iliodromiti S, Rehal A, et al. (2021) SARS-CoV-2 and the subsequent development of preeclampsia and preterm birth: evidence of a dose-response relationship supporting causality. Am J Obstet Gynecol 225(6): 689-693.e1.
20. Chen YP, Pfab T, Slowinski T, Richter CM, Godes M, et al. (2006) Impact of genetic variation of tumor necrosis factor-alpha on gestational hypertension. Chin Med J (Engl) 119(9): 719-724.
21. Lin Y, Wang L, Yan Y, Zhou W, Chen Z, et al. (2019) A meta-analysis of tumor necrosis factor-α and FAS/FASL polymorphisms with risk of pre-eclampsia. Hypertens Pregnancy 38(1): 20-31.
22. Raguema N, Ben AGM, Zitouni H, Ben LD, Seda O, et al. (2022) Contribution of -1031T/C and -376G/A tumor necrosis factor alpha polymorphisms and haplotypes to preeclampsia risk in Tunisia (North Africa). J Reprod Immunol 149: 103461.
23. Molvarec A, Jermendy A, Nagy B, Kovács M, Várkonyi T, et al. (2008) Association between tumor necrosis factor (TNF)-alpha G-308A gene polymorphism and preeclampsia complicated by severe fetal growth restriction. Clin Chim Acta 392(1-2): 52-57.
24. Mikkelsen E, Huppertz B, Singh R, Rasvn K, Hatt L, et al. (2023) mRNA and protein expression in human fetal membrane cells: potential biomarkers for preterm prelabor rupture of the fetal membranes? Int J Mol Sci 24(21): 15826.
25. Huppertz B, Weiss G, Moser G (2014) Trophoblast invasion and oxygenation of the placenta: measurements versus presumptions. J Reprod Immunol 101-102: 74-79.
26. Huppertz B (2023) Placental physioxia is based on spatial and temporal variations of placental oxygenation throughout pregnancy. J Reprod Immunol 158: 103985.
27. Kumer K, Premru ST, Fabjan VV, Tul N, Fabjan T, et al. (2018) Peripheral arterial tonometry and angiogenic biomarkers in preeclampsia. Hypertens Pregnancy 37(4): 197-203.
28. (2020) American College of Obstetricians and Gynecologists, Gestational Hypertension and Preeclampsia: ACOG Practice Bulletin Summary, Number 222. Obstet Gynecol 135(6): 1492-1495.