RT-qPCR detection of SARS-CoV-2 RNA in placentas of women with spontaneous abortion: a retrospective pilot study

Abstract

Background

Concerns about the possible effects of SARS-CoV-2 on pregnancy have been raised because ACE2 receptors are expressed in placental cells, and previous studies have suggested potential associations with adverse outcomes such as spontaneous abortion and preterm birth. This retrospective pilot study aimed to detect SARS-CoV-2 RNA in archived FFPE placental tissues from women who experienced spontaneous abortion.

Methods

This cross-sectional study analyzed 83 paraffin blocks of placentas from mothers who had spontaneous abortions between winter 2021 and summer 2022. RNA was extracted, assessed for quality and quantity, and RT-qPCR was employed to identify the SARS-CoV-2 genome. Histopathological evaluations were also performed, with results analyzed using SPSS version 26.

Results

Among the 83 samples, 35 mothers (42.2%) were aged 30 to 40, with ages from 17 to 57. The most common symptom was bleeding (57.83%). Maternal SARS-CoV-2 test returned negative for 73 (87.9%) and positive for 10 (12.1%). Half of the abortions occurred in the first trimester, and 25.3% of mothers had hypertension, while 9.6% had hyperthyroidism. Three placental samples (3.6%) tested positive for SARS-CoV-2 RNA and exhibited histopathological findings consistent with the placentitis triad previously reported in SARS-CoV-2-associated cases.

Conclusion

In this retrospective pilot study, SARS-CoV-2 RNA was detected in 3.6% of archived FFPE placental tissues from spontaneous abortion cases, and all positive samples showed histopathological features consistent with the placentitis triad described in previous reports. However, given the small number of positive cases and the limitations of the study design, these findings should be considered descriptive and hypothesis-generating, and no causal relationship can be inferred. Further prospective studies with larger cohorts are needed to clarify the relationship between SARS-CoV-2 infection and pregnancy outcomes.

Introduction

In late 2019, SARS-CoV-2 emerged as the causative agent of COVID-19, rapidly evolving into a global pandemic with significant morbidity, mortality, and strain on healthcare systems worldwide [1, 2]. Pregnant women are one of the categories that are most vulnerable to SARS-CoV-2 infections. As a result, it is vital to understand how the virus may affect embryonic development. Managing these patients is critical, as poor maternal and neonatal outcomes have been linked to both pandemic-related healthcare interruptions and the virus’s direct impacts [3, 4]. According to the data, SARS-CoV-2 infection rates in pregnant women range from 3% to 20%. The World Health Organization reports that 80% of cases show mild or no symptoms, while a few can result in severe complications. SARS-CoV-2 enters host cells via angiotensin-converting enzyme 2 (ACE2) receptors and the TMPRSS2 co-factor, both of which are expressed in placental tissue, supporting the biological plausibility of placental infection [5–7].

Emerging evidence suggests that SARS-CoV-2 can cross the placental barrier via receptors including ACE2 and Neuropilin-1, with viral presence in placental villi linked to early pregnancy losses and adverse fetal outcomes. While confirmed placental infections are rare, studies on virus-infected placental tissue have produced conflicting results [8, 9]. However, the frequency of placental infection and its relationship to adverse pregnancy outcomes, including spontaneous abortion, remain incompletely understood [2, 10]. In light of this knowledge gap, this retrospective pilot study aims to detect SARS-CoV-2 RNA in archived FFPE placental tissue from women with spontaneous abortion and to generate preliminary, hypothesis-driving data on a possible association between viral placental presence and pregnancy loss.

Methods

Study design and participants

This cross-sectional retrospective study analyzed 83 paraffin-embedded placental block samples obtained from mothers who experienced spontaneous abortion at Shahid Rahimi Hospital in Lorestan province, Khorramabad, Iran, between December 2021 and August 2022. This period coincided with the peak spread of the Omicron variant. Formalin-fixed paraffin-embedded (FFPE) placental tissue samples from spontaneous abortions were used. The inclusion criteria for this study comprised women who had experienced spontaneous abortion and had an established record of SARS-CoV-2 testing. Conversely, patients with incomplete data within hospital records were excluded from the study.

