Non-invasive transabdominal measurement of placental oxygenation: a step toward continuous monitoring.

This study aimed to assess transabdominal placental oxygenation levels non-invasively. A wearable device was designed and tested in 12 pregnant women with an anterior placenta, 5 of whom had maternal pregnancy complications. Preliminary results revealed that the placental oxygenation level is closely related to pregnancy complications and placental pathology. Women with maternal pregnancy complications were found to have a lower placental oxygenation level (69.4% ± 6.7%) than those with uncomplicated pregnancy (75.0% ± 5.8%). This device is a step in the development of a point-of-care method designed to continuously monitor placental oxygenation and to assess maternal and fetal health.

Many studies of the placenta have been based on the examination of the organ ex-vivo [32][33][34] or in the study of the anatomy and function in animals [35][36][37]. Non-invasive assessment of placental anatomy and function is considered an important priority. Imaging with ultrasound, thus far, is the predominant non-invasive method which provides information about the dimensions, location, gross anatomical lesions, as well as impedance to flow of the fetal circulation [38][39][40][41][42]. However, it does not provide quantitative assessment of placental oxygenation which is key for fetal survival. Functional magnetic resonance imaging (fMRI) can provide information about oxygenation, but it is not portable, is expensive and at this point cannot be used at the patient's bedside. Thus, a non-invasive method to assess placental oxygenation at the bedside is needed to investigate placental function during pregnancy to assess fetal well-being and the respiratory function of the placenta.
Near-Infrared Spectroscopy (NIRS) is an optical method that non-invasively measures bloodoxygenated and deoxygenated hemoglobin and tissue oxygenation in deep-tissue layers such as brain and muscle. This method has been used widely by many researchers to assess tissue hemodynamics, specifically, changes in blood hemoglobin concentrations in the brain [43]. It has also been used previously for placental oxygenation measurements [44][45][46][47]. However, results from these studies have been conflicting. While Matsuo et al. [44] reported a decreased placental oxygenation capacity in women with preeclampsia, Hasegawa et al. [45] found an increased tissue oxygen index in mother of small for gestational age babies with severe preeclampsia. This inconsistency could be due to an unknown placental reduced-scattering coefficient used in the calculation of oxygenation or differences in patient populations. A major challenge in the assessment of placental oxygenation using NIRS derives from the organ's anatomical location. To reach the placenta, the light emitted from a light source at the maternal abdomen surface must travel through several tissue layers (e.g., skin, adipose tissue, and the uterine wall), each having varying thicknesses on an individual basis. To address this challenge, the thickness of all layers lying above the placenta ought to be measured and the NIRS device should include multiple source-detector separations, which can probe different depth layers.
A novel depth-resolved NIRS device that features six source-detector distances ranging from 10-60 mm was designed. The NIRS device was capable of observing changes in the optical properties of a placenta-mimicking phantom at a depth of 25 mm. By using an intensity of backscattered light from various tissue depths along with the thickness of tissue layers acquired from ultrasound imaging, we measured the in-vivo placental oxygenation in anterior placentas during the third trimester of pregnancy. In a separate study, the placental reduced scattering coefficients as a function of NIR wavelengths were reported for the first time [48]. These values were then used to correct for the scattering effects in tissue oxygenation calculation.

Measurement device
Tissue oxygenation was measured using an in-house custom-made flexible NIRS device ( Fig. 1(a)). The device consists of six dual wavelength LEDs (760 nm and 840 nm) (L760/840-05A, Ushio Semiconductors, Japan) arranged in a single row with an LED-to-LED spacing of 10 mm. Two large-area silicon photodiodes (S12915-66R, Hamamatsu photonics, Japan) were placed, one at each end of the LED array. The center-to-center distance between the photodiode and the closest LED was 10 mm. Six LEDs and two photodiodes formed a range of six source-detector separations (SDSs) from 10 to 60 mm to target different depths. The flexible part of the circuit board was encapsulated in a thick layer of black silicone (Sylgard 184 silicone elastomer, Dow Silicones Corporation, USA and India ink) and the control board was enclosed in a 3D printed box.
The photodiode output was amplified by using a two-stage amplifier and digitized by using a 16-bit Analog to Digital Converter (ADC), which allowed us to detect light at a low-intensity level. Each SDS and wavelength combination was operated one at a time by switching the respective LED ON and OFF sequentially. The LED intensity was controllable with 0 to 50 mA current through each LED (the maximum intensity was 20 mW), which is useful when choosing an optimum optical power for the different ranges of skin melanin. In this experiment, the intensity of all LEDs was set to be equal. The device sampling rate was 0.5 Hz. A Bluetooth module (BT832, Fanstel, USA) was included in the circuit to provide wireless data transmission. The control system was designed using a microcontroller (ATMEGA32U4, Microchip Technology Inc, USA) operating at 16 MHz clock frequency. The firmware for the controller was developed using an Arduino programming environment to acquire and process simple commands via serial interface from a computer or a cell phone. The command set included settings related to the LED selection, LED current, ADC gains and the data acquisition rate. Data collected from the NIRS device were transferred to a computer through a lab-designed, MATLAB-based graphical user interface via a Bluetooth or USB connection.

