Raman Spectroscopy of the Junctional Zone Reveals Metabolic Alterations in IVF-Conceived Mouse Placentas

Reza K. Oqani¹, Arda Inanc^2,3, Muhammet Baki Karakus², Begum Durkut-Kuzu⁴, Melike Ucak⁴, Mehmet Burcin Unlu^5,6, Bora Akgun^2,3, Emin Maltepe¹, Ciler Celik-Ozenci^7,8 and Paolo Rinaudo⁹
Affiliations: ¹Department of Pediatrics, University of California, San Francisco, USA; ²Department of Physics, Bogazici University, Istanbul, Turkey; ³Center for Life Sciences and Technologies, Bogazici University, Istanbul, Turkey; ⁴Graduate School of Health Sciences, Reproductive Medicine, Koc University, Istanbul, Turkey; ⁵Faculty of Engineering, Ozyegin University, Istanbul, Turkey; ⁶Faculty of Aviation and Aeronautical Sciences, Ozyegin University, Istanbul, Turkey; ⁷Department of Histology and Embryology, School of Medicine, Koc University, Istanbul, Turkey; ⁸Koc University Research Center for Translational Medicine, Istanbul, Turkey; ^{9 1}Department of Obstetrics and Gynecology, University of California, San Francisco, USA.

Abstract Text:

Placental development is essential for fetal growth and pregnancy success, with molecular and structural differences significantly influencing its function. Placentae resulting from IVF are larger and exhibit changes in the transport of amino acids and glucose, although their metabolic activity remains unexplored.

Raman spectroscopy, a label-free and non-invasive technique, allows for biochemical characterization of placental tissues at a molecular level. In this study, we employed a novel Raman micro-spectroscopy method to compare the metabolic signatures in histologic slides of E12.5 placentas generated by natural conception (FB group) versus those produced by in vitro fertilization (IVF group).

IVF conceptuses were generated by superovulating CF-1 female mice with 5IU PMSG and 5 IU hCG 48 hours later. MII oocytes were fertilized with capacitated sperm from B6D2F1 males and cultured in potassium simplex optimization medium with amino acids (KSOMaa) until the blastocyst stage.  Control concepti were generated by flushing blastocysts (FB group) at E3.5 blastocysts. Blastocyst-stage embryos (16 embryos per recipient) were non-surgically transferred into one uterine horn of pseudo-pregnant CD-1 females. At E12.5, placentas from recipients were collected, weighed, and fixed in 4% formaldehyde for Raman spectroscopy.

A custom-built Raman micro-spectroscopy setup was used to scan placental tissues. A 785 nm diode laser was focused using a 40x, 0.6 NA objective lens onto tissues adhered to stainless steel slides. Raman-scattered photons were collected and coupled to a spectrometer. Ten placental samples (five FB, five IVF) were raster scanned using a motorized XY stage controlled via MATLAB, with 5 µm steps and 1 s integration per spectrum. The RamanSPy library and the OPTICS clustering algorithm were used for spectrum preprocessing and edge detection of tissue regions, respectively. Significant spectral differences between the FB and IVF groups were differentiated using Bonferroni-corrected Mann-Whitney U tests (p < 0.001).

Spectral analysis revealed increased Raman intensity in the junctional zone of IVF placentas at various wavelengths, which was confirmed by corresponding H&E images, with Raman successfully distinguishing between the decidua and labyrinth zone; the junctional zone, primarily composed of glycogenic cells and trophoblasts, plays a crucial role in nutrient storage and trophoblast invasion. The increase at 930–940 cm⁻¹ and 1040–1120 cm⁻¹, corresponding to carbohydrates and glycogen, indicates altered energy metabolism. The heightened 1000 cm⁻¹ peak in IVF samples suggests increased phenylalanine content, while the 1360–1380 cm⁻¹ signals indicate nucleic acid and tryptophan enrichment, which may reflect differential gene expression patterns. In contrast, IVF placentas exhibited lower Raman intensity at 1280–1310 cm⁻¹ (Amide III, lipids), 1420–1450 cm⁻¹ (fatty acids, proteins), 1640–1680 cm⁻¹ (Amide I, secondary protein structures), and 1735–1740 cm⁻¹ (phospholipids), suggesting alterations in protein folding, membrane composition, and lipid and phospholipid metabolism.

These findings align with histological and transcriptomic evidence of molecular perturbations in IVF placentas. The observed reduction in lipid-associated signals in IVF placentas could compromise membrane integrity and cell signaling, while increased carbohydrate and nucleic acid-related signals may indicate altered trophoblast proliferation and differentiation. These results provide new insights into the molecular underpinnings of IVF-associated placental adaptations and may have implications for understanding the long-term developmental outcomes of IVF conceptions.