Demographic and clinical information of the patients is presented in Table 1, including age, history of abortion, vaccination status, trimester of pregnancy, presence of underlying disease, and symptoms of abortion. The ethics code IR.LUMS.REC.1402.101 was obtained from Lorestan University of Medical Sciences for conducting this study. Ethics approval was obtained prior to commencement of laboratory analyses and research use of the archived tissue; the FFPE blocks were collected during routine clinical care and subsequently accessed for research purposes under this approval.

Table 1.

Comparison of study variables in mothers with positive and negative SARS-CoV-2 test results with a history of spontaneous abortion

Variable	SARS-CoV-2 Negative n = 73	SARS-CoV-2 Positive n = 10	P-valueǂ
Age, mean ± SD	31.6 ± 8.3	34.4 ± 9.8	0.61
Age group, (%)
≤ 20	7 (9.6%)	-	0.7
20–30	26 (35.6%)	4 (40%)
30–40	31 (42.5%)	4 (40%)
≥ 40	9 (12.3%)	2 (20%)
History of miscarriage, n(%)
0	48 (65.8%)	2 (20%)	0.005
1	20 (27.4%)	5 (50%)
2	3 (4.1%)	3 (30%)
> 2	2 (2.7%)	-
Vaccination, n (%)
No	41 (56.2%)	8 (80%)	0.15
Yes	32 (43.8%)	2 (20%)
Trimester, n (%)
First	33 (45.2%)	9 (90%)	0.02
Second	27 (37%)	1 (10%)
Third	13 (17.8%)	-
Underlying condition, n (%)
Nothing	45 (61.6%)	9 (90%)	0.19
Hypertension	20 (27.4%)	1 (10%)
Hyperthyroid	8 (11%)	-
Symptoms, n (%)
Bleeding
No	36 (49.3%)	1 (10%)	0.01*
Yes	37 (50.7%)	9 (90%)
Abdominal pain
No	31 (42.5%)	5 (50%)	0.65*
Yes	42 (57.5%)	5 (50%)
Nausea
No	53 (72.6%)	10 (100%)	0.05*
Yes	20 (27.4%)	-
Amniotic fluid leakage
No	62 (84.9%)	10 (100%)	0.18*
Yes	11 (15.1%)	-
Backache
No	67 (91.8%)	10 (100%)	0.34*
Yes	6 (8.2%)	-

ǂ The significance level of P < 0.05 was indicated in bold

*The Fisher Exact test was used to evaluate the P-value

RNA extraction from FFPE tissue

For this research, FFPE tissue sections with a thickness of 6–8 micrometers were cut using a microtome, and 4–5 layers (200–300 milligrams) were used for RNA extraction from the paraffin-embedded tissue samples. Strict adherence to hygiene protocols, including the use of gloves and alcohol, was maintained throughout the process to prevent contamination. In brief, to eliminate paraffin from the tissue samples, xylene was applied three times, followed by washing with ethanol and phosphate-buffered saline (PBS). After the removal of paraffin, Proteinase K was employed for enzymatic digestion of proteins, and the samples were incubated overnight to ensure complete digestion. Subsequently, TRIzol reagent (Sinaclon, Iran) was added to each sample tube, followed by the addition of chloroform to facilitate phase separation. RNA was then precipitated using isopropanol, and the precipitated samples were washed with 75% cold ethanol. Finally, after drying the RNA pellet, either distilled water or a wash buffer was introduced to dissolve the RNA for downstream applications.

Examination of RNA quality and quantity, and molecular assays

Following RNA extraction from the aborted placental tissue, the quality and quantity of the extracted RNAs were evaluated. The NanoDrop (Thermo Scientific 2000c, United States) spectrophotometer was utilized to assess both the concentration and purity of the RNA samples. The presence of the RNase P gene was analyzed as an internal control to verify the adequacy of RNA extraction and the absence of PCR inhibition. The purity of the RNA was determined by examining the optical density (OD) ratios, specifically the absorbance ratio at 260 nm to 280 nm, with values ranging from 1.8 to 2.0 considered acceptable for high-quality RNA.

