Pregnancies complicated by preeclampsia (PE), fetal growth restriction (FGR), or maternal diabetes (GDM) exhibit various patterns of placental histopathological lesions. These lesions include infarcts, fibrinoid deposition, syncytial knots, thrombosis, villitis, chorangiosis, villous dysmaturity and overall abnormalities in placental size and were recently standardized by the Amsterdam Criteria (Khong et al., 2016). While there is no single pathognomonic lesion for any of these syndromes, certain patterns are considered to be suggestive of specific maternal-fetal conditions (Table 1).

Placental histopathology reveals lesions common to both normal and pathological pregnancies. As summarised in Table 1, many findings—such as infarcts, fibrinoid deposition, and syncytial knots—occur in normal placentas and are increased to a variable degree across various obstetric syndromes including PE, FGR, and GDM (Veerbeek et al., 2014; Salafia et al., 1992; Huynh et al., 2015; Byrne et al., 2025; Lai et al., 2024). While they may support clinical impressions, these lesions lack specificity and often provide only circumstantial evidence. Histological interpretation must therefore consider clinical context, gestational age, and possible therapeutic interventions. For instance, reduced placental weight in PE may reflect both the disease itself and medically indicated preterm delivery (Sebire et al., 2005). The true diagnostic strength of placental histopathology lies in the identification of infections. Acute chorioamnionitis, characterised by neutrophilic infiltration of membranes and cord, can be reliably diagnosed microscopically and is of high clinical relevance (Redline, 2015). In such cases, histology offers clarity where clinical signs may be subtle or ambiguous.

In summary, while its value in distinguishing villous or vascular and metabolic syndromes is limited, histopathology remains essential for diagnosing intrauterine infection. PE, FGR, and GDM are syndromes which are diagnosed primarily by clinical, but not by histopathologic parameters. For histopathologists and clinicians, this is a challenging situation. Efforts were therefore made to arrive at more focused and, especially, quantitative morphometric evaluations of placental structure to identify specific histological core correlates of the pathogenesis of the main obstetric syndromes.

Early quantitative placental morphology research relied on Stereology, in which randomly sampled, haematoxylin–eosin (HE)-stained, 2D histological sections are analysed to infer 3D structure of the sample (Mayhew, 2006). For example, FGR placentas showed significantly reduced volumes of all villous types along with smaller exchange surface areas (Mayhew et al., 2003). Such findings established a baseline understanding that FGR is associated with an “impoverished” villous tree in terms of bulk structure.

However, classical Stereology still depended on HE-stained 2D histological sections and on skilled human observers to identify the three classical villous types. Moreover, many aspects of villous 3D architecture—such as branching angles, connectivity, and spatial organisation—could principally not be assessed from 2D sections (Haeussner et al., 2016).

By the mid-2010s, researchers began critically evaluating the reliability of these 2D histology-based classifications. Haeussner et al. tested whether identifying villous types on 2D sections correlates with the villus’s actual position in the 3D placental tree (Haeussner et al., 2015). They found high inter-observer variability and poor correlation with actual 3D positions. This study demonstrated that the classical approach was inconsistent and often inaccurate, highlighting the need for more objective criteria and genuine three-dimensional analysis (Haeussner et al., 2015; Haeussner et al., 2016). The present review focuses on advanced three-dimensional morphological techniques that have been applied to human placentas since these fundamental challenges were first articulated.

Stereology is an excellent method for obtaining three-dimensional data of organs, including the placenta, from microscopic thin sections. It is a multi-step procedure designed to ensure that every part of the placenta has an equal chance of being placed under the microscope and getting analysed. The essential principle of Stereology lies in random sampling throughout all stages of histological tissue sampling, tissue preparation, and tissue sectioning. By adhering strictly to random processing, the results can be considered representative of the entire organ (Howard and Reed, 2004).

The application of Stereology to the placenta was pioneered, recommended, and refined primarily through the work of Terry Mayhew, among others (Mayhew et al., 1984; Mayhew et al., 1986; Mayhew and Burton, 1988; Mayhew et al., 1990; Mayhew et al., 1994; Mayhew and Wadrop, 1994; Jackson et al., 1987). To distinguish between different parts of the villous tree, HE sections were traditionally used. The stromal core of villous profiles were then classified as one of three histological villous types: terminal villi, intermediate villi, or stem villi (Demir et al., 1997; Kohnen et al., 1996; Sen et al., 1979; Kaufmann et al., 1979). This approach remained the standard for villous typing until it was challenged for being observer-dependent and subject to considerable inter-observer variability (Haeussner et al., 2015). Such variability inevitably led to inconsistencies in the accurate and consistent allocation of stereological findings to specific villous subtypes.