Phantom experiment
A phantom, mimicking the maternal tissue and the placenta experiment was designed to test the sensitivity of each SDS to changes in light absorption of the placenta layer. The phantom consisted of 4 layers, with 3 solid layers on the top and 1 liquid layer at the bottom ( Fig. 1(b)). The solid phantoms were made of polydimethylsiloxane (PDMS, Sylgard TM 184 silicone elastomer, Dow Silicones Corporation, USA), Titanium dioxide powder (TiO 2 , Atlantic Equipment Engineers, USA), and India ink. The amount of ink and TiO 2 were determined based on the study by Ayers et al. [49] to obtain the desired absorption coefficient (µ a ) and reduced scattering coefficients (µ s ′ ) at 650 nm. The first phantom layer was designed to mimic the optical properties of skin with µ a = 2 mm −1 (high melanin level), µ s ′ = 1.5 mm −1 [50] and thickness = 2 mm. The second phantom layer mimicked the optical properties of adipose tissue with µ a = 0.03 mm −1 , µ s ′ = 1.5 mm −1 [50]. Thickness of the second layer varied from 4 mm to 20 mm (in 2 mm increment). The third phantom layer mimicked the optical properties of uterine muscle tissue with µ a = 0.1 mm −1 , µ s ′ = 0.9 mm −1 [50] and thickness = 3 mm. The liquid phantom was a mixture of water, 1% intralipid, and varied amounts of India ink (various µ a ). The NIRS device and the phantoms were arranged as shown in Fig. 1(b). Initially, the experiment started without the presence of India ink in the liquid phantom. The ink was then gradually injected into the liquid phantom (which represents placental tissue) at the bottom. NIRS intensities were recorded at three ink concentrations (5, 15, and 25 µl per 100 ml of liquid phantom). For each addition of ink, the liquid phantom was thoroughly stirred to mix the ink evenly. This procedure was repeated for all adipose tissue phantom thicknesses ranging from 4 mm to 20 mm.

In-vivo experiment
To assess accuracy and validate the performance of the custom-made NIRS device, in-vivo oxygenation measurement was performed in two human subjects at multiple parts of the body including both arms, calves, and abdomen using the wearable NIRS device and a time-domain NIRS system (TRS-41 system, Hamamatsu photonics, Japan). During the experiment, the subjects sat quietly in a comfortable chair and the two device/system were attached securely to their body parts to minimize motion artifacts. The time-domain NIRS system emits and collects light at three wavelengths 760, 800, and 830 nm with a source-detector separation of 30 mm. At a body part, measurements were taken multiple times. Oxygenation levels measured from the two device/system were compared to evaluate the accuracy of the wearable NIRS device in measuring in-vivo oxygenation.

Participants and experimental procedure
Measurements on volunteer subjects were performed at the Center for Advanced Obstetrical Care and Research of the Perinatology Research Branch, located at the Detroit Medical Center (DMC, Detroit, Michigan, USA) and all procedures followed the guidelines and regulations described in a protocol approved by the Wayne State University Human Investigations Committee Institutional Review Board (090717MP4E). Inclusion criteria included females aged 18 years or older with a singleton pregnancy and a gestational age greater than 28 weeks with an anterior placenta diagnosed by ultrasound examination. This study recruited participants through a regular visit at the DMC where a physician introduced the protocol to patients. Each patient, who agreed to participate in the study, was referred to our clinical research coordinator for initial screening, which included a review of health and pregnancy information. Patients who provided written informed consent to participate subsequently underwent placental tissue oxygenation assessment.
At the beginning of each examination, the experimental procedures were reviewed with the participant and oral informed consent was confirmed. A total of 12 volunteers (Table 1) participated in this study. During the examination, the participant lied down in an examination bed in a supine position. Blood oxygen saturation and the thickness of the skin, adipose, and uterine tissue above the placenta were measured using an FDA-approved, medical-use pulse oximeter (Nellcor Oximax N-600X SpO2, Medtronic, Minnesota, USA) and ultrasound machine, respectively. The anatomical thickness was measured at the upper, middle, and lower parts of the placenta.
The tissue oxygenation was measured after an ultrasound examination and at the same positions as the thickness measurements. The NIRS device was placed and held by a nurse practitioner on the abdomen of the participant at the upper, middle, and lower parts of the placenta for approximately 30 seconds per position. During that time, measurements were taken continuously with a sampling rate of 0.5 Hz, which resulted in 15 data points. These data points were then averaged for each position to minimize the effect of motion artifacts. Based on the clinical history, the participants were classified into two groups: one group with maternal pregnancy complications and one group with uncomplicated pregnancy. Those patients with short cervix, hypertension and polyhydramnios (Table 1) were categorized in the group with pregnancy complications.
Where ε is the molar extinction coefficient of HbO and HHb, i = HbO and HHb, j = 760 nm and 840 nm. Tissue oxygenation saturation level (StO 2 ) was derived from HbO and HHb by StO 2 = HbO/(HbO + HHb) × 100%.