Ten microliters (equivalent to 700 nanograms) of the RNA sample were used to detect the SARS-CoV-2 virus genome using the One-step RT-PCR technique. The One-step multiplex Real-time PCR (Viga SARS-CoV-2, ROJE TECHNOLOGIES, Iran) for SARS-CoV-2 identification was performed using specific reagent concentrations: 5 µl of Q-ROMAX 4X, 4 µl of Pro Mix, 1 µl of RTase (Recombinant Reverse Transcriptase, RNase H), and 10 µl of isolated RNA, resulting in a final reaction volume of 20 µl. For the inclusion of positive and negative controls, equivalent volumes of the respective controls were substituted for the extracted RNA after adding the other reagents. The thermal protocol for this reaction involves several stages. Initially, the temperature is set to 50 °C for 20 min to allow for cDNA synthesis. This is followed by a temperature increase to 95 °C for 3 min to activate the polymerase enzyme. Subsequently, 45 cycles are conducted, which include denaturation at 95 °C for 10 s, followed by annealing and amplification of nucleic acids at 60 °C for 40 s. During this phase, fluorescence is assessed across the green, yellow, and orange channels, yielding essential data for the identification of the SARS-CoV-2 virus. The N and RdRp genes were designated as target genes, while the RNase P gene served as an internal control for RNA extraction efficiency and reaction inhibition. Samples with a cycle threshold (Ct) value of less than 40 were classified as positive. For fluorescent detection, the FAM channel (green) was utilized for the RdRp gene, the Texas Red channel (orange) for the N gene, and the HEX channel (yellow) for the RNase P gene.

Histopathological evaluation

A histological examination of placenta samples from both SARS-CoV-2-positive and -negative cases was conducted to evaluate any abnormalities. Samples were examined using a light microscope at magnifications of 40× and 100× following hematoxylin and eosin (H&E) staining, enabling a comparative analysis of histological characteristics. All slides were reviewed by a dedicated pathologist (Z. Haghighatian) with expertise in placental pathology. The pathologist was blinded to the RT-qPCR results and assessed the placental slides for the classic triad of SARS-CoV-2 placentitis (trophoblast necrosis, chronic histiocytic intervillositis, and increased perivillous fibrin) without knowledge of the viral RNA status.

Statistical analysis

In this study, the demographic information of the patients was collected from their medical records, and the data analysis was performed using SPSS version 26 software. Qualitative variables were reported as frequency and percentage, while quantitative variables were reported as mean and standard deviation. The chi-square test and Fisher’s exact test were used for qualitative variables. In all the tests, a significance level of P < 0.05 was considered.

Results

This study examined 83 pregnant women with a history of spontaneous abortion, of whom 73 (88%) had a negative SARS-CoV-2 test result, while 10 (12%) tested positive for SARS-CoV-2. The mean age of all participants was 31.93 ± 8.50 years, with the youngest being 17 years old and the oldest being 57 years old. Patients were divided into four age groups: ≤20, 20–30, 30–40, and > 40 years. The age distribution among the groups was as follows: 7 cases (8.4%) in the ≤ 20 years age range, 30 cases (36.1%) in the 20 to 30 years range, 35 cases (42.2%) in the 30 to 40 years range, and 11 cases (13.3%) in the > 40 years age group. The most common clinical symptoms included bleeding (57.83%), abdominal pain (27.71%), nausea (9.64%), amniotic fluid leakage (3.61%), and back pain (1.20%). In cases where multiple symptoms were reported, the first symptom of spontaneous abortion was considered. 59% of patients did not participate in the COVID-19 vaccination program, while 41% did participate. The timing of abortion was categorized into three trimesters: first, second, and third. Based on this categorization, 50.6% of patients experienced spontaneous abortion in the first trimester, 33.7% in the second trimester, and 15.7% in the third trimester. 65.1% of patients had no underlying medical conditions. High blood pressure was found in 25.3% of patients and hyperthyroidism in 9.6%. Additionally, 60.2% of patients had no history of miscarriage, while the remaining patients had experienced one (30.1%), two (7.2%), or three (2.4%) previous miscarriages.