Subsequent studies sought to address this limitation by employing immunohistochemical markers to identify whether the perivascular stroma of villous profiles was positive or negative for γ -SMA (e.g., Barapatre et al. (2021); Barapatre et al. (2024)), a modification which had already been recommended by Haeussner et al. (2015). This immunohistochemical characterization allowed villous profiles to be classified into just two categories: C-villi and NC-villi. Notably, this classification does not correspond exactly to the traditional histological classifications of terminal, intermediate, or stem villi. Most of the studies reviewed here employ such immunohistochemistry-guided Stereology of the placenta (IHC-guided Stereology, Figure 1). IHC-guided Stereology has also been extended through the inclusion of second-order stereological parameters, particularly the branching index. This dimensionless value represents the percentage of concave villous surfaces observed in villous profiles. It is interpreted as a proxy for branching points, as concave villous surfaces in such profiles are predominantly associated with sectioning through branching regions rather than through straight segments of villi.

In contrast, the recently introduced method of three-dimensional (3D) microscopy (Haeussner et al., 2014; Haeussner et al., 2017) uses whole-mount preparations of small peripheral placental villous bushes, thereby eliminating the need for sectioning (Figure 2). The innovative section-free microscopic approach of 3D microscopy to the villous tree is illustrated in Figure 2. It allows direct recognition of branching points and of whole villi—rather than partial profiles—including branching angles, diameters, lengths, and surface areas (Figure 3a–z; Table 2). At higher magnification, the nuclei of individual villous trophoblast cells can be mapped directly onto the villous surface. Immunohistochemical staining, particularly of villous trophoblast, is feasible with such preparations. Software originally developed for tracing dendritic trees in neurons is applied to generate in silico reconstructions of peripheral branches via a digitised camera lucida procedure (Figure 3a–z). While 3D microscopy complements Stereology and provides unique data, it focuses specifically on peripheral branches, and its findings cannot be considered representative of the entire villous tree.

Multiple studies employing advanced stereological and three-dimensional microscopic techniques have detailed the morphological alterations in placentas from pregnancies complicated by FGR.

A principal observation is that FGR placentas are not merely smaller versions of normal placentas, but instead display distinct structural pathologies. Quantitative analyses show a significant reduction in the volume of C-villi, which include stem villi characterised by perivascular myofibroblasts. In contrast, NC-villi, representing more peripheral branches, exhibit no statistically significant volume reductions. Vessel volumes are markedly reduced in both compartments, indicating compromised vascularisation throughout the villous tree (Barapatre et al., 2021).

At the cellular level, FGR placentas demonstrate a marked increase in the density of PCNA-negative nuclei—representing post-proliferative syncytial nuclei—without a corresponding change in the density of proliferative (PCNA-positive) nuclei. This suggests that trophoblast proliferation is unaltered, while nuclear clearance or syncytial passage time may be impaired (Figure 4). These nuclei tend to accumulate at the villous surface, particularly in peripheral branches, and show increased spatial density, as evidenced by reduced nearest-neighbour distances. These structural reorganisations, undetectable by standard histology, are discernible through three-dimensional microscopy (Haeussner et al., 2017; Barapatre et al., 2019).

Furthermore, the sexual dimorphism normally observed in placental development—manifested by higher syncytial nuclear densities in female placentas and greater inter-nuclear spacing in males—is absent in FGR (Figure 4). This loss of dimorphism suggests a convergent pathological adaptation to growth restriction that overrides physiologically distinct developmental pathways. In normal pregnancies, such dimorphic features are thought to influence fetal vulnerability to intrauterine stress and subsequent postnatal health outcomes (Barapatre et al., 2019).

Branching complexity is also significantly altered in FGR placentas. Using novel 3D reconstruction methods, studies have shown that branching angles and hierarchical organisation of the villous tree correlate with fetoplacental weight ratios, a clinical indicator of placental efficiency. In FGR, this complexity is reduced, implying not only quantitative insufficiency but also a qualitatively aberrant developmental trajectory (Haeussner et al., 2014).