Phantom experiments
For a given adipose thickness, the backscattered light intensity at different SDSs decreases when the amount of ink added to the liquid phantom increases. This demonstrated that the NIRS device is sensitive to the changes in optical absorption at the fourth phantom layer. The device displayed such sensitivity with all adipose phantom thicknesses (4 mm to 20 mm, data not shown). Figure 2(a), b represent the intensity at 4 SDSs (30, 40, 50, and 60 mm) in the phantom experiment with an adipose thickness of 20 mm. The inverse relationship between the photodetector output and amount of ink added to the solution was observed at both wavelengths. With 2 mm skin, 20 mm adipose and 3 mm uterine wall, the liquid phantom (mimicking placenta) was 25 mm away from the measurement surface, which means that our NIRS device is sensitive to the optical signal at least 25 mm below the abdomen surface.

In-vivo oxygenation
Oxygenation levels at the arms, calves, and abdomen of two subjects measured by the wearable NIRS device and time-domain NIRS system (TRS system) are presented in Fig. 2(c). The wearable NIRS device yields close oxygenation levels with the time-domain NIRS system at different body parts (R 2 = 0.94, Fig. 2(c)). An averaged error of 2.7% ± 1.8% was found between the two device/system. The agreement between measurements from the wearable NIRS device and a well-established TRS system validates a high accuracy of our device in measuring in-vivo tissue oxygenation.

Pregnancy outcomes and placental pathology
Eleven out of twelve participants delivered at the DMC. After delivery, pregnancy outcomes including mode of delivery, gestational age, labor status at delivery, birth weight, Apgar scores at 1 minute and at 5 minutes and neonatal complications were recorded (Table 2). In addition, the placentas of 10 of the 11 participants were delivered to the pathology department at the DMC to inspect for lesions. Five placentas were found to have chronic or acute lesions, four of which belonged to participants with maternal pregnancy complications ( Table 2).

Tissue thickness
The thicknesses of the skin, adipose, and uterus above the placenta varied depending on measurement positions, but no significant difference between different positions was found (Fig. 3). Mean thicknesses of the skin, adipose, and uterine tissue across subjects were 2.2 ± 0.6 mm, 14.5 ± 7.8 mm, and 6.2 ± 2.0 mm, respectively. The total thickness of the three tissues layers above placenta was 23.0 ± 8.9 mm.

Pregnancy with and without maternal complications
The antenatal diagnosis of the participants indicates that 3 participants had hypertension, 1 participant had polyhydramnios, and 1 participant had a short cervix (Table 1). These 5 participants were assigned as a group with maternal pregnancy complications while the other  7 participants were categorized as a group with an uncomplicated pregnancy. The tissue oxygenation level was divided into two groups accordingly. For each patient, three oxygenation levels at the three measurement sites above the placenta are reported. A two-way ANOVA was used to compare oxygenation levels based on both measurement positions (middle n = 12, upper n = 12, lower n = 12) and maternal complication status (No complication n = 7, with complication n = 5). The result showed a significantly higher oxygenation level in the group with an uncomplicated pregnancy (75.0% ± 5.8%) compared to those with pregnancy complications (69.4% ± 6.7%) (F(1,30) = 7.8, p = 0.009). Significant difference in the mean oxygenation levels at the middle (69.1% ± 6.8%), upper (74.5% ± 6.3%), and lower (74.3% ± 5.9%) parts of the placenta (F(2,30) = 3.5, p = 0.044) was further observed (Fig. 4(a)). However  Correlation analysis, using a two-tailed Pearson correlation, revealed no relation between the oxygenation levels and adipose thickness (r = 0.03, p = 0.87). This lack of correlation combined with homogenous tissue thickness at different measurement sites indicates that significant difference in obtained oxygenation level at different measurement sites are due to changes in placental oxygenation level based on existence of maternal complications.