Regarding maternal SARS-CoV-2 status, there was no statistically significant difference in mean age or vaccination status between the groups. However, statistically significant differences were observed in the occurrence of bleeding (p = 0.01) and nausea (p = 0.05), with higher rates in the SARS-CoV-2 positive group. The timing of spontaneous abortions also differed significantly (p = 0.02), with 90% of SARS-CoV-2 positive mothers experiencing first-trimester miscarriages compared to 45.2% in the negative group. A statistically significant difference between groups was also observed in previous abortion history (p = 0.005). Among SARS-CoV-2 positive mothers, 50% had a history of one previous abortion, whereas 65.8% of SARS-CoV-2 negative mothers reported no previous abortion history (Table 1). RT-qPCR analysis of 83 placental tissue samples from spontaneous abortions was performed to detect SARS-CoV-2. All samples showed amplification of the internal control (RNase P gene), confirming assay validity. Three samples (3.6%) were positive for SARS-CoV-2 RNA, with amplification of the N and RdRp genes at Ct values < 40; The RdRp Ct values reported in the SARS-CoV-2 RNA-positive samples were 26, 31, and 33, consistent with the detection of viral RNA in placental tissue. A lower Ct value (e.g., 26) is generally associated with a higher amount of target RNA, although any Ct value below 40 was considered a positive result in this study (Table 3). Maternal age showed no significant association with SARS-CoV-2 placental positivity (p = 0.42). While two of three positive cases were in the 30–40 year age group, the age distribution of negative samples was broader, with 41.2% in the 30–40 year group and 37.5% in the 20–30 year group. Furthermore, 66.7% (2/3) of SARS-CoV-2 positive placental samples were from mothers who tested negative for SARS-CoV-2 (Table 2). Among the 80 SARS-CoV-2 negative placental samples, 88.7% were from mothers who also tested negative (p = 0.24; Table 2). None of the mothers with SARS-CoV-2 positive placentas had a documented history of COVID-19 vaccination. In contrast, 42.5% of mothers with SARS-CoV-2 negative placentas reported COVID-19 vaccination, while 57.5% did not (p = 0.14). This difference was not statistically significant. Gestational age at spontaneous abortion was similar between groups: 66.7% (2/3) of SARS-CoV-2 positive placentas were associated with first-trimester abortions, and 33.3% with second-trimester abortions; whereas, for SARS-CoV-2 negative placentas, these proportions were 50%, 33.7%, and 16.3% for first, second, and third trimesters, respectively (p = 0.72). Among mothers with SARS-CoV-2 positive placentas, 66.7% had a history of hypertension; the remaining had no reported pre-existing conditions. No significant difference in pre-existing conditions was observed between the groups (p = 0.23). All mothers with SARS-CoV-2 positive placentas presented with vaginal bleeding and abdominal pain. In total, 10 mothers (12.1%) had a positive SARS-CoV-2 RT-qPCR, whereas 3 placental samples (3.6%) were positive (Table 2), and the characteristics of these three placenta-positive cases are summarized in Table 3. Histological examination of the three SARS-CoV-2 RNA-positive placentas (3/3, 100%) showed findings consistent with the placentitis triad previously described in SARS-CoV-2-associated cases; however, given this very small number, these observations should be regarded as preliminary (Fig. 1). In contrast, SARS-CoV-2 RNA-negative placental samples (0/80, 0%) demonstrated normal placental architecture, without evidence of the placentitis triad or other significant histological abnormalities, a finding that is consistent with but does not confirm viral causality.

Table 3.

Characteristics and RT-qPCR results of the three SARS-CoV-2 placenta-positive cases

Patient	Age (years)	Maternal SARS-CoV-2 Status	Symptoms	COVID-19 Vaccination	Trimester of Abortion	Underlying Condition	Prior Abortions (n)	Placental SARS-CoV-2 Result	RT-qPCR Ct Values (per gene)
									RdRp	N Gene	RNase P (Internal Control)
Case 1	35	Positive	Bleeding	No	First	None	1	Positive	26	28	20
Case 2	32	Negative	Bleeding	No	Second	Hypertension	1	Positive	31	30	18
Case 3	41	Negative	Bleeding	No	First	Hypertension	0	Positive	33	34	22

Abbreviations: Ct cycle threshold, RdRp RNA-dependent RNA polymerase gene (FAM/green channel), N gene nucleocapsid gene (Texas Red/orange channel), RNase P internal RNA quality control gene (HEX/yellow channel). Ct values <40 were considered positive

Table 2.