Together, these findings characterise FGR placentation as a condition involving disrupted villous growth, altered syncytial structure, and impaired nuclear dynamics. Sexual dimorphism is effectively neutralised, and the implementation of high-resolution 3D techniques provides a mechanistic insight into the impaired exchange function and increased perinatal risk associated with FGR.

A detailed stereological and 3D-microscopy-based investigation of placentas from late-onset PE, stratified by fetal sex, has revealed a complex pattern of trophoblast remodelling (Barapatre et al., 2024). A key observation is the significantly increased proliferative activity, evidenced by elevated PCNA-positive nuclear density, in female PE placentas relative to male PE placentas and controls. Concomitantly, a marked reduction in PCNA-negative nuclei in female PE placentas suggests accelerated trophoblast turnover in this group. This dynamic shift in nuclear composition is mirrored by nearest-neighbour density measures, underscoring the robustness of these findings. No such changes are present in male PE placentas, indicating a sex-specific trophoblast response to the preeclamptic environment. The sexual dimorphism observed in nuclear distribution in control placentas is absent in PE, suggesting disrupted trophoblast architecture across both sexes (Figure 4).

In addition to nuclear changes, PE placentas show significant alterations in villous morphology. The volume of non-contractile villi and their intravillous fetal vessels is reduced across both sexes, reflecting compromised exchange capacity. While this reduction is statistically significant in males, female PE placentas show a comparable trend that does not reach significance, implying a subtler but present effect. The branching index of the villous tree is significantly decreased, indicating impaired arborisation, with the reduction more pronounced in females.

These findings demonstrate that PE induces both quantitative and structural changes in the placenta, with clear sexual dimorphism. These alterations distinguish PE from related conditions such as FGR, where such sex-specific structural changes are less evident. The sex-dependent nature of trophoblast and villous adaptations may influence clinical outcomes, including gestational duration, maternal morbidity, and offspring health trajectories.

Placental alterations in well-controlled gestational diabetes mellitus (GDM) have been investigated using integrated stereological and 3D microscopic techniques (Barapatre et al., 2025). Although gross clinical parameters—including gestational age, birth weight, placental weight, and placental-to-birth weight ratios—do not significantly differ between GDM and control pregnancies, distinct microanatomical changes are evident.

A primary finding is a pronounced reduction in the branching index of both C-villi and NC-villi components, indicating a global simplification of the villous tree architecture. This effect appears independent of fetal sex and suggests impaired villous development affecting both early (stem villi) and late (terminal villi) forming structures.

Trophoblast dynamics further reveal sex-dependent effects (Figure 4). GDM placentas, particularly those of female fetuses, show increased surface density of PCNA-positive nuclei and decreased density of PCNA-negative nuclei. These changes are associated with reduced inter-nuclear distances among proliferative nuclei and increased spacing among non-proliferative nuclei—hallmarks of heightened turnover and abbreviated syncytial residence. Such imbalance likely promotes excess shedding of trophoblast material into maternal circulation, consistent with elevated placenta-derived exosome levels reported in GDM.

While overall villous and vascular volumes remain unaltered, subtle changes such as decreased standard deviation of diffusion distances in NC-villi are observed in females, suggesting functional compensation. These adaptations, though subclinical, highlight the capacity of the placenta to structurally accommodate metabolic stress. The predominance of changes in female placentas mirrors patterns seen in PE (Figure 4) and underscores the critical role of fetal sex in shaping placental resilience.

In conclusion, even in the absence of macrosomia or overt pathology, GDM induces significant trophoblast and villous changes. These alterations may represent an early, sex-specific adaptive response to metabolic dysregulation, with potential implications for the increased incidence of comorbid conditions such as PE.

Classifying placental villi presents a morphological challenge that has been mitigated through the use of immunohistochemical markers, notably γ -smooth muscle actin ( γ -SMA). This technique enables a binary distinction between “contractile” ( γ -SMA-positive) and “non-contractile” ( γ -SMA-negative) villi, reflecting their position within the branching hierarchy of the villous tree (Buehlmeyer et al., 2019; Barapatre et al., 2021; Kohnen et al., 1995; Kohnen et al., 1996; Graf et al., 1997). This innovation enhances objectivity in villous classification and reduces observer variability associated with HE-based typing (Haeussner et al., 2015). While total villous volume reduction in FGR was already known (Mayhew et al., 2003), it is now possible to localise this loss to the C-villi, sparing the NC-villi.