Placenta with and without lesions
Based on the placental pathology, the tissue oxygenation levels were divided into two groups, placenta-with-lesions and placenta-without-lesions groups. A two-way ANOVA was performed to compare placental oxygenation levels at different measurement positions above the placenta (middle n = 10, upper n = 10, lower n = 10) and presence of placental lesions (no lesion n = 5, with lesions n = 5). Since the placenta pathology data of subject 11 and 12 were not available, the oxygenation levels of the two subjects were not included in the lesion analysis. The statistical test indicated a significantly lower oxygenation level in those with presence of placental lesions (68.7% ± 5.6%) group than those without lesions (74.2% ± 5.8%) group (F(1,24) = 7.7, p = 0.010) across all measurement sites. No significant difference in the mean oxygenation levels at the different measurement sites (F(2,24) = 3.2, p = 0.061) were observed ( Fig. 4(b)). The interaction between the lesion presence and measurement position was not significant (F(2,24) = 0.2, p = 0.822).

Discussion
In this study, we designed a flexible NIRS device that measures the optical properties of the anterior placental tissue as deep as 25 mm below the measurement surface. Anterior placenta is associated with an increased risk of dysfunctional labor and postpartum problems [52]. The NIRS device has been used to measure the transabdominal placental oxygenation in 12 participants. The results indicate a close relation between the placental oxygenation and pregnancy complications as well as placental pathology. Women with maternal pregnancy complications had a significantly lower placental oxygenation level than the group with uncomplicated pregnancy. We further found that patients whose placenta was lesion-free presented a significantly higher placental oxygenation level than patients whose placenta had lesions. These results suggest that placental oxygenation might infer placental complications. It is also possible that presence of lesions can increase the regional blood flow causing further placental complications. Furthermore, we noticed that the placental oxygenation levels vary depending on the measurement location, thus suggesting the importance of multi-site repeated measurements to obtain accurate results. Our results further show that the tissue thickness across measurement sites were homogenous. Moreover, we did not find a significant correlation between thickness of adipose tissue and oxygenation level. Together these results indicate the independency of the measurements from maternal tissue layers and that observed differences in oxygen level at different measurement sites are based on changes in placental oxygenation. Finally, by incorporating the reduced scattering coefficient of the placental tissue in the analysis in Eq. (1), we were able to eliminate the dependence of the placental oxygenation levels on the scattering effects.
The average thickness of tissues above the placenta in the study was 23.0 mm. Based on our validation experiments using phantom and in-vivo study, our device is capable of obtaining placental oxygenation at up to 25 mm below the abdomen surface. However, due to tissue thickness variability, particularly adipose thickness, the placenta can be more than 25 mm below the abdomen surface, which is above the probing limit of the current wearable NIRS device. Under such conditions, increasing source-detector separation as well as upgrading device components such as photodetectors will be necessary to improve the penetration depth.
The structural flexibility of our NIRS device ensures good contact with the skin on a curved abdominal surface. In addition, the incorporation of multiple source-detector separations enables us to acquire a signal from different depths. In general, a longer source-detector separation is preferred since it can detect a signal at deeper layers. However, in the case of a highly absorbing tissue, the detector at a far separation distance may not capture sufficient backscattered light. In our experiments, in 10 African American participants, the signals measured at 60 mm (in some cases, 50 mm) separation were discarded due to a very low signal-to-noise ratio.
A limitation of the current study is that the multi-layer tissues (skin, adipose, uterus, placenta) were considered as a homogenous medium and only transabdominal placental oxygenation was measured. Future studies will incorporate anatomical thickness measurements with the acquired signals from multiple source-detector separations and use advanced data processing algorithms to separate placental oxygenation from maternal tissue oxygenation.

Conclusion
This study presents the design of a flexible, affordable NIRS device, which is capable of continuously monitoring placental oxygenation levels. Our results suggest the possibility of the relationship between the placental oxygenation level and pregnancy complications and placental pathology. However, the sample size used in this study is small (12 participants). Further study that includes more subjects should be conducted to draw a reliable correlation between placental oxygenation and pregnancy health.