Results of SARS-CoV-2 genome identification in the placental tissue of miscarriage samples

Variable	Overall Cohort (n = 83)	SARS-CoV-2 Placenta Negative (n = 80)	SARS-CoV-2 Placenta Positive (n = 3)	P-value
RT-qPCR Results
SARS-CoV-2 RNA detected, n (%)	3 (3.6)	0 (0)	3 (100)	< 0.001*
Ct value range	–	–	26–33	–
Maternal SARS-CoV-2 Test Concordance
Concordant (both positive or both negative)	72 (86.7)	71 (88.7)	1 (33.3)	0.24
Discordant (placenta+/mother − or placenta−/mother+)	11 (13.3)	9 (11.3)	2 (66.7)
Trimester of Abortion, n (%)
First	42 (50.6)	40 (50.0)	2 (66.7)	0.72
Second	28 (33.7)	27 (33.8)	1 (33.3)
Third	13 (15.7)	13 (16.3)	0 (0)
Age Group, n (%)
≤ 20 years	7 (8.4)	6 (7.5)	1 (33.3)	0.42
20–30 years	30 (36.1)	29 (36.3)	1 (33.3)
30–40 years	35 (42.2)	34 (42.5)	1 (33.3)
≥ 40 years	11 (13.3)	11 (13.8)	0 (0)
Underlying Condition, n (%)
None	54 (65.1)	52 (65.0)	1 (33.3)	0.23
Hypertension	21 (25.3)	19 (23.8)	2 (66.7)
Hyperthyroidism	8 (9.6)	8 (10.0)	0 (0)
COVID-19 Vaccination, n (%)
No	49 (59.0)	46 (57.5)	3 (100)	0.14
Yes	34 (41.0)	34 (42.5)	0 (0)
Prior Abortions, n (%)
0	50 (60.2)	48 (60.0)	2 (66.7)	–
1	25 (30.1)	24 (30.0)	1 (33.3)
2	6 (7.2)	6 (7.5)	0 (0)
> 2	2 (2.4)	2 (2.5)	0 (0)
Histopathological Findings
Placentitis triad present, n (%)	3 (3.6)	0 (0)	3 (100)	< 0.001*
Normal architecture, n (%)	80 (96.4)	80 (100)	0 (0)

* Fisher’s exact test used. Ct, cycle threshold; Ct values < 40 were considered positive. Concordant cases are those in which maternal and placental SARS-CoV-2 results are the same (both positive or both negative). Discordant cases are those in which maternal and placental results differ (placenta negative/maternal positive or placenta positive/maternal negative)

Fig. 1.

Histopathological examination of placental tissue from SARS-CoV-2 negative and positive cases. Hematoxylin and eosin (H&E) staining at 40× and 100× magnification. A SARS-CoV-2 negative placental sample showing normal placental architecture without significant pathological changes. B-D SARS-CoV-2 positive placental samples demonstrating the characteristic "placentitis triad" associated with SARS-CoV-2 infection, consisting of trophoblast necrosis (black arrows), chronic histiocytic intervillositis with inflammatory cell infiltration in the intervillous space (yellow arrows), and massive perivillous fibrin deposition (black circles). Images were acquired using a Leica light microscope equipped with LAS EZ software

Discussion

The present study detected SARS-CoV-2 RNA in 3.6% (3/83) of archived FFPE placental samples from women with spontaneous abortion during the Omicron variant peak in Iran (winter 2021–summer 2022). The mean patient age of 31.93 years and the predominance of the 30–40 age group (42.2%) are consistent with a study conducted in Turkey in 2021, where a similar age distribution was reported among mothers experiencing spontaneous abortion [11]. However, it is important to emphasize from the outset that the cross-sectional, retrospective design of this study does not permit causal inference between SARS-CoV-2 RNA detection in placental tissue and spontaneous abortion; the low positivity rate (3.6%) and the absence of statistically significant associations for most clinical variables indicate that these findings should be considered hypothesis-generating and interpreted with caution, particularly regarding vertical transmission and direct fetal effects.

The highest proportion of spontaneous abortions occurred in the first trimester (50.6%), consistent with a UK cohort study reporting a 1.7-fold higher miscarriage risk among COVID-19-infected women in the first trimester compared to non-infected controls [12]. A substantial proportion of patients had comorbidities independently associated with miscarriage risk: 25.3% had hypertension and 9.6% had hyperthyroidism rates higher than those reported in comparable studies (8.6% and 5.8%, respectively) representing important confounders that cannot be disentangled from any potential viral contribution in this retrospective setting [13]. Consistent with a Canadian cohort study reporting no statistically significant association between SARS-CoV-2 vaccination and miscarriage risk [14], vaccination status did not differ significantly between groups in our cohort.