This stereological toolkit is effectively complemented by whole-mount 3D microscopy. Although this technique is limited to the analysis of peripheral villous branches, it remains the sole light microscopy method that enables a detailed, three-dimensional morphological analysis. Whole-mount specimens extend up to several hundred micrometres in depth and are analysed through a computer-assisted camera lucida method that digitally reconstructs a 3D model of the villous tree. Quantitative parameters such as branching angles, diameters, surface areas, lengths, and volumes can be extracted and spatially assigned to terminal and preterminal positions. Trophoblast phenotyping in these peripheral regions is achieved via immunohistochemical cell cycle markers. Proliferative trophoblast (PCNA-positive) and post-proliferative (PCNA-negative) compartments can be distinguished, allowing further calculations of nuclear surface densities and inter-nuclear distances—metrics not accessible via traditional histology.

Recent innovations in 3D microscopy, including confocal imaging, have further transformed placental analysis. These technologies allow direct visualisation and quantification of villous architecture—including branching angles, tortuosity, and connectivity—that cannot be captured through 2D approaches (Haeussner et al., 2016). These metrics provide critical insights into placental function, particularly under pathological conditions.

The villous branching index (BI), formerly known as the Concavity Index (CI), can be assessed on standard HE sections, although this precludes association with C- or NC-villi. The method hinges on the premise that branches between villous nodes exhibit rounded profiles. Upon random histological sectioning, these profiles present as convex, straight, or concave. Only branching points yield concave profiles. BI is a second-order stereological measure indicating the percentage of surface area comprising concave villous profiles. A higher BI reflects a greater density of villous branching points (Tables 3, 4; Barapatre et al. (2024); Barapatre et al. (2021)).

FGR, PE, and GDM are associated with a lower BI than healthy controls, indicating reduced branching complexity.

• FGR Placentas: These placentas exhibit a significantly reduced frequency of concave villous profiles, indicating diminished branching. The villous tree is developmentally altered, not merely miniaturised. No sex differences were observed in BI in FGR placentas (Barapatre et al., 2021).

Often referred to as the “mirror of the prenatal period,” the placental functional microarchitecture is now revealed in unprecedented detail through modern histological techniques. Traditional Stereology and histopathology laid the groundwork, but were limited in resolution and specificity. Recent research by Haeussner, Barapatre, Buehlmeyer, Lahti-Pulkkinen and colleagues (Haeussner et al., 2014; Haeussner et al., 2015; Haeussner et al., 2017; Barapatre et al., 2021; Barapatre et al., 2019; Barapatre et al., 2024; Lahti-Pulkkinen et al., 2018) illustrates how the integration of immunohistochemistry and 3D microscopy refines our understanding. By delineating central versus peripheral villi, one can identify specific compartments affected by pathology. Quantification of nuclear and branching characteristics in 3D exposes regulatory phenomena previously undetectable in 2D. Stratifying morphological data by sex or linking placental metrics to offspring outcomes further embeds placental pathology within personalised medicine.

Importantly, these studies do not merely describe phenotypes but offer mechanistic insights. For instance, the identification of reduced volume of the more centrally located C-villi in FGR redirects attention to early placental development, implicating mesenchymal and vasculogenic defects (Tables 3, 4; Barapatre et al. (2021)). The emergence of sex-specific placental responses in PE suggests hormonal or genetic modulation, potentially informing risk assessment (Barapatre et al., 2024). Correlations between placental structure and child neurodevelopment (Lahti-Pulkkinen et al., 2018) imply that placental structural features could serve as a proxy for intrauterine conditions, encouraging examination even in pregnancies without overt complications.

From a methodological perspective, these findings support the practical feasibility of integrating quantitative histology into routine diagnostics. Modern computing enables scalable application of systematic sampling, point counting, and 3D reconstruction. With reasonable sample sizes, rigorous and reproducible quantitative analyses are achievable. Routine pathology may soon incorporate selected immunohistochemical stains (e.g., γ -SMA, PCNA) and appropriate, possibly simplified tools to objectively quantify placental function.

In summary, while current histological standards (e.g., Amsterdam criteria, Khong et al. (2016)) provide a vital framework for placental evaluation, the innovations discussed herald a new era of “enhanced placental histology” (Figure 4; Table 3). Here, subtle microstructural alterations with macroscale implications can be detected and quantified, advancing both scientific understanding and clinical practice. Future integration of these methods promises to bridge the remaining gaps between placental structure, function, and long-term health outcomes.