The placental detection rate of 3.6% is consistent with other retrospective series: Horn et al. [15] demonstrated that placental SARS-CoV-2 infection occurs in fewer than 10% of SARS-CoV-2-positive mothers, manifesting as acute placental failure with massive perivillous fibrin deposition and trophoblast necrosis. Konstantinidou et al. [16] identified SARS-CoV-2 placentitis in 6 of 165 (3.6%) placentas from COVID-19-affected pregnancies, all associated with stillbirth, collectively supporting the rarity of placental viral invasion despite maternal infection. Biological plausibility for placental infection is supported by the confirmed expression of ACE2 and TMPRSS2 in syncytiotrophoblast and cytotrophoblast cells, with direct immunohistochemical evidence of focal SARS-CoV-2 RNA detection in syncytiotrophoblast [17]. Transplacental transmission, while possible, remains rare, estimated at approximately 1.1–5.2% of cases [18, 19].

All three SARS-CoV-2 RNA-positive placentas demonstrated the complete placentitis triad — trophoblast necrosis, chronic histiocytic intervillositis, and massive perivillous fibrin deposition, consistent with the foundational characterization by Bouachba et al. [20] and the multicenter validation by Schwartz et al. [21] encompassing 68 perinatal deaths (64 stillbirths and 4 neonatal deaths). The triad’s apparent specificity for SARS-CoV-2 infection was further established by Remoué et al., subsequently confirmed the apparent specificity of this triad for SARS-CoV-2 infection in a retrospective analysis of 8,857 pre-COVID placentas (2002–2012), none of which demonstrated the complete constellation, yielding a cumulative incidence of 0% (95% CI: 0–0.04%) before SARS-CoV-2 emergence [22]. Broader meta-analytic data confirm that SARS-CoV-2-infected pregnancies are associated with maternal vascular malperfusion (30.7%), perivillous fibrin deposition (32.7%), and chronic inflammatory pathologies (25.65%), supporting a plausible mechanistic link between viral placental infection and adverse pregnancy outcomes [23], although direct causality cannot be established in this retrospective study. The triad was present in all three SARS-CoV-2 RNA-positive placentas and entirely absent in the 80 negative samples; these observations are consistent with but do not confirm a causal viral role, and should be interpreted as hypothesis-generating given the very small number of positive cases.

Although the placentitis triad has been most prominently reported in association with the Delta variant, emerging evidence confirms that Omicron variants retain placental tropism. Essaidi-Laziosi et al. demonstrated that Omicron BA.1, BA.2, and BA.5 can infect and replicate in primary first-trimester trophoblasts, albeit with lower replicative efficiency than Delta [24]. Shanes et al. reported placentitis cases during the Omicron period [25], and Radan et al. confirmed placental SARS-CoV-2 replication with histiocytic intervillositis during Omicron dominance [26]. Variant-specific genotyping of the three positive samples was not performed, owing to the well-recognized challenges of whole-genome sequencing from FFPE-extracted RNA and funding constraints; however, based on epidemiological probability during the Omicron BA.1/BA.2 peak in Iran, it is reasonable to attribute the observed findings to this variant.

The maternal-placental discordance (2/3 placenta-positive cases had negative maternal swabs) is consistent with published evidence and likely reflects one or more of the following: earlier maternal infection with subsequent upper respiratory clearance before the time of testing ; Omicron clears the upper respiratory tract significantly faster than Delta (mean 13.24 vs. 17.30 days); false-negative nasopharyngeal RT-qPCR results (reported sensitivity 70–95% depending on timing and variant); or infection via a transient and undetected viremic phase, given that approximately 35–43% of SARS-CoV-2 infections are asymptomatic or pauci-symptomatic [27–29]. Together, these factors underscore that maternal swab negativity does not reliably exclude placental SARS-CoV-2 infection, and prospective studies with serial maternal and placental sampling are needed to clarify the dynamics of maternal-placental viral discordance [30, 31].

This study has several limitations that should be acknowledged. First, and most critically for the interpretation of our molecular findings, the detection of SARS-CoV-2 RNA relied solely on RT-qPCR performed on FFPE-extracted RNA, without orthogonal confirmation by immunohistochemistry (IHC), in situ hybridization (ISH), or subgenomic RNA detection; although these approaches would have strengthened the validity of our findings, funding and resource constraints prevented their application. FFPE processing is well known to cause RNA fragmentation and chemical modification, increasing the risk of false-positive signals from degraded viral RNA. To address RNA integrity, the RNase P gene was included as an internal control in all 83 samples, with amplification confirmed in every case with Ct values below 40, providing uniform evidence of extractable RNA quality across the cohort. Nevertheless, a positive RNase P signal does not exclude partial viral RNA degradation, nor does it distinguish residual non-replicating viral RNA from active infection. The absence of IHC or ISH confirmation limits localization of viral protein or RNA to specific placental cell types, and the absence of subgenomic RNA detection prevents conclusions regarding active viral replication. These constraints must be weighed centrally in interpreting the molecular findings, and future investigations combining RT-qPCR with IHC and ISH on fresh-frozen tissue are essential to confirm productive placental infection. Second, several potential confounding factors including hypertension, thyroid disorders, and other comorbidities independently associated with miscarriage risk, could confound the relationship between SARS-CoV-2 and spontaneous abortion. Third, information on relevant exposures (smoking, BMI, medications, detailed obstetric history) was incomplete or not systematically recorded, limiting adjustment for these variables. Fourth, the timing of SARS-CoV-2 infection relative to gestational age was not always precisely known, and asymptomatic or past infections may have been missed, potentially leading to misclassification of maternal infection status, particularly in discordant cases where the placenta tested positive but the maternal swab was negative. Fifth, selection bias cannot be excluded, as only women with available FFPE blocks and relatively complete records were included. Finally, variant-specific genotyping was not performed due to FFPE RNA quality constraints and funding limitations, preventing variant-stratified analysis of placental pathology. The overall sample size and especially the very small number of SARS-CoV-2-positive placentas, substantially reduces statistical power, increases the risk of type II error and unstable estimates, and precludes definitive attribution. Together, these limitations support a cautious and hypothesis-generating interpretation, and future studies with larger prospective cohorts, fresh tissue analysis, multimodal diagnostic confirmation, and variant sequencing are needed to provide robust evidence on the impact of SARS-CoV-2 on placental pathology and pregnancy outcomes.

Conclusion

In this retrospective cross-sectional study of 83 FFPE placental samples from spontaneous abortions during the Omicron variant peak (winter 2021–summer 2022), SARS-CoV-2 RNA was detected in 3.6% (3/83) of tissues by RT-qPCR (Ct values: 26, 31, 33), with all three positive placentas demonstrating the “placentitis triad” (trophoblast necrosis, chronic histiocytic intervillositis, massive perivillous fibrin deposition) absent in negative samples; 66.7% (2/3) were associated with first-trimester and 33.3% (1/3) with second-trimester abortions. Although ACE2 receptor expression provides biological plausibility and recent literature confirms SARS-CoV-2 can induce such histopathological alterations, viral RNA detection does not establish direct causality. This represents the first systematic investigation of SARS-CoV-2 in archived FFPE placental tissue from spontaneous abortion cohorts; however, several substantial limitations preclude definitive attribution: small sample size (n = 3), absence of immunohistochemical/in situ hybridization confirmation, potential FFPE RNA degradation, incomplete clinical data and retrospective design preventing assessment of viral replication markers. We cannot definitively determine whether the placentitis triad is SARS-CoV-2 specific or a non-specific inflammatory pattern. These findings are hypothesis-generating, warranting larger prospective studies with fresh tissue analysis, immunohistochemistry, in situ hybridization, and subgenomic RNA detection to validate the association between SARS-CoV-2 infection and placental histopathology.

Authors’ contributions

Reza Parnian: Sample collection, RNA extraction and RT-qPCR experiments, data analysis, and manuscript writing, Mania Seyfi: Contributed to laboratory procedures including RNA extraction and RT-qPCR assay optimization; critically reviewed the manuscript, Zahra Haghighatian: Sample acquisition, histopathological evaluation and interpretation of placental slides; critically reviewed the manuscript, Zahra Heydarifard: Conceptualization, study design and methodology, data interpretation, manuscript writing and critical revision, and supervision of the project, Sayad Khanizadeh: Contributed to data interpretation and critical revision of the manuscript for important intellectual content, Gholamreza Talei: Contributed to data interpretation and critical revision of the manuscript for important intellectual content. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work.

Funding

None.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study was conducted in accordance with the Declaration of Helsinki and national and institutional standards and it was approved in the Ethics Committee of Lorestan University of Medical Sciences with the approval code IR.LUMS.REC.1402.101. Patients signed written informed consents and accepted the publication of clinical data for research